Open Access

Evaluation of bacteriocinogenic activity, safety traits and biotechnological potential of fecal lactic acid bacteria (LAB), isolated from Griffon Vultures (Gyps fulvus subsp. fulvus)

  • Sara Arbulu1,
  • Juan J. Jiménez1,
  • Loreto Gútiez1,
  • Cristina Campanero1,
  • Rosa del Campo2,
  • Luis M. Cintas1,
  • Carmen Herranz1 and
  • Pablo E. Hernández1Email author
BMC MicrobiologyBMC series – open, inclusive and trusted201616:228

https://doi.org/10.1186/s12866-016-0840-2

Received: 11 May 2016

Accepted: 15 September 2016

Published: 29 September 2016

Abstract

Background

Lactic acid bacteria (LAB) are part of the gut microbiota and produce ribosomally synthesized antimicrobial peptides or bacteriocins with interest as natural food preservatives and therapeutic agents. Bacteriocin-producing LAB are also attractive as probiotics. Griffon vultures (Gyps fulvus subspecies fulvus) are scavenger birds that feed almost exclusively on carrion without suffering apparent ill effects. Therefore, griffon vultures might be considered a reservoir of bacteriocin-producing lactic acid bacteria (LAB) with potential biotechnological applications.

Results

Griffon vulture feces were screened for LAB with antimicrobial activity, genes encoding bacteriocins, potential virulence determinants, susceptibility to antibiotics, genotyping and characterization of bacteriocins. In this study, from 924 LAB evaluated 332 isolates (36 %) showed direct antimicrobial activity against Gram-positive bacteria only. The molecular identification of the most antagonistic 95 isolates showed that enterococci was the largest LAB group with antimicrobial activity (91 %) and E. faecium (40 %) the most identified antagonistic species. The evaluation of the presence of bacteriocin structural genes in 28 LAB isolates with the highest bacteriocinogenic activity in their supernatants determined that most enterococcal isolates (75 %) encoded multiple bacteriocins, being enterocin A (EntA) the largest identified (46 %) bacteriocin. Most enterococci (88 %) were resistant to multiple antibiotics. ERIC-PCR and MLST techniques permitted genotyping and recognition of the potential safety of the bacteriocinogenic enterococci. A multiple-step chromatographic procedure, determination of the N-terminal amino acid sequence of purified bacteriocins by Edman degradation and a MALDI TOF/TOF tandem MS procedure permitted characterization of bacteriocins present in supernatants of producer cells.

Conclusions

Enterococci was the largest LAB group with bacteriocinogenic activity isolated from griffon vulture feces. Among the isolates, E. faecium M3K31 has been identified as producer of enterocin HF (EntHF), a bacteriocin with remarkable antimicrobial activity against most evaluated Listeria spp. and of elevated interest as a natural food preservative. E. faecium M3K31 would be also considered a safe probiotic strain for use in animal nutrition.

Keywords

Lactic acid bacteria (LAB) Antimicrobial activity Bacteriocins Enterococci Virulence traits Probiotics

Background

Lactic acid bacteria (LAB) are part of the gut microbiota of mammals and birds with an important role in environmental, food and clinical microbiology. Many LAB produce ribosomally synthesized antimicrobial peptides or bacteriocins which attract considerable interest as natural and nontoxic food preservatives [1], and as therapeutic agents for human and veterinary applications and in the animal production field [2, 3]. Bacteriocin-producing LAB are also attractive vectors for delivery of therapeutic peptides and proteins and as probiotics [46].

Most bacteriocins produced by LAB are synthesized as biologically inactive precursors or prepeptides containing an N-terminal extension that is cleaved off during export to generate their biologically active or mature form. They are generally divided into two main classes. Class I consists of the lanthionine-containing post-translationally modified bacteriocins or lantibiotics, while class II consists of the bacteriocins containing non-modified amino acids. The class II bacteriocins may be further subdivided into the pediocin-like (class IIa) bacteriocins, the two-peptide (class IIb) bacteriocins, the cyclic (class IIc) bacteriocins, and the non-pediocin-like one peptide linear (class IId) bacteriocins [7, 8]. However, additional subgroups have been suggested for leaderless peptides, circular bacteriocins, linear peptides derived from large proteins, and the glycosylated bacteriocins [9].

Environmental sources as well as wild and game animals are a powerful source of bacteriocin-producing LAB [1012]. Griffon vultures (Gyps fulvus subspecies fulvus) belong to the Old World vultures group, a diverse mix of colonial cliff-nesting scavenger birds that play an essential ecological role as garbage collectors and recyclers. Their feeding habits are based almost exclusively on carrion, preferentially of mammals. Consequently, their gut microbiota may derive from ecological and evolutionary strategies for carrion exploitation [13]. Symbiotic relationships among animal hosts and bacteria that confer protection against pathogens are widespread in nature and are considered a driving force in evolution [14]. Therefore, griffon vultures might be considered as potential reservoirs of bacteriocin-producing LAB, with potential biotechnological applications. This work constitutes a first approach on the evaluation of the antimicrobial activity and safety aspects of bacteriocinogenic LAB isolated from griffon vulture feces.

Methods

Microbiological analysis, indicator strains and bacteriocinogenic assays

Fresh fecal samples from griffon vultures (Gyps fulvus subspecies fulvus) were collected from the Parque Natural del Alto Tajo (Molina de Aragón, Guadalajara, Spain), and placed into sterile disposable plastic tubes at 4 °C. Samples were 10-fold diluted in sterile peptone water (Oxoid Ltd., Basingstoke, UK) and homogenized in a stomacher. Aliquots from decimal dilutions of the homogenates were spread on duplicate plates of (i) Triptycase Soya Agar (TSA; Oxoid) at 37 °C, 48 h for aerobic mesophilic counts, (ii) on de Man, Rogosa, and Sharpe (MRS) plates (Oxoid) at 37 °C, 48 h for growth of LAB, and (iii) on Slanetz and Bartley (SB) plates (Oxoid) and Kanamycin Aesculin Azide (KAA) plates with the kanamycin selective supplement (Oxoid) at 37 °C, 48 h for growth of the enterococci. The direct antimicrobial activity of randomly selected isolates was screened by the stab-on-agar test (SOAT) [15] against three Gram-positive and four Gram-negative indicator bacteria. Next, cell-free supernatants of isolates producing halos of inhibtion larger than 7 mm or antimicrobial activity against, at least, three Gram positive indicator strains were evaluated for their antimicrobial activity by an agar diffusion test (ADT), against a larger number of Gram-positive indicators. Finally, the most active isolates were also evaluated for the antimicrobial activity of their supernatants by a microtiter plate assay (MPA) [15] against Listeria spp. strains. In the MPA test, one bacteriocin unit (BU) is defined as the reciprocal of the highest dilution of the bacteriocin causing 50 % growth inhibition (50 % of the turbidity of the control culture without bacteriocin). Supernatants were subjected to proteolytic treatment with proteinase K (Sigma-Aldrich GmbH, Madrid, Spain), at 10 mg/ml for 37 °C during 2 h, to ascertain the protein nature of their antagonistic activity. After proteinase inactivation by heat treatment (100 °C, 10 min), samples were assayed for residual antimicrobial activity by ADT, as described above, using Pediococcus damnosus CECT4797 as the indicator microorganism. Strains with antimicrobial activity in their supernatants and susceptible to proteinase treatment were considered Bac+ and selected for further characterization. Indicator strains and specific bacterial growth conditions used in this study are shown in Table 1.
Table 1

Indicator species and specific bacterial growth conditions used in this study

Organism

Origin/Referencea

Growth conditions

Medium

T (°C)

Gram-positive

   

Enterococcus faecalis

   

  BFE 1071

[54]

MRS

37

  DAC9

DNBTA

MRS

37

  DBH9

DNBTA

MRS

37

  DBH18

DNBTA

MRS

37

  INIA 4

INIA

MRS

37

  JH2-2

HRC

MRS

37

  P4

IFR

MRS

37

  V583

LMG

MRS

37

Enterococcus faecium

   

  L50

DNBTA

MRS

37

  M3K31

This work

MRS

37

  P13

DNBTA

MRS

37

  T136

DNBTA

MRS

37

Enterococcus hirae DCH5

DNBTA

MRS

37

Lactobacillus sakei 2714

NCDO

MRS

37

Lactococcus lactis BB24

DNBTA

MRS

37

Listeria grayii 931

CECT

BHI

37

Listeria innocua 910

CECT

BHI

37

Listeria ivanovii 913

CECT

BHI

37

Listeria seeligeri 917

CECT

BHI

37

Listeria welshimeri 919

CECT

BHI

37

Listeria monocytogenes 911

CECT

BHI

37

Listeria monocytogenes 935

CECT

BHI

37

Listeria monocytogenes 936

CECT

BHI

37

Listeria monocytogenes 939

CECT

BHI

37

Listeria monocytogenes 4031

CECT

BHI

37

Listeria monocytogenes 4032

CECT

BHI

37

Pediococcus damnosus 4797

CECT

MRS

32

Pediococcus pentosaceus FBB61

ATCC

MRS

32

Gram-negative

   

Aeromonas salmonicida 3776

LMG

TSB

28

Campylobacter jejuni 33560

ATCC

BHI + 1 % horse serum

37

Campylobacter jejuni 11168

NCTC

BHI + 1 % horse serum

37

Yersinia ruckeri 3279

LMG

TSB

28

aAbbreviations as: ATCC American Type Culture Collection, VA, USA, CECT Colección Española de Cultivos Tipo, Valencia, Spain, DNBTA Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain, HRC Servicio de Microbiología, Hospital Universitario Ramón y Cajal, Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain, IFR Institute of Food Research, Norwich, UK, INIA Instituto Nacional de Investigación y Tecnologıía Agraria y Alimentaria, Madrid, Spain, LMG Laboratorium voor Microbiologie, University of Ghent, Ghent, Belgium, NCDO, National Collection of Dairy Organisms, Aberdeen, Scotland, UK; NCTC National Collection of Type Type Cultures, Salisbury, UK

PCR analysis, DNA sequencing and other DNA manipulations

PCR amplifications were performed from total bacterial DNA obtained using the InstaGene matrix (Bio-Rad laboratories Inc., Hercules, CA, USA) in 25 or 50 μl reaction mixtures containing MyTaq mix buffer (Bioline Reagents Ltd., London, UK), 0.7 μM of each primer and 1 μl of purified DNA. Oligonucleotide primers were obtained from Sigma Genosys Ltd. (Cambridge, UK). Samples were subjected to PCR amplification in an Eppendorf Mastercycler thermal cycler (Eppendorf, Hamburg, Germany). When needed, the resulting PCR fragments were purified using the NucleoSpin Extract II kit (Macherey-Nagel, Düren, Germany) and sequenced at the Unidad de Genómica (Parque Científico de Madrid, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Spain).

Genus and species identification, and detection of bacteriocin structural genes and potential virulence factors

From the 332 LAB isolates showing direct antimicrobial activity, 95 of them were taxonomically identified by PCR amplification and sequencing of genes encoding 16S rRNA (16S rDNA) with primers plb16/mlb16 [16], and the gene encoding superoxide dismutase (sodA) with primers d1/d2 [17]. Genus and species identification was performed by nucleotide BLAST analysis using the NCBI platform.

The presence of bacteriocin structural genes of previously described bacteriocins, was evaluated in 28 LAB isolates with the highest antimicrobial activity in their supernatants. A total of 21 bacteriocin structural genes were analysed including (i) the pediocin-like class IIa bacteriocins avicin A (AviA), bacteriocin 31 (Bac31), bacteriocin MC4-1 (BacMC4-1), enterocin A (EntA), enterocin SE-K4 (EntSE-K4), enterocin P (EntP), hiracin JM79 (HirJM79), mundticin L (MunL) and pediocin PA-1 (PedA-1); (ii) the (ii) two-peptide class IIb bacteriocins enterocin 1071A-1071B (Ent1071A-Ent1071B) and enterocin XA-XB (EntXA-EntXB); (iii) the circular class IIc bacteriocin enterocin AS-48 (EntAS-48); (iv) the class IId non-pediocin-like one peptide linear bacteriocins including the leaderless bacteriocins enterocin L50 (EntL50A-EntL50B), enterocin JS (EntJSA-EntJSB) and enterocin Q (EntQ), and other small heat-stable linear bacteriocins such as enterocin B (EntB), enterocin 96 (Ent96), enterocin IT (EntIT), enterocin V583 (EntV583) and brevicin 925A (BreB); as well as the large bacteriolysin enterolysin A (EnlA). The specific oligonucleotide primers, PCR conditions, positive control strains and references concerning each of the bacteriocin structural genes, are shown in Additional file 1: Table S1.

The presence of genes coding potential virulence factors was evaluated in the nine most active bacteriocinogenic E. faecalis isolates, by using primer pairs and PCR conditions designed for detection of genes cylL LcylL S (cytolysin precursor), cylM (postranslational modification of cytolysin), cylB (transport of cytolysin), cylA (activation of cytolysin), ace (adhesin to collagen), agg (aggregation substance), esp (enterococcal surface protein), efaAfm and efaAfs (cell wall adhesins of E. faecium and E. faecalis, respectively) and gelE-sprE and sprE (gelatinase and serine protease E), as previously described [10, 18].

Safety assessment of E. faecium M3K31

The safety assessment of the E. faecium M3K31 isolate was determined according to guidelines established by the European Food Safety Authority (EFSA) [19], including the evaluation of (i) ampicillin resistance, (ii) determination of esp, a putative glycosyl hydrolase (hly Efm ) and identification of the insertion sequence IS16 [20].

Production of gelatinase, caseinolytic and hemolytic activity, and antibiotic susceptibility testing

For production of gelatinase, single colonies of the most active nine bacteriocinogenic E. faecalis isolates, previously grown on MRS agar (Oxoid), were streaked onto Todd-Hewitt agar (Oxoid) containing 30 g of gelatin (Oxoid) per liter, grown overnight at 37 °C, and placed at 4 °C for 5 h before examination of zones of turbidity around the colonies. The caseinolytic activity of the isolates was evaluated by streaking the colonies onto TSA agar (Oxoid) containing 1.5 % bovine skim milk powder (Oxoid) and overnight growth at 37 °C. A clear zone of hydrolysis within 24 h of growth was considered positive. For investigation of their haemolytic activity, the strains streaked on Columbia agar supplemented with 5 % (v/v) horse blood (COH, BioMérieux, Madrid, Spain) were grown at 37 °C for 1 to 2 days. Haemolysis was evidenced by the formation of clear zones surrounding the colonies on blood agar plates. The antibiotic susceptibility of the 27 selected enterococci with the highest antimicrobial activity in their supernatants was determined by overlaying antibiotic-containing disks (Oxoid) on the Diagnostic Sensitivity Test Agar (Oxoid), following the Clinical and Laboratory Standards Institute (CLSI) guidelines [21]. The antibiotics tested were ampicillin (10 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), gentamicin (120 μg), nitrofurantoin (300 μg), norfloxacin (10 μg), penicillin G (10 IU), rifampicin (5 μg), teicoplanin (30 μg), tetracycline (30 μg), and vancomycin (30 μg). Inhibition zone diameters were measured after overnight incubation of the plates at 37 °C. Resistance phenotypes were recorded as recommended by the CLSI [21]. E. faecalis ATCC29212 and Staphylococcus aureus ATCC25923, were used as control strains.

Enterobacterial repetitive intergenic consensus sequences (ERIC-PCR) and multilocus sequence typing (MLST) analysis

The clonal relationship among the 9 E. faecalis and 14 E. faecium isolates from griffon vultures feces with the highest antimicrobial activity in their supernatants and from other enterococci from food, environmental and clinical origin was evaluated by ERIC-PCR, as previously described [20]. The resulting patterns were interpreted after constructing dendrograms using the unweighted-pair group method with arithmetic mean (UPGM) and the similarity on the Dice’s coefficient, analyzed with the Phoretix v5.0 software (Nonlinear Dynamics Ltd., UK). For MLST analysis, purified genomic DNA from selected enterococcal isolates was used for PCR-amplification of internal fragments of seven housekeeping genes, as previously described [22]. The resulting PCR products were purified with an ExoSAP-IT PCR clean up reagent (USB Europe GmbH, Staufen, Germany) and sequenced in a ABI Prism 377 automated sequencer (Applied Biosystems, Foster City, CA, USA) at the Servicio de Microbiología, Hospital Universitario Ramón y Cajal, and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid (Spain). Clusters of related sequence types (STs) were grouped into clonal complexes (CCs) by using eBURST (http://www.mlst.net).

Purification of bacteriocins

The peptides responsible of the antimicrobial activity of E. faecium M1M10, E. faecium M3K31 and E. faecium T136 (producer of EntA) were purified to homogeneity by using a multichromatographic procedure, as previously described [23, 24]. Briefly, 1-L cultures of supernatants from the early stationary phase were subjected to precipitation with ammonium sulphate (50 %, w/v), desalted by gel filtration (PD columns) and further subjected to cation-exchange (SP Sepharose Fast Flow) and hydrophobic-interaction (Octyl-Sepharose CL-4B) chromatographies, followed by reverse phase chromatography (PepRPC HR 5/5) in a fast-protein liquid chromatography system (ÄKTA, RP-FPLC). The most active fractions from the last chromatographic step were combined and rechromatographied on the reverse-phase column to obtain the purified bacteriocin. All the chromatographic columns and equipment were from GE Healthcare (Madrid, Spain).

Mass spectrometry analysis and amino acid sequencing

Purified peptide fractions from the last RP-FPLC step were subjected to matrix-assisted laser desorption–ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) at the Unidad de Proteómica, Universidad Complutense de Madrid (Madrid, Spain). MALDI-TOF MS analyses were performed in a 4800 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA, USA), operated in 1 KV reflector mode. All mass spectra were calibrated externally using a standard peptide mixture (AB Sciex, Foster City, CA, USA).

MALDI TOF/TOF tandem mass spectrometry (MS) was used to determine the partial amino acid sequence of the purified peptide produced by E. faecium M3K31. Acquisition of the MS data was performed on an Ultraflex MALDI-TOF/TOF (Bruker Daltonics Inc. Billerica, MA, USA) instrument operated in reflection mode with delayed extraction, at the Proteomics Core Facility of the Norwegian University of Life Sciences, Ås (Norway). MS/MS spectra of selected peptides were recorded using the LIFT ion optics of the mass spectrometer. Recorded spectra were processed in flexAnalysis software (v3.3, Bruker Daltonics) and mass lists submitted to database searches (via BioTools software, v 3.2, Bruker Daltonics) were performed using an in-house Mascot server (v.2.1). Manual annotation of MS/MS spectra (de novo sequencing) was performed in flexAnalysis. The sequences generated were searched against NCBI/taxonomy Firmicutes using protein BLAST.

For N-terminal amino acid sequencing, the purified peptide from E. faecium M1M10 was subjected to automatic Edman degradation and sequence on polyvinylidene difluoride membranes (PVDF) in a Procise 494 HT Sequencing System (Applied Biosystems Inc., Foster City, CA, USA) at the Centro de Investigaciones Biológicas (CIB, Madrid, Spain).

Results

Identification of isolates with antimicrobial activity

In this study, 406 randomly selected isolates from griffon vultures feces grown on MRS plates, 418 isolates grown in SB plates and 100 isolates grown on KAA plates, were evaluated for their direct antimicrobial activity (SOAT) against 3 Gram-positive indicators (Pediococcus damnosus CECT4797, Lactococcus lactis BB24 and Listeria innocua CECT910) and 4 Gram-negative bacteria (Yersinia ruckeri LMG3279, Aeromonas salmonicida LMG3776, Campylobacter jejuni ATCC33560 and Campylobacter jejuni NCTC11168). From the 924 LAB evaluated, 332 isolates (36 %) showed direct antimicrobial activity against, at least, one of the Gram-positive bacterial indicators tested. However, no evidence for direct antimicrobial activity was shown against any of the four Gram-negative bacteria tested, including the two C. jejuni strains. From this initial screening, 95 LAB isolates with the largest halos of inhibition were identified by PCR amplification and sequencing of genes encoding 16S rDNA and sodA. From these results 38 E. faecium (40 %), 30 E. faecalis (31 %) 1 E. hirae (1 %), 1 E. mundtii (1 %), 5 Lactobacillus brevis (5 %) and 1 Lactobacillus plantarum (1 %) strains were identified. The remaining 19 isolates were identified as Enterococcus spp. (20 %).

From the above cited 95 LAB isolates, a set of 28 isolates comprising 9 E. faecalis, 16 E. faecium, 1 E. hirae, 1 E. mundtii and 1 Lb. brevis were tested for antimicrobial activity in their supernatants by an agar diffusion test (ADT). Among the E. faecalis isolates, E. faecalis M2M6 and E. faecalis M3M42 were active against a number of Gram-positive bacteria, but none of the isolates were active against any of the Gram-negative indicators tested (Table 2). When supernatants from E. faecium, E. hirae, E. mundtii and Lb. brevis were tested for their antimicrobial activity most E. faecium and the Lb. brevis isolate were active against most Gram-positive indicators, with no antagonistic activity observed for E. hirae M4S3. It is noteworthy to observe the high antimicrobial activity of E. faecium M3K31 against most of the bacterial Gram-positive indicators tested. However, none of the enterococcal and the Lb. brevis supernatants evaluated were active against C. jejuni ATCC33560 (Table 3). Supernatants of the most antagonistic LAB isolates (3 E. faecalis, 5 E. faecium, 1 E. mundtii, and 1 Lb. brevis) were also evaluated against 11 Listeria spp. by a microtiter plate assay (MPA). The supernatant of E. faecium M3K31 was remarkably active against most of the Listeria spp. evaluated (Table 4).
Table 2

Antimicrobial activitya of supernatants from selected E. faecalis isolates

Isolate

Indicator microorganismsb

E. faecalis

E. faecium

P. damnosus

4797

L. lactis

BB24

Y. ruckeri

3276

A. salmonicida

3276

C. jejuni

33560

BFE 1071

DAC9

DBH9

DBH18

INIA4

JH2-2

P4

V583

T136

L50

     

E. faecalis

               

AS10

-

-

-

-

-

-

-

9.7

-

-

7.2

-

-

-

-

M1M32

-

-

-

-

-

-

-

7.4

-

-

8.9

-

-

-

-

M1S19

-

-

-

-

-

-

-

-

-

-

7.2

-

-

-

-

M1S20

-

-

-

-

-

-

-

-

-

-

6.9

-

-

-

-

M2M6

9.2

14.1

11.5

-

-

-

9.6

11.1

12.0

-

16.8

-

-

-

-

M2M39

-

-

-

-

-

-

-

-

-

-

8.2

-

-

-

-

M3M42

9.4

13.6

10.0

-

-

-

8.6

8.3

12.6

-

10.3

-

-

-

-

M3S1

-

-

-

-

-

-

-

-

-

-

6.7

-

-

-

-

PM2-13

-

-

-

-

-

-

-

8.5

-

-

12.2

-

-

-

-

aAntimicrobial activity determined by ADT. Results as the diameter of the inhibition halos in millimeters (mm). (−) Antimicrobial activity not detected. Most of the data are means from two independent determinations in triplicate

bSource of indicator microorganisms indicated in Table 1

Table 3

Antimicrobial activitya of supernatants from isolated E. faecium and other lactic acid bacteria (LAB)

Isolate

Indicator microorganismsb

E. faecalis

E. faecium

E. hirae DCH5

L. lactis BB24

L. sakei 2714

L. innocua 910

L. monocytogenes 4032

P. damnosus 4797

P. pentosaceus FBB61

C. jejuni 33560

DBH18

INIA4

JH2-2

V583

L50

M3K31

P13

T136

        

E. faecium

 AS 41

11.5

7.6

11.7

12.8

15.2

12.7

13.4

-

13.6

-

17.9

12.1

13.4

20.4

-

-

 BS15

12.2

7.3

11.3

14.6

14.2

14.4

14.0

-

14.0

-

18.9

12.7

13.5

21.3

9.3

-

 CS14

11.5

7.1

12.4

12.6

16.9

14.6

15.0

-

14.2

-

18.8

12.1

12.4

22.9

8.6

-

 CS46

11.7

8.6

13.5

13.6

16.8

16.9

14.4

-

14.5

-

19.2

11.6

12.5

23.0

8.7

-

 M1M10

10.0

-

11.3

13.0

15.1

13.0

14.8

-

14.3

-

15.2

12.4

12.3

21.0

-

-

 M1M26

9.4

-

11.8

13.3

15.5

12.6

14.1

-

14.6

-

15.2

11.8

11.6

20.9

-

-

 M2M33

-

-

-

-

-

-

-

-

-

-

8.2

-

-

12.2

-

-

 M2S31

9.4

-

11.1

11.5

14.2

13.8

14.4

-

-

-

14.5

11.3

12.4

16.8

-

-

 M3M31

11.0

7.0

11.8

13.9

16.4

14.6

14.9

-

14.1

-

15.8

12.3

13.2

20.9

9.7

-

 M3M32

11.6

8.8

12.8

14.9

16.1

17.9

16.0

-

15.1

-

8.4

11.9

13.0

22.9

10.3

-

 M3K31

16.3

15.5

14.3

16.4

16.9

-

18.7

17.8

16.8

18.2

18.0

15.3

17.0

26.4

19.2

-

 M4M2

-

-

-

-

-

-

-

-

-

-

-

-

-

10.1

-

-

 PM1-27

11.2

-

10.7

14.0

13.9

12.7

15.8

-

-

-

19.0

11.1

13.6

17.2

-

-

 PM1-32

13.0

7.5

11.7

15.5

15.0

12.9

15.5

-

-

-

18.3

11.7

13.9

17.3

10.3

-

 PM1-36

12.2

-

11.8

14.5

13.9

12.1

14.5

-

-

-

18.9

12.5

13.8

17.6

9.1

-

 PM1-47

13.1

7.8

12.4

14.0

15.6

13.5

15.2

-

-

-

18.5

11.6

12.9

19.5

9.6

-

E. hirae

 M4S3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

E. mundtii

                

 M2M33

-

-

-

-

-

-

-

-

-

-

8.2

-

-

12.2

-

-

L. brevis

                

 PM1-26

11.1

-

10.0

14.6

15.1

12.5

14.0

-

-

-

17.0

11.1

12.6

16.0

-

-

aAntimicrobial activity determined by ADT. Results as the diameter of the inhibition halos in millimeters (mm). (−) Antimicrobial activity not detected. Most of the data are means from two independent determinations in triplicate

bSource of indicator microorganisms indicated in Table 1

Table 4

Antimicrobial activitya of supernatants from selected LAB against Listeria spp.b

Strain

L. grayii 931

L. innocua 910

L. ivanovii 913

L. seeligeri 917

L. welshimeri

919

L. monocytogenes

911

935

936

939

4031

4032

E. faecalis

 M2M6

0.50

0.06

0.05

0.12

0.06

0.06

0.05

0.06

0.06

0.06

0.06

 M3M42

0.49

0.06

0.05

0.12

0.06

0.06

0.06

0.06

0.11

0.57

0.06

 PM2-13

0.10

0.13

1.05

0.29

0.27

0.22

0.15

0.29

0.15

0.15

0.27

E. faecium

 M1M10

9.1

2.6

16.3

2.2

4.3

34.5

8.5

18.6

3.9

3.5

16.3

 M3M31

3.6

3.8

97.9

8.3

19.8

4.1

7.9

18.3

8.2

5.1

12.5

 M3M32

3.8

1.9

288.4

35.0

20.1

7.7

7.8

34.9

14.6

4.2

34.7

 M3K31

27.6

30.7

1.1 × 106

3.0 × 105

1.7 × 103

12.2

1.1 × 103

6.0 × 103

3.9 × 104

1.7x106

8.6 × 106

 PM1-27

1.9

2.3

9.3

7.5

3.7

4.8

3.8

8.7

4.4

1.9

8.4

E. mundtii

 M2M33

NA

0.24

0.23

0.17

0.19

0.17

8.74

0.07

0.08

0.08

0.16

L. brevis

           

 PM1-26

3.8

1.9

29.1

4.4

4.7

5.0

3.8

9.4

4.4

1.8

8.6

aAntimicrobial activity measured by MPA and expressed as 103 × BU/mL. NA, No antimicrobial activity detected. Most of the data are means from two independent determinations in triplicate

bSource of indicator microorganisms indicated in Table 1

Evaluation of bacteriocin structural genes

Purified genomic DNA of the 28 selected LAB isolates was subjected to PCR amplification to determine the presence of structural genes coding for 21 previously described bacteriocins. All isolates encoded, at least, one described bacteriocin gene except E. hirae M4S3. From the E. faecalis isolates evaluated all of them encoded entV583, 6 isolates enlA, 5 isolates ent1071A-ent1071B, 2 isolates ent96 and 2 more isolates encoded entJSA-entJSB. Five bacteriocin-producing genes (for ent96, ent1071A-ent1071B, entJSA-entJSB, entV583, enlA) were detected in E. faecalis M2M6 and three (for ent1071A-ent1071B, entV583, enlA) in E. faecalis M1S20. Among the E. faecium isolates, the entA gene was detected in 13 (86.6 %) out of the 15 evaluated isolates and, associated with the entB gene, in 7 (53.8 %) of the isolates (Table 5). The ent96, entK4, entP and entXA-entXB and hirJM79 bacteriocin-producing genes were shown to have a lower incidence (3.5 to 17.8 %) in the evaluated isolates while the aviA, bac31, bacMC4, entAS48, entIT, entL50A-entL50B, entQ and munL structural genes, could not be detected in any of the evaluated isolates. E. faecium M3K31 was shown to encode only the entP gene (Table 5).
Table 5

PCR amplification of bacteriocin structural genes from selected bacteriocinogenic lactic acid bacteria (LAB) isolates

Isolate

aviA

bac31

bacMC4

breB

ent96

ent1071A-ent1071B

entA

entAS-48

entB

entIT

entJSA-entJSB

entK4

entL50A-entL50B

entP

entQ

entV583

entXA-entXB

enlA

hirJM79

munL

pedA-1

E. faecalis

 AS10

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

 M1M32

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

+

-

+

-

-

-

 M1S19

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

+

-

+

-

-

-

 M1S20

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

+

-

+

-

-

-

 M2M6

-

-

-

-

+

+

-

-

-

-

+

-

-

-

-

+

-

+

-

-

-

 M2M39

-

-

-

-

+

+

-

-

-

-

-

-

-

-

-

+

-

+

-

-

-

 M3M42

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

+

-

-

-

-

-

 M3S1

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

+

-

-

-

 PM1-27

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

 PM2-13

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

E. faecium

 AS41

-

-

-

-

-

-

+

-

+

-

-

-

-

-

-

-

+

-

-

-

-

 BS15

-

-

-

-

-

-

+

-

+

-

-

-

-

-

-

-

-

-

-

-

-

 CS14

-

-

-

-

-

-

+

-

+

-

-

-

-

-

-

-

-

-

-

-

-

 CS46

-

-

-

-

-

-

+

-

+

-

-

-

-

-

-

-

+

-

-

-

-

 M1M10

-

-

-

-

+

-

+

-

+

-

-

+

-

-

-

-

+

-

-

-

-

 M1M26

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

+

-

-

 M2S31

-

-

-

-

-

-

+

-

+

-

-

-

-

-

-

-

-

-

-

-

-

 M2M31

-

-

-

-

-

-

+

-

+

-

-

-

-

-

-

-

+

-

-

-

-

 M3M32

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

+

-

-

-

-

 M3K31

-

-

-

-

-

-

-

-

-

-

-

-

-

+

-

-

-

-

-

-

-

 M4M2

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 PM1-27

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 PM1-36

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

+

-

-

 PM1-37

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

 PM1-47

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

E. hirae

 M4S3

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

E. mundtii

 M2M33

-

-

-

-

-

-

-

-

+

-

-

+

-

-

-

-

-

-

-

-

-

L. brevis

 PM1-26

-

-

-

+

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

Potential virulence factors, antibiotic susceptibility and hemolytic activity

When the previously selected 9 bacteriocinogenic E. faecalis isolates were evaluated for potential virulence factors the presence of the cylL L -cylL S genes, encoding the two peptide cytolysin (hemolysin-bacteriocin) precursor, was detected in three isolates. However, only E. faecalis PM2-13 showed the presence of the cylLMAB genes for expression of cytolysin. Accordingly, only E. faecalis PM2-13 showed β-hemolytic activity when streaked on blood agar plates. Only two E. faecalis isolates encoded agg but none encoded the esp gene. Most E. faecalis isolates encoded ace, gelE and sprE but E. faecalis M1S9 and E. faecalis M3S1 did not encode any of the three cited genes (Table 6). All the evaluated E. faecalis strains hydrolysed gelatin and bovine casein, except those isolates not encoding gelE-sprE.
Table 6

PCR amplification of genes related to potential virulence in E. faecalis

Isolate

Virulence determinants

ace

agg

cylA

cylB

cylL L -cylL S

cylM

efaAfm

efaAfs

esp

gelE

sprE

E. faecalis

 AS10

+

-

-

-

-

-

-

+

-

+

+

 M1M32

+

+

-

-

-

+

-

+

-

+

+

 M1S19

-

+

+

+

+

-

-

+

-

-

-

 M1S20

+

-

-

-

-

-

-

+

-

+

+

 M2M6

+

-

-

-

-

-

-

+

-

+

+

 M2M39

+

-

-

-

-

-

-

+

-

+

+

 M3M42

+

-

-

-

-

+

-

+

-

+

+

 M3S1

-

-

+

+

+

-

-

+

-

-

-

 PM2-13

+

-

+

+

+

+

-

+

-

+

+

Antibiotic susceptibility of the 27 selected bacteriocinogenic enterococci revealed that all of them (100 %) were resistant to at least one of the tested antibiotics. Furthermore, 85 % of the isolates were resistant to rifampicin, 77 % to tetracycline, 50 % to erythromycin, 44 % to cyprofloxacin, 29 % to chloramphenicol, 28 % to nitrofurantoin, 29 % to gentamycin and ampicillin, 25 % to penicillin, and 7 % to vancomycin. However, E. faecalis M3M42 and E. faecium M3K31 were only resistant to rifampicin while E. faecalis M2M6 was sensitive to all antibiotics tested. All isolates were sensitive to teicoplanin. The genotypic evaluation of the antibiotic resistance profile of the enterococci evaluated was not pursued in this study.

Safety assessment of E. faecium M3K31

The sensitivity of E. faecium M3K31 to ampicillin resulted in a minimum inhibitory concentration (MIC) ≤ 2 mg/L and, thus, susceptible to ampicillin according to EFSA guidelines [19]. This isolate also showed the absence of the virulence markers esp, hyl Efm and IS16.

Molecular genotyping of selected enterococcal strains by ERIC-PCR and MLST analysis

The ERIC-PCR fingerprints of the nine most antagonistic E. faecalis strains isolated from griffon vultures feces and those from other E. faecalis strains from different food, environmental and clinical origin, revealed 2 different clusters (50 % similarity). The first cluster included all the isolates from griffon vulture feces and other E. faecalis strains, mostly from human clinical origin. The second cluster included only E. faecalis strains from different origins (Fig. 1a). The ERIC-PCR dendrogram showing the genetic relatedness between E. faecium isolates from different origins could be also divided into two clusters. One cluster contained 13 out of the 14 most antagonistic E. faecium isolates from griffon vultures showing 60 % similarity. The second cluster grouped all the E. faecium isolates from different origins including E. faecium M4M2, isolated from griffon vultures. Among the griffon vulture isolates only E. faecium BS15 and E. faecium CS14 showed an almost identical genotype (Fig. 1b).
Fig. 1

Dendrogram of ERIC-PCR showing the relatedness amongst the E. faecalis (a) and E. faecium (b) isolates from griffon vulture feces and enterococcal isolates from other food, environmental and clinical origin. Source abbreviations as indicated in Table 1

Comparison of the resulting allelic profiles of E. faecalis M3M42 and M1S19 in the E. faecalis MLST database permitted their annotation as sequence types ST167 and ST76, respectively. On the other hand, E. faecium M1M10, E. faecium M3K31 and E. faecium PM1-27 were annotated as sequence types ST22, ST76 and ST670, respectively, in the E. faecium MLST database. Comparative eBURST analysis of the resulting sequence types (STs) permitted their location into clusters of related STs (Additional file 1: Figure S1).

Purification of bacteriocins, mass spectrometry analysis and amino acid sequencing

Two of the bacteriocinogenic enterococci isolated from griffon vulture feces, E. faecium M1M10 and E. faecium M3K31 with high antimicrobial activity in their supernatants and encoding several or a single bacteriocin structural gene, respectively, were selected for purification of the bacteriocins being produced. Purification of the antimicrobial activity of E. faecium M1M10, encoding five bacteriocin structural genes, permitted a 1397-fold increase of its specific activity and a 32 % recovery of the initial antimicrobial activity. MALDI-TOF MS of the purified peptide showed a major peak of 5519.8 Da, suggesting the peptide was purified to homogeneity (Fig. 2a). Furthermore, determination of the N-terminal amino acid sequence of the purified peptide by Edman degradation permitted identification of its 15 N-terminal amino acids as ENDHRMPNELNRPNN, which unambigously correspond to the N-terminal sequence of the enterocin B (EntB). However, EntB has a calculated theoretical molecular mass of 5465.2 Da. Purification of the antimicrobial activity of E. faecium M3K31, encoding the entP gene, permitted an 897-fold increase of its specific activity and a 11 % recovery of the initial antimicrobial activity. MALDI-TOF MS analysis of the purified peptide showed a major peak of 4328.1 Da (Fig. 2b). Furthermore, the de novo amino acid sequencing of the resulting peptide by MALDI TOF/TOF tandem MS permitted identification of the carboxy-terminal (y-ion series) 27-residue peptide SVDWGKAIGIIGNNAAANLTTGGKAGW and the amino-terminal (b-ion series) 14-residue peptide AAANLTTGGKAGWK (Fig. 2c), identical to the C-terminal amino acid sequence of enterocin HF (EntHF) (GenBank accession numbers P86183 and KJ442693), and with a theoretical molecular mass of 4330.9 Da. Primers devised from the nucleotide sequence of EntHF from GenBank accession number KJ442693 and PCR amplifications and sequencing of the resulting fragments, confirmed that E. faecium M3K31 encoded entHF.
Fig. 2

MALDI-TOF MS analysis of purified bacteriocins from E. faecium M1M10 (a) and E. faecium M3K31 (b). MALDI TOF/TOF tandem MS analysis of the purified bacteriocin from E. faecium M3K31 (c). The carboxy-terminal (y-ion series) shows the 27-residue peptide SVDWGKAIGIIGNNAAANLTTGGKAGW (top line) and the amino-terminal (b-series) shows the 14-residue peptide AAANLTTGGKAGWK (bottom line). Numbers indicate molecular mass in daltons

Antimicrobial activity of purified enterocin A (EntA) and enterocin HF (EntHF) against Listeria spp

The sensitivity of several Listeria spp. to purified EntA, produced by E. faecium T136 and a potent antilisterial class IIa bacteriocin and to EntHF, produced by E. faecium M3K31, is shown in Table 7. Purified EntHF showed a higher specific antimicrobial activity against Listeria spp. than purified EntA. Furthermore, L. ivanovii CECT913 was the most sensitive indicator strain for both bacteriocins.
Table 7

Antimicrobial activitya of chromatographically purified enterocin HF and enterocin A produced by E. faecium M3K31 and E. faecium T136, respectively, against Listeria spp. and P. damnosus CECT4797b

Purified bacteriocins

L. grayii 913

L. innocua 910

L. ivanovii 913

L. seeligeri 917

L. welshimeri 919

L. monocytogenes

P. damnosus 4797

911

935

936

939

4031

4032

Enterocin HF

1,168

4

8.3 × 109

582

464

9

364

1,118

155

55

2,455

73

Enterocin A

8

9

19,000

10

5

10

17

8

10

2

14

5

Most of the data are means from two independent determinations in triplicate

aAntimicrobial activity measured by MPA and expressed as BU/ng of purified bacteriocin

bSource of indicator microorganisms indicated in Table 1

Discussion

Among other features, most bacteria produce compounds that inhibit competing and pathogenic microorganisms, improving host health [7, 25]. Since griffon vultures feed regularly on carcasses from dead birds and mammals, it may be hypothesized that vultures could benefit themselves hosting bacteriocin-producing LAB to combat bacterial pathogens. In this study, 36 % of the evaluated LAB isolates showed direct antimicrobial activity. The enterococci comprised the largest LAB group with antimicrobial activity (91 %) with E. faecium (40 %) as the most identified antagonistic species. Enterococci with antimicrobial activity have been previously identified from LAB isolated from mammals, birds and fish [1012], but not with this elevated percentage of isolation. Enterococci are also the most common species in cloacal and pharyngeal samples of Euroasian griffon vultures [26] and in Turkey and Black vultures whereas enterococcal symbionts living in the hoopoe (Upua epops) uropygial gland produce bacteriocins active against Gram-positive pathogens [27]. Thus, LAB with antimicrobial activity isolated from griffon vulture feces mainly contains bacteriocinogenic enterococci.

In this work none of the LAB isolated from griffon vultures showed antagonistic activity against any of the four Gram-negative indicator bacteria evaluated, including two C. jejuni strains. Since griffon vultures are not a reservoir for Campylobacter spp. [26, 28], perhaps bacteria different than LAB may inhibit or control their presence in this reservoir. Several nonribosomal lipopeptides produced by Bacillus and Paenibacillus spp. have shown antagonistic activity against C. jejuni [29]. Thus, these bacteria would be further evaluated for antimicrobial activity against C. jejuni when identified from animal and food reservoirs [30]. When selected LAB were evaluated for antimicrobial activity in their supernatants, the E. faecalis isolates (Table 2) were less active than E. faecium and other LAB isolates (Table 3), which has been observed from enterococci from other sources [1012, 31]. But of interest is the high antimicrobial activity of E. faecium M3K31 against most bacterial indicators, including L. lactis BB24 (Table 3) and most of the Listeria spp. evaluated (Table 4).

In this study, most selected bacteriocinogenic LAB isolates encoded a variable number of bacteriocin structural genes (Table 5). However, the presence of structural bacteriocin-producing genes, either alone or in combination with others, seems to be common in bacteriocinogenic enterococci from human, animal, clinical, food, agricultural and environmental sources [1012]. Furthermore, is also difficult to find a correlation between the number of bacteriocin structural genes and the antagonistic activity and antimicrobial spectrum of the isolates. It may be hypothesized that some bacteriocin-producing genes may be silent, that modifications in the promoter region may affect transcription, and that immunity/regulation/transport of the synthesized bacteriocin may also be impaired. The production of bacteriocins is also regulated by environmental factors such as the temperature [32].

The enterococci are LAB with a beneficial role in the sensory characteristics of fermented foods and have been successfully used as starter and adjunct cultures, and permitted as probiotics [33]. However, the enterococci are also gastrointestinal (GI) tract colonizers responsible of nosocomial and, to a lesser extent, community acquired infections [34]. E. faecalis harbours significantly more virulence determinants than E. faecium [10, 18] and, therefore, we screened the bacteriocinogenic E. faecalis isolates for presence of virulence determinants (Table 6). In this study, only E. faecalis PM2-13 amplified all genes for expression of cytolysin. Insertion/deletion events leading to truncated or absent cyl genes and possible sequence divergences may explain the difficulties to tackle genes involved in the production of active cytolysins. Most E. faecalis isolates encoded ace, gelE and sprE. Protease expression seems to be strain specific and not representative of clinical isolates although regulatory genes must be also active, to permit protease expression [35]. None of the E. faecalis evaluated encoded esp and this is considered a positive attribute since Esp is supposed to promote primary attachment to surfaces and escape from the immune system.

Enterococci are commonly resistant to macrolides, cephalosporins and tetracycline and often exhibit high-level resistance to gentamicin [36]. Vultures rely greatly on food which usually derives from medicated livestock that could lead vultures to the acquisition of antimicrobial-resistant bacteria, modification of the normal microbiota and acquisition of pathogenic bacteria. But, in this study several E. faecalis and E. faecium isolates did not show a large antimicrobial resistance. Moreover, E. faecium M3K31 was susceptible to ampicillin and did not encode the virulence markers esp, hyl Efm and IS16 being considered, according to EFSA guidelines [19], a safe probiotic for use in animal nutrition.

The virulence of the enterococci also reflects a selection for specific variants or clones with enhanced pathogenic potential [37]. The epidemiological typing of E. faecalis and E. faecium has mainly been performed by ERIC-PCR and MLST analysis. As expected, E. faecalis strains from griffon vulture feces showed a distinct genetic relatedness among them and from other strains from food, clinical and environmental origin when evaluated by ERIC-PCR (Fig. 1a). However, the E. faecium strains formed a more conserved, but also distinct group as compared to enterococcal isolates from different origins (Fig. 1b). These results suggest the reliability of ERIC-PCR for genotyping enterococci and, very likely, for their specific monitoring.

Previous MLST studies have demonstrated the association between specific clonal complexes and human nosocomial infections for E. faecalis [38, 39] and E. faecium [34]. Indeed, the majority of hospital-derived isolates of E. faecalis cluster in two clonal complexes, CC2 and CC9. However, in E. faecium the sequence types ST17, ST18, ST78 and ST192 which were previously designated clonal complex CC17, constitutes a hospital-associated clade genetically distinct from most commensal isolates [34, 37]. In this study, E. faecalis M3M42 and E. faecalis M1S19 were identified with the sequence types ST167 and ST76, respectively, none of them included into the clonal complexes CC2 and CC9 represented by sequence types ST6 and ST9, respectively (Additional file 1: Figure S1). In this context, E. faecalis M3M42 sensitive to most antibiotics, free of most virulence determinants and encoding gelE-sprE could be considered a potential strain for production of bioactive peptides with antihypertensive and antioxidant activity, during its growth on bovine skim milk [40, 41]. The MLST analysis of the E. faecium isolates showed that while E. faecium M1M10 (ST22) and E. faecium PM1-27 (ST670) remain close to strains from a hospital-associated clade the strain of E. faecium M3K31 (ST176) remains distant from those of the clonal complex CC17 and, thus, is considered a safe isolate (Additional file 1: Figure S1).

For bacteriocinogenic isolates the possibility exists for their antimicrobial activity being mediated by still unknown or not yet described bacteriocins or by regulation of the production of multiple encoded bacteriocins. In this study, MALDI-TOF MS analysis and N-terminal amino acid sequencing of the purified supernatant of E. faecium M1M10, encoding several bacteriocin structural genes, permitted the identification of a major peptide fragment (Fig. 2a) unambigously recognized as the N-terminal sequence of EntB [42]. The difference between the obtained and the calculated molecular mass of te EntB may be adscribed to still unknown modifications. Furthermore, the absence of other major peptide fragments in the purified activity of E. faecium M1M10, may imply that: (i) other encoded bacteriocins are produced in much lower concentrations, (ii) their structural genes remain silent and/or (iii) their production is regulated by still unknown mechanisms.

On the other hand, MALDI-TOF MS analysis and the de novo amino acid sequencing by MALDI TOF/TOF tandem MS of the major peptide from the purified supernatant of E. faecium M3K31 (Fig. 2b), encoding entP and with antimicrobial activity against L. lactis BB24, a LAB species with low or non-inhibitory activity by class IIa bacteriocins [9], permitted the identification of a peptide identical to the C-terminal amino acid sequence of the bacteriocin EntHF. The difference between the measured and the calculated molecular mass of EntHF, suggests the existence of a disulfide bond linking the two known cysteine residues in the molecule. In this context, mature EntHF is 91 % identical to mundticin KS/enterocin CRL35 [43, 44] and 90 % identical to mundticin L [45] and avicin A [46]. These bacteriocins display a high antilisterial activity, and enterocin CRL35 has even been postulated as a promising alternative agent for the in vivo prevention of Listeria spp. infections [47]. The absence of the bacteriocin EntP in the purified peptide fraction from E. faecium M3K31, imply that further studies on the regulation of the expression of bacteriocin structural genes in bacteriocinogenic enterococci would be pursued.

The specific antimicrobial activity of purified EntA and EntHF against Listeria spp. was higher for EntHF as compared to EntA (Table 7). Thus, although EntA is one of the most potent class IIa bacteriocins [48], EntHF is even more potent than EntA against Listeria spp. The determination of the three-dimensional (3D) structure of EntHF, produced by E. faecium M3K31, suggest that apparently the β-sheet-like N-terminal domain mediates initial binding to the target cell whereas the helix-containing C-terminal half penetrates into the hydrophobic core of target-cell membranes, docks to domains of specific protein receptors and causes dead of sensitive cells [49]. Modifications in the amino acid composition of the C-terminal end of class IIa bacteriocins may alter the manner and/or extent to which bacteriocins interact with putative or cognate receptors and even with cognate immunity proteins in the membrane of target cells [50, 51]. This would explain why the antimicrobial spectra of similar class IIa bacteriocins and the susceptibility of sensitive cells to a given bacteriocin, varies much more than would be expected from their known amino acid sequences. Accordingly, EntHF may be considered a bacteriocin of elevated potential biotechnological interest as a natural food preservative and a therapeutic antimicrobial agent for human and veterinary applications. Furthermore, because EntHF is a primary metabolite of a linear peptidic nature, it may be considered suitable for the development of novel bacteriocin quimeras with increased target specifity and antimicrobial activity by peptide bioengineering.

Within LAB, enterococci are increasingly used as probiotics. Most probiotics enhance intestinal barrier function, display immunomodulatory activity and exert protective effects due to production of antimicrobial compounds including bacteriocins [4, 52]. Moreover, the elucidation of the draft genome of E. faecium M3K31 has confirmed the existence in this strain of the EntHF biosynthetic cluster, the enterocin P structural and immunity genes, and a gene encoding the putative antimicrobial peptide SRCAM 602 [53]. Accordingly, E. faecium M3K31 producing EntHF and free of defined virulence markers, would be also considered a safe probiotic for further biotechnological applications including animal nutrition.

Conclusions

This study has determined that enterococci is the largest LAB group with bacteriocinogenic activity, isolated from griffon vulture feces. However, the absence of LAB with antagonistic activity against Gram-negative bacteria suggest that bacteria, other than LAB, would be investigated for their activity against this bacterial group. Most enterococci encoded multiple bacteriocins although is production seems to be regulated by still unknown and deficiently evaluated mechanisms. Moreover, E. faecium M3K31 has been identified as producer of EntHF, a bacteriocin with remarkable antimicrobial activity against most evaluated Listeria spp. and elevated interest as a natural food preservative. E. faecium M3K31, absent of defined virulent markers, would be also considered a safe probiotic for use in animal nutrition.

Abbreviations

ADT: 

Agar diffusion test

BLAST: 

Basic local alignment search tool

CC: 

Clonal complex

CLSI: 

Clinical and laboratory standards institute

EFSA: 

European food safety authority

ERIC-PCR: 

Enterobacterial repetitive intergenic consensus sequences- Polymerase chain reaction

KAA: 

Kanamycin aesculin azide

LAB: 

Lactic acid bacteria

MALDI TOF/TOF MS: 

MALDI TOF tandem mass spectrometry

MALDI-TOF MS: 

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

MIC: 

Minimum inhibitory concentration

MLST: 

Multilocus sequence typing

MPA: 

Microtiter plate assay

MRS: 

de Man, Rogosa and Sharpe

NCBI: 

National center for biotechnology information

PCR: 

Polymerase chain reaction

RP-FPLC: 

Reverse phase-Fast protein liquid chromatography SB, Slanetz and Bartley

SOAT: 

Stab-on-agar test

TSA: 

Trypticase soy agar

UPGM: 

Unweighed-pair group method

Declarations

Acknowledgements

We acknowledge the forest police officers of the Parque Natural del Alto Tajo (Guadalajara, Spain) and, specially, to Mr. Cristóbal Mateo González for collecting the griffon vulture feces. The authors are also grateful to Prof. IF Nes, Prof. DB Diep and Dr. M Skaugen from the Norwegian University of Life Sciences (Norway) for help in the amino acid sequencing of EntHF. SA and JJJ held a fellowship (FPI) from the MINECO, LG held a fellowship (FPU) from the Ministerio de Educación y Ciencia (MEC) and CC held a predoctoral fellowship from the Universidad Complutense de Madrid (UCM), Spain.

Funding

This work was partially supported by Project AGL2012-34829 from the Ministerio de Economía y Competitividad (MINECO), by Grant GR314 from the BSCH-UCM, and by Grant S2013/ABI-2747 from the Comunidad de Madrid (CAM).

Availability of data and materials

All data generated and analysed during this stud are included in this published article [and its supplementary information files].

Authors’ contributions

SA carried out the microbiological and genetic analyses, purification and biochemical characterization of the bacteriocins, participated in the design of the experiments and prepