Molecular and probiotic characterization of ... - Wiley Online Library

24 downloads 887 Views 304KB Size Report
Certain LAB have also been described to exhibit pro- biotic activity because they .... agar plates were seeded with a bacterial lawn of each indi- cator strain at a ...
Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Molecular and probiotic characterization of bacteriocinproducing Enterococcus faecium strains isolated from nonfermented animal foods S.V. Hosseini1*, S. Arlindo1, K. Bo¨hme1, C. Ferna´ndez-No1, P. Calo-Mata1,2 and J. Barros-Vela´zquez1,2 1 Department of Analytical Chemistry, Nutrition and Food Science, LHICA, School of Veterinary Sciences, University of Santiago de Compostela, Lugo, Spain 2 Biotechnology Laboratory, College of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain

Abstract

Keywords aquaculture, bacteriocins, enterocins, Enterococcus, food biopreservation, nonfermented foods, probiotics. Correspondence Jorge Barros-Vela´zquez, Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago, Campus Universitario, E-27002 Lugo, Spain. E-mail: [email protected] *Present address Department of Fisheries and Environmental Sciences, Faculty of Natural Resources, University of Tehran, PO Box 31585-4314, Karaj, Tehran, Iran.

2009 ⁄ 0119: received 20 January 2009, revised 23 February 2009 and accepted 11 March 2009 doi:10.1111/j.1365-2672.2009.04327.x

Aims: The characterization of four novel bacteriocin-producing enterococcal strains, isolated from nonfermented animal foods, was carried out with a view to evaluate their potential application as probiotics in raw and processed foodstuffs. Methods and Results: 16S rRNA sequencing and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis allowed the identification and intra-specific grouping of Enterococcus faecium strains, which inhibited the growth of four relevant food-borne pathogenic and spoilage species. Enterococcus faecium strains exhibited remarkable probiotic profiles, being able to survive to pH 3Æ0 and to the presence of bile salts, pancreatin and pepsin. Enterococcus faecium strains evaluated did not exhibit bile salt hydrolase or haemolytic activity, but showed good adhesion properties, also exhibiting sensitivity to clinically relevant antimicrobial agents. Conclusions: In our study, DNA sequencing of the 16S rRNA gene and RAPDPCR analysis were equally discriminatory for typing E. faecium strains. This study also confirmed the potential tolerance and survival of E. faecium strains isolated from nonfermented animal foods to the gastrointestinal tract. Significance and Impact of the Study: This study represents the first report on potential probiotic E. faecium strains isolated from nonfermented meat and fish. Their moderate heat resistance opens the way to their potential use as probiotics in minimally processed foods subjected to moderate heat processing.

Introduction The presence of spoilage and pathogenic micro-organisms in foods is a major concern for the food industry, the administration and consumers. This has moved food technologists to develop a wide variety of preservation processes aimed at destroying microbial cells or at delaying their growth (Marth 1998). However, the increasing demand of consumers for safe but minimally processed fresh products, together with the increasing concerns about the use of certain chemical preservatives, has raised a significant interest to the development and use of novel 1392

natural bio-preservation methods (Cotter et al. 2005). In this sense, the ability of lactic acid bacteria (LAB) to inhibit the growth of undesirable bacteria is well known and has been appreciated by man for more than 10 000 years. Among LAB metabolites, bacteriocins – ribosomallysynthesized, small, cationic, amphiphilic antibacterial peptides – are the subject of intense research because of their antibacterial activity against food-borne pathogenic and spoilage bacteria (Todorov and Dicks 2005). Certain LAB have also been described to exhibit probiotic activity because they are components of the natural microflora of almost all organisms, are rarely pathogenic

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

S.V. Hosseini et al.

and present antagonistic properties against pathogenic micro-organisms (Klein et al. 1998). According to the definition of the World Health Organization (WHO), probiotics are living micro-organisms which, when administered in adequate amounts, confer a health benefit on the host (Gilliland et al. 2001). Mattila-Sandholm et al. (2002) reported that the utilization of probiotics in food stimulated the growth of desirable micro-organisms, crowding out potentially harmful bacteria and reinforcing the body’s natural defense mechanisms. Owing to their perceived health benefits, probiotic bacteria have been increasingly included in yoghurts and fermented milk during the past two decades (Succi et al. 2005). Recent studies have also supported a role for probiotics as a part of a healthy diet for humans and animals and may be an avenue to provide a safe and cost-effective barrier against microbial infection (Parvez et al. 2006). Likewise, the risks associated to the use of antimicrobial agents for the therapeutic treatment of sick animals and fish has been a controversial issue in the last decade. This has caused an increasing demand for environment-friendly feeding practices in farms and aquaculture facilities, this provoking that the use of probiotics in animal nutrition is now widely accepted (Doyle 2006; Wang et al. 2008). The characterization of a LAB strain as a probiotic culture should involve the investigation of its inhibition pattern, its safety and both its functional and technological characteristics. Prior to inducing any effect, most living probiotic cultures are orally administered. Accordingly, probiotic cultures should be resistant or able to survive the specific conditions of the gastrointestinal tract (GIT), such as the presence of proteolytic enzymes and low pH values prevailing in the stomach, and to bile salts and pancreatic juices in the gut (Verschuere et al. 2000). Probiotic cultures should also be antagonistic to pathogenic bacteria by producing antimicrobial substances and, obviously, they must be safe for human use and maintain their viability and beneficial properties during manufacturing processes (Schillinger et al. 2005). Little information is available about the potential application as probiotics of bacteriocin-producing enterococci isolated from nonfermented foods. In our laboratory, we have undertaken the isolation and preliminary characterization of E. faecium bacterocinogenic strains from raw meat and raw fish (Arlindo et al. 2006; Campos et al. 2006). Such strains were isolated after long storage refrigeration periods, thus indicating their resistance to low temperatures. The antimicrobial activities exhibited by these strains had been previously reported to be sensitive to proteinase K and not linked to hydrogen peroxide or lactic acid production, thus suggesting the proteinaceous nature of the substance(s) responsible for the inhibition (Arlindo et al. 2006; Campos et al. 2006).

Probiotic E. faecium strains

Therefore, the objective of the present investigation was to perform a complete characterization of four selected novel food-grade enterococcal strains exhibiting antilisterial and anti-staphylococcal activity, both in their genetic and probiotic profiles, with a view to elucidate their potential application as probiotics in raw or processed foods destined for human and animal consumption. Materials and methods Bacterial strains and culture conditions The bacteriocin-producing LAB strains used in this study belonged to the collection of the Laboratory of Food Hygiene and Control (LHICA) at the University of Santiago de Compostela. Strains LHICA 28Æ4, LHICA 34Æ5 and LHICA 40Æ4 were isolated from vacuum-packaged beef while strain LHICA 46 was isolated from refrigerated turbot (Psetta maxima). The nisin-producing Lactococcus lactis ATCC 11454 strain was considered as a positive control in bioassays aimed at detecting bacteriocin production. All LAB were maintained as frozen stocks at )80C. Before experimental use, LAB strains were recovered in de Man Rogosa Sharpe (MRS) broth (Oxoid, Ltd, London, UK) and plated on MRS agar (Oxoid). When required, a 48-h culture of each strain was centrifuged at 7000 rev min–1 for 15 min and the culture supernatant was sterilized by filtration through 0Æ22 lm pore membrane (Millex GS; Millipore, St. Quentin, France) and kept at 4C for assaying bacteriocin production against eight indicator strains corresponding to five different gram-positive species (Table 2). Such indicator strains included reference strains from international culture collections as well as pathogenic and ⁄ or specific spoilage micro-organisms isolated at our laboratory (LHICA) and involved in food intoxication or food spoilage events. All indicator strains were maintained as frozen stocks at )80C and were recovered in Mueller– Hinton broth (Oxoid) without shaking at 37C for 48 h prior to being used as bacterial lawns in susceptibility disc diffusion assays, as previously described (Campos et al. 2006). Molecular characterization of Enterococcus faecium strains and their bacteriocins Phenotypic and genetic identification of enterococcal strains by nucleotide sequencing of the 16S rRNA gene All four LAB strains considered in this study were phenotypically identified as members of the genus Enterococcus based on the following criteria (Schleifer and Kilpper-Ba¨lz 1984): Gram-positive, catalase-negative, facultatively anaerobic chain-forming cocci able to ferment glucose

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

1393

Probiotic E. faecium strains

S.V. Hosseini et al.

and exhibiting the ability to grow at 10 and 45C in media containing 6Æ5% NaCl at pH 9Æ6, and in Chromocult enterococci-selective agar (Merck, Darmstadt, Germany). Phenotypic characterization was also performed by the miniaturized API 20 STREP tests (BioMe´rieux, Marcy L’Etoile, France). Total genomic DNA from the enterococcal strains was isolated from the pellets of 1Æ5 ml of overnight cultures after spinning at 7500 rev min)1 (10 min), as described elsewhere (Campos et al. 2006). Bacterial DNA was purified from each pellet by means of the DNeasy tissue minikit (Qiagen, Valencia, CA, USA), based on the use of microcolumns. The concentration of purified DNA extracts was determined by measuring the fluorescence developed after mixing with the Hoechst 33258 reagent (Sigma, St Louis, MO, USA) on an LS50 fluorimeter (Perkin Elmer, Wellesley, MA, USA). The universal primer set p8FPL ⁄ p806R was used for the amplification of c. 800 bp fragment of the 16S rRNA bacterial gene (McCabe et al. 1995). All amplification assays comprised 100 ng of template DNA, 25 ll of a master mix (BioMix, Bioline, London, UK) – this including the reaction buffer, dNTP, magnesium chloride and Taq DNA polymerase – double-distilled water (Genaxis, Montigny le Bretonneaux, France) and 25 pmol of each oligonucleotide primer to achieve a final volume of 50 ll. All PCR assays were carried out in duplicate on a MyCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). PCR products were processed in 2Æ5% horizontal agarose (MS-8; Pronadisa, Madrid, Spain) gels. Prior to sequencing, the PCR products were purified by means of the ExoSAP-IT kit (GE Healthcare, Amersham Biosciences, Uppsala, Sweden). Direct sequencing was performed with BigDye Terminator ver. 3.1 Cycle Sequencing Kit (Applied Biosystems). The same primers used for PCR were considered for the sequencing of both strands of the PCR products, respectively. Sequencing reactions were analysed in an automatic sequencing system (ABI 3730XLDNA Analyzer; Applied Biosystems). All nucleotide sequences were carefully reviewed by eye, using the Chromas software (Griffith University, Queensland, Australia) and were compared among them by alignment of sequences using the ClustalW software and with known sequences in the NCBI database by using the Blast tool. Genetic characterization of Enterococcus faecium strains by random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis Intra-specific comparison of strains was accomplished by RAPD-PCR analysis. This was performed using 200 ng of template DNA and 14 pmol of M13 primers (5¢-GAG GGTGGCGGTTCT-3¢; Huey and Hall 1989). Amplifica1394

tion reactions were: initial denaturation at 94C for 5 min, followed by 33 cycles of 94C ⁄ 60 s, annealing at 45C ⁄ 60 s, extension at 72C ⁄ 60 s and a final extension step at 72C ⁄ 15 min. The PCR products were separated and visualized by means of 1Æ5% agarose electrophoresis. To check reproducibility, the PCR assays were performed at least thrice for each strain. The molecular weight (MW) of each PCR product was estimated by comparison with a DNA ladder by means of the 1D software (TDI, Madrid, Spain). The presence or absence of each DNA band in each MW range was recorded as ‘ones’ or ‘zeros’ in the positive and negative reactions, respectively, and used to prepare cluster dendrograms with the Statgraphycs software (Statpoint Inc, Herndon, VA, USA). Genetic identification of enterocins produced by Enterococcus faecium strains The identification of enterocins produced by E. faecium strains was performed by PCR using the primers described for the following bacteriocins: EntA, EntB, EntL50 and EntP (Du Toit et al. 2000); EntP(2), EntQ and EntL50 (Cintas et al. 2000); open reading frame (ORF; Kawamoto et al. 2002) and Mun1 (Saavedra et al. 2004). Such primers allow the amplification of the most relevant IIa class bacteriocins produced by enterococci. Nucleotide sequencing of the enterocin genes were performed as described before. Probiotic evaluation of bacteriocin-producing Enterococcus faecium strains Inhibition spectra of Enterococcus faecium strains against pathogenic and spoilage bacteria Antimicrobial activity of LAB strains over indicator bacterial strains was screened by means of a standardized agar disc diffusion method. Briefly, Mueller–Hinton (Oxoid) agar plates were seeded with a bacterial lawn of each indicator strain at a concentration of 105 CFU ml)1. Then, 20 ll portions of each culture supernatant from each LAB strain – grown at 30C ⁄ 24 h in MRS broth without shaking – were placed on 6 mm sterile discs (Oxoid), which had previously been placed on the agar plates. The plates were incubated overnight at 37C, antimicrobial activity being detected as translucent halos in the bacterial lawn surrounding the discs. Negative controls were prepared by adjusting the pH of the MRS broth (Oxoid) aliquots to 3Æ5 to mimic the pH value of the LAB cultures grown at 30C ⁄ 24 h. Assay of acid tolerance Bacterial cells from overnight MRS cultures were harvested by centrifugation and washed with sterile phosphate buffer saline [PBS buffer, per litre:

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

S.V. Hosseini et al.

di-potassium hydrogen phosphate (Merck), 1Æ41 g; potassium di-hydrogen phosphate (Merck), 0Æ26 g; sodium chloride (Merck), 8Æ0 g; pH 7Æ2]. Centrifugation ⁄ washing procedures were repeated thrice. The bacterial cells were finally re-suspended in sterile PBS buffer adjusted to pH 2Æ0, 3Æ0 or 4Æ0, respectively, and incubated at 37C for 3 h. The viable bacterial counts were then determined in brain heart infusion (BHI) agar (Liofilchem, Italy). Assay of bile salt hydrolase (BSH) and bile salts tolerance Bacterial cells from overnight MRS cultures were harvested by centrifugation, washed and re-suspended in freshly prepared PBS (pH 8Æ0) supplemented with 0Æ5%, 1Æ0% or 2Æ0% (w ⁄ v) oxgall (Sigma), or with 0Æ5%, 1Æ0% or 2Æ0% (w ⁄ v) taurodeoxycholic acid (Sigma). After an incubation period of 4 h at 37C, the viable cell counts were determined in BHI agar. For the BSH assay, overnight bacterial cultures of each E. faecium strain were streaked on MRS agar, supplemented with 0Æ5% (w ⁄ v) taurodeoxycholic acid or oxgall, respectively, and incubated for 24 and 48 h at 37C. The bacterial hydrolysis of bile salts was visualized as altered colony morphology as compared with the control MRS plates, and by the visualization of precipitation zones around the colonies. Haemolytic activity Overnight MRS cultures were seeded on Columbia agar plates (Oxoid) containing 5% (v ⁄ v) sheep blood (Oxoid) and incubated for 48 h at 37C. The zones around the colonies suggested the type of haemolysis: green zone for a-haemolysis, clear zone for b-haemolysis and no halo for c-haemolysis. Bacterial resistance to pepsin and pancreatin Bacterial cells from overnight MRS cultures were collected, washed and re-suspended in either PBS (pH 8Æ0) supplemented with either 1 mg ml)1 of pancreatin (Sigma) or in PBS buffer (pH 2Æ0 and 3Æ0) supplemented with 3 mg ml)1 of pepsin, respectively. Resistance of the E. faecium strains to such enzymes was assessed by determining the initial viable cell counts in BHI agar and after incubation at 37C for 3 h (pepsin) or at 37C for 4 h (pancreatin). Sensitivity to antimicrobial agents Antibiotic susceptibility testing was performed in Mueller–Hinton plates (Oxoid) by the agar diffusion disc method recommended by the National Committee for Clinical Laboratory Standards (NCCLS 1998). Antimicrobial discs were purchased from BioMe´rieux. The following antimicrobial agents were tested: ampicillin (10 lg), cefazolin (30 lg), clindamycin (2 lg), cloram-

Probiotic E. faecium strains

phenicol (30 lg), doxycyclin (30 lg), erythromycin (5 lg), fosfomycin (50 lg), gentamycin (10 lg), oxacillin (1 lg), penicillin (10 IU), rifampicin (30 lg), streptomycin (10 lg), sulfamide (200 lg), tetracycline (30 lg) and vancomycin (30 lg). Briefly, freshly prepared cultures of the E. faecium strains were adjusted to a 0Æ5 value of the McFarland scale and seeded on Mueller–Hinton agar plates. Then, discs containing each antimicrobial agent were placed on the surface of the plate. Antibiotic resistance was assessed by measuring the diameter around the discs (mm) after 24 h of incubation at 37C, according to the NCCLS (1998) recommendations. Bacterial adhesion to stainless steel plates Adhesion of E. faecium strains was evaluated as previously described (Giaouris et al. 2005; Paramithiotis et al. 2006) with a slight modification. Briefly, the stainless steel plates (2Æ5 cm · 0Æ8 cm · 0Æ5 mm) were initially soaked in acetone overnight. Afterwards, the plates were immersed for 45 min in detergent solution at 50C under agitation. The plates were then thoroughly washed in tap water followed by several washes in distilled water and were allowed to air dry. Then, the dry plates were autoclaved at 121C for 15 min. Overnight bacterial cultures (0Æ5 ml in MRS broth) of each E. faecium strain were transferred to a glass tube containing 4Æ5 ml of the MRS broth and a stainless steel plate. After 24 h of incubation at 37C each plate was aseptically removed, washed with 10 ml of sterile 1% peptone water (Oxoid) and placed for 5 min into a glass tube containing 5 ml of sterile 1% peptone water in order to remove the loosely adhered bacteria. The plates were washed again with 10 ml of sterile 1% peptone water, placed inside a glass tube containing 6 ml of sterile 1% peptone water and vortexed for 3 min in order to create a suspension of the enterococcal cells adhered to the surface of the plate. The cell numbers of this suspension were determined on MRS agar (Oxoid) after incubation at 37C for 24 h. The results were compared with the total initial cell numbers and the results were expressed as the percentage of adhered cells for each strain. Assay of bacterial heat resistance The assay of heat resistance in the enterococcal strains was performed according to the method described by Stopforth et al. (2008) with a slight modification. Briefly, 0Æ1 ml of a 109 CFU ml)1 suspension of each strain was aseptically placed into sterile capillary tubes and subjected to instantaneous heating in a water bath at four different temperatures – 60, 65, 70 and 80C – during four different incubation times – 30, 60, 180 and 300 s. After cooling, the cell suspensions were transferred onto BHI

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

1395

Probiotic E. faecium strains

S.V. Hosseini et al.

agar plates, incubated at 37C for 24 h and the viable counts were determined.

E. faecium LHICA284

(a)

E. faecium EU003448 70

E. faecium LHICA404 E. faecium LHICA345

Statistical analysis

E. faecium AB326300

All assays were performed in triplicate. Means and SD were calculated for each assay. One-way analysis of variance (anova) was used to explore the significance of differences owing to the parameters and levels tested. All tests were performed using Excel and spss version 13.5 (SPSS Inc., Chicago, IL, USA). A confidence interval of 95% (P < 0Æ05) was considered.

E. faecium LHICA46 E. faecium DQ672262 0·0001

(b)

M

1

2

3

4

1476 bp 1230 bp 984 bp 738 bp

Results 492 bp

Identification of bacteriocinogenic LAB strains As stated before, all four strains were gram-positive, catalase-negative, facultatively anaerobic chain-forming cocci, able to ferment glucose and grow at 10 and 45C in media containing 6Æ5% NaCl, pH 9Æ6. Additionally, the four strains were able to grow in chromocult enterococciselective agar. Once checked that the four LAB studied belonged to the Enterococcus genus, their genomic DNA was purified and a fragment of their 16S rRNA was amplified and sequenced. The alignment of the 16S rRNA sequences showed that all four strains exhibited very high homology with respect to other E. faecium strains deposited in the GenBank. These results allowed the classification of strains LHICA 28Æ4, 34Æ5, 40Æ4 and 46 as belonging to the species E. faecium. However, as it can be observed in Fig. 1a, strains LHICA 28Æ4, 34Æ5 and 40Æ4 on the one hand, and strain LHICA 46 on the other hand, were grouped into two different clusters together with other previously described E. faecium strains. Further genetic intra-specific characterization of the E. faecium isolates was performed by RAPD-PCR with M13 primers. The results revealed two different profiles: one shared by strains LHICA 28Æ4, 34Æ5 and 40Æ4, and a different profile characteristic of strain LHICA 46 (Fig. 1b). Cluster analysis assigned 100% homology among the three former E. faecium strains, all of them isolated from meat, while strain LHICA 46, isolated from turbot, only exhibited 40% homology with respect to them, a result that was in agreement with nucleotide sequencing and phylogenetic analyses. Although strains 28Æ4, 34Æ5 and 40Æ4 seemed to be alike according to genetic analyses, several phenotypic differences were assessed for strain 28Æ4 with respect to the other two related strains (Table 1). Thus, up to 5 of the 20 biochemical tests considered in the API 20 STREP provided a different phenotype for strain 28Æ4 as compared with the other two similar strains, the latter two exhibiting identical phenotypes. 1396

Figure 1 (a) Phylogenetic relationships according to the nucleotide sequences of the 16S rRNA fragments, among the four Enterococcus faecium strains described in this work, as compared with a selection of type E. faecium strains identified with their GenBank accession numbers; (b) random amplified polymorphic DNA-polymerase chain reaction of E. faecium strains with M13 primers; lane M, MW marker; lane 1, E. faecium LHICA 28Æ4; lane 2, E. faecium LHICA 34Æ5; lane 3, E. faecium LHICA 40Æ4; and lane 4, E. faecium LHICA 46.

Table 1 Phenotypic differences among Enterococcus faecium strains 28Æ4, 34Æ5 and 40Æ4 Test

Phenotype

28Æ4

34Æ5

40Æ4

ESC b-GAL ADH ARA MAN

Esculin hydrolysis b-galactosidase production Arginine dihydrolase production Arabinose utilization Mannitol utilization

+ + + + +

) ) ) ) )

) ) ) ) )

Microbial inhibition spectra and identification of bacteriocins produced by Enterococcus faecium strains All four strains were tested for their antimicrobial activity against at least four gram-positive species (Table 2), these including relevant pathogenic and specific spoilage bacteria. Thus, Bacillus cereus ATCC 11040, Carnobacterium maltaromaticum, Listeria monocytogenes and Staphylococcus aureus were sensitive to all four E. faecium strains. Interestingly, E. faecium LHICA 46 also exhibited antimicrobial activity against other B. cereus and Bacillus subtilis strains, a result that was not observed for all the other three LAB strains. Total DNA from the four bacteriocin-producing enterococcal strains was subjected to amplification with

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

S.V. Hosseini et al.

Probiotic E. faecium strains

Table 2 Antimicrobial activity of culture supernatants from strains of Enterococcus faecium LHICA 28Æ4, 34Æ5, 40Æ4 and 46 on Gram-positive pathogenic and specific spoilage micro-organisms

Bacillus cereus ATCC 11040 B. cereus CLHICA 2383 Bacillus subtilis CLHICA 0702721 B. subtilis CLHICA 6A Carnobacterium maltaromaticum CLHICA 2383 C. maltaromaticum CLHICA 2384 Listeria monocytogenes ATCC 11994 Staphylococcus aureus ATCC 35845

Control*

28Æ4

12Æ0 14Æ0 14Æ0 14Æ0 0 13Æ0 9Æ7 9Æ5

9Æ0 0 0 0 14Æ0 12Æ0 11Æ0 8Æ0

± ± ± ±

0Æ1 0Æ1 0Æ1 0Æ1

± 0Æ1 ± 0Æ7 ± 0Æ5

34Æ5 ± 0Æ5

± ± ± ±

9Æ0 0 0 0 15Æ0 18Æ0 10Æ0 8Æ0

0Æ1 0Æ1 1Æ0 0Æ5

40Æ4 ± 1Æ0

± ± ± ±

10Æ0 0 0 0 15Æ0 15Æ0 11Æ0 8Æ0

0Æ1 0Æ1 1Æ0 0Æ1

46 ± 0Æ5

± ± ± ±

11Æ0 14Æ0 9Æ0 11Æ0 9Æ0 9Æ0 11Æ0 9Æ5

0Æ1 0Æ1 1Æ0 1Æ0

± ± ± ± ± ± ± ±

0Æ5 0Æ1 1Æ5 0Æ1 1Æ0 0Æ5 1Æ0 1Æ0

Results are expressed as diameters of the inhibition zone and SD in mm. *The nisin-producing strain, Lactococcus lactis ATCC 11454, was included as a positive control.

primers targeted to the enterocins genes, as stated before. Positive amplifications were obtained for all four strains only for enterocin P primers (Fig. 2b). Interestingly, while strains E. faecium LHICA 28Æ4, 34Æ5 and 40Æ4 yielded a 132 bp PCR product with primers EntP(2), strain E. faecium LHICA 46 produced a 432 bp fragment with such primers. Sequencing of the PCR products confirmed the presence of the structural gene encoding enterocin P in strains E. faecium LHICA 28Æ4, 34Æ5 and 40Æ4, all three sequences exhibiting 98% homology as compared with the previously described enterocin P genes (Fig. 2c). The larger fragment amplified from strain LHICA 46 with EntP(2) primers clustered in a different branch confirming its lack of homology with respect to enterocin P genes and shared 95% homology with the hypothetical protein EfaeDRAFT 0811 from E. faecium deposited in the GenBank under accession number ZP_00604146 (data not shown).

(a)

1

(b)

M

3

2

1

2

3

4

M

4

500 bp 400 bp 200 bp 100 bp

(c) 10 E. faecium AF005726 5 E. faecium GM1 AY728265 9 E. faecium LHICA404 46 E. faecium LHICA284 34 E. faecium LHICA345 E. faecium ATB197a AY633748 E. faecium AB075741 E. faecium LHICA46

1

Characterization of the probiotic profile of Enterococcus faecium strains The effect of low pH on the viability of all four E. faecium strains is shown in Table 3. Significant (P < 0Æ05) decreases in the bacterial numbers were observed after 3 h of exposure to low pH. However, although no viable cells were detected at pH 2Æ0, the reduction ranges after 3 h of exposure to pH 3Æ0 and 4Æ0 were 6Æ07–9Æ27% and 0Æ49–6Æ13%, respectively. The highest resistance was observed in strains E. faecium LHICA 46 and 28Æ4 at pH 3Æ0 and 4Æ0, respectively. Exposure to different concentrations of oxgall and taurodeoxycholic acid significantly (P < 0Æ05) affected the viability of strains E. faecium LHICA 28Æ4 and 34Æ5. Nevertheless, all four strains were able to grow in high numbers in the presence of oxgall (Table 4) and taurodeoxycholic acid (Table 5), and the effect of bile salts differed depending on the E. faecium strain consid-

Figure 2 (a) 1–4, inhibition of Carnobacterium maltaromaticum CLHICA 2383 by Enterococcus faecium strains 28Æ4, 34Æ5, 40Æ5 and 46, respectively. (b) Amplification of bacteriocins produced by E. faecium strains with EntP(2) primers; lane M, MW marker; lane 1, E. faecium LHICA 28Æ4; lane 2, E. faecium LHICA 34Æ5; lane 3, E. faecium LHICA 40Æ4; and lane 4, E. faecium LHICA 46. (c) Phylogenetic relationships according to the nucleotide sequences of the bacteriocin genes, among the four bacteriocinogenic E. faecium LHICA strains described in this work, as compared with the previously described bacteriocinproducing E. faecium strains identified with their GenBank accession numbers.

ered, with strains E. faecium LHICA 40Æ4 and 46 exhibiting the highest resistance to both oxgall and taurodeoxycholic acid. Remarkably, none of the E. faecium strains under study hydrolysed taurodeoxycholic acid or oxgall. Likewise, none of the E. faecium strains was able to hydrolyse sheep blood, all of them proving to be nonhaemolytic.

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

1397

Probiotic E. faecium strains

S.V. Hosseini et al.

Table 3 Effect of low pH on the viability of Enterococcus faecium strains pH 2Æ0

pH 3Æ0

0h E. E. E. E.

faecium faecium faecium faecium

28Æ4 34Æ5 40Æ4 46

8Æ13 8Æ06 8Æ21 8Æ26

± ± ± ±

0Æ11 0Æ05 0Æ14 0Æ07

3h

0h

0 0 0 0

8Æ08 8Æ19 8Æ18 8Æ24

pH 4Æ0 3h

± ± ± ±

0Æ06a 0Æ05a 0Æ07a 0Æ05a

0h

7Æ43 7Æ43 7Æ67 7Æ74

± ± ± ±

0Æ10b 0Æ18b 0Æ11b 0Æ06b

8Æ13 8Æ22 8Æ32 8Æ10

3h ± ± ± ±

0Æ03a 0Æ04a 0Æ04a 0Æ02a

8Æ09 8Æ12 7Æ81 7Æ75

± ± ± ±

0Æ05a 0Æ05b 0Æ03b 0Æ07b

± ± ± ±

0Æ06b 0Æ03a 0Æ07a 0Æ02a

± ± ± ±

0Æ02b 0Æ03b 0Æ07a 0Æ08a

All results are expressed as log CFU ml)1. Values in the same row followed by a different letter are significantly different (P < 0Æ05).

Table 4 Effect of oxgall concentration on the viability of Enterococcus faecium strains 0Æ5%

1%

0h E. E. E. E.

faecium faecium faecium faecium

28Æ4 34Æ5 40Æ4 46

8Æ03 8Æ12 8Æ18 7Æ53

4h ± ± ± ±

0Æ05a 0Æ04b 0Æ03a 0Æ05a

7Æ85 8Æ41 8Æ11 7Æ51

2%

0h ± ± ± ±

0Æ08b 0Æ02a 0Æ04a 0Æ04a

8Æ11 7Æ98 8Æ25 7Æ60

4h ± ± ± ±

0Æ02a 0Æ05b 0Æ03a 0Æ01a

7Æ49 8Æ33 8Æ24 7Æ52

0h ± ± ± ±

0Æ06b 0Æ01a 0Æ05a 0Æ03b

8Æ10 7Æ90 8Æ24 7Æ48

4h ± ± ± ±

0Æ02a 0Æ1a 0Æ02a 0Æ03b

7Æ34 7Æ96 8Æ26 7Æ56

All results are expressed as log CFU ml–1. Values in the same row followed by a different letter are significantly different (P < 0Æ05).

Table 5 Effect of taurodoxycholic acid concentration on the viability of Enterococcus faecium strains 0Æ5%

1%

0h E. E. E. E.

faecium faecium faecium faecium

28Æ4 34Æ5 40Æ4 46

8Æ31 8Æ24 7Æ93 7Æ75

4h ± ± ± ±

a

0Æ07 0Æ02a 0Æ04a 0Æ03b

8Æ18 7Æ76 7Æ82 7Æ93

2%

0h ± ± ± ±

b

0Æ03 0Æ06b 0Æ06a 0Æ04a

8Æ34 8Æ17 7Æ92 7Æ73

4h ± ± ± ±

a

0Æ02 0Æ02a 0Æ06a 0Æ01a

8Æ21 7Æ65 7Æ90 8Æ37

0h ± ± ± ±

b

0Æ07 0Æ04b 0Æ11a 0Æ55a

8Æ06 8Æ25 7Æ86 7Æ57

4h ± ± ± ±

a

0Æ05 0Æ05a 0Æ03a 0Æ07b

7Æ89 7Æ80 7Æ86 7Æ79

All results are expressed as log CFU ml)1. Values in the same row followed by a different letter are significantly different (P < 0Æ05).

As no viability had been observed for any of the strains after exposure to pH 2Æ0, the addition of pepsin at this pH value had no detectable effect. In contrast, all four E. faecium strains survived well when exposed to pepsin at pH 3Æ0, the viability rates being in the range of 89– 94% (Table 6). Moreover, all four E. faecium strains proved to be resistant to pancreatin at pH 3Æ0, with viability rates in the range of 96–98% (Table 6). Among them, strains E. faecium LHICA 28Æ4 and 46 proved to be the most sensitive to pepsin and pancreatin, respectively. The antibiotic susceptibility patterns for E. faecium strains are shown in Table 7. On the one hand, all four strains were sensitive to cloramphenicol, doxycyclin, penicillin, rifampicin, tetracycline and, interestingly, also to vancomycin. On the other hand, all strains exhibited resistance to cefazolin, clindamycin, oxacillin and sulfamide. Only strain E. faecium LHICA 46 was not sensitive to ampicillin, gentamycin and streptomycin. All four E. faecium strains were able to adhere to stainless steel plates (Fig. 3). Strain E. faecium LHICA 46 1398

proved to exhibit the highest adhesion rates as c. 59% of the bacterial cells successfully adhered to steel plates in vitro. The adhered population for the other E. faecium strains was above 55%, except for E. faecium 40Æ4, which exhibited the lowest adhesion rates (43Æ6%). The effect of moderate heating on the viability of the E. faecium strains is shown in Fig. 4. In global terms, all strains decreased significantly (P < 0Æ05) their numbers as temperature or time of treatment increased. Thus, as it can be observed in Fig. 4, none of the E. faecium strains was able to survive to 80C or even 5 min at 75C. In contrast, all strains resisted well for 5 min at 60C and survived well for an exposure of 3 min at 65C. Discussion Four E. faecium strains isolated from meat and fish were characterized in their genetic and probiotic profiles in this study. The antimicrobial activities exhibited by these strains had been previously described as sensitive to

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 1392–1403

S.V. Hosseini et al.

Probiotic E. faecium strains

Table 6 Effect of pepsin and pancreatin on the viability of Enterococcus faecium strains

Pepsin (pH 3Æ0) 0h E. E. E. E.

faecium faecium faecium faecium

28Æ4 34Æ5 40Æ4 46

Pancreatin (pH 8Æ0) 3h

8Æ92 9Æ03 8Æ98 8Æ73

± ± ± ±

0Æ02a 0Æ02a 0Æ02a 0Æ04a

7Æ97 8Æ25 8Æ46 8Æ06

0h ± ± ± ±

0Æ12b 0Æ23b 0Æ11b 0Æ08b

8Æ76 8Æ90 8Æ56 8Æ89

4h ± ± ± ±

0Æ01a 0Æ03a 0Æ01a 0Æ03a

8Æ57 8Æ86 8Æ96 8Æ54

± ± ± ±

0Æ01b 0Æ01a 0Æ03b 0Æ01b

All results are expressed as log CFU ml)1. Values in the same row followed by a different letter are significantly different (P < 0Æ05).

Table 7 Antimicrobial susceptibility testing of Enterococcus faecium strains

Antimicrobial agent

28Æ4

34Æ5

40Æ4

46

S

R

Ampicillin Cefazolin Clindamycin Cloramphenicol Doxycyclin Erythromycin Gentamycin Fosfomycin Oxacillin Penicillin Rifampicin Streptomycin Sulfamide Tetracycline Vancomycin

S R R S S MS S R R S S S R S S

S R R S S R S S R S S S R S S

S R R S S R S R R S S S R S S

MS R R S S S MS R R S S R R S S

‡19Æ0 ‡18Æ0 ‡15Æ0 ‡23Æ0 ‡19Æ0 ‡22Æ0 ‡10 ‡14 ‡13Æ0 ‡17Æ0 ‡19Æ0 ‡9Æ0 ‡17Æ0 ‡19Æ0 ‡17Æ0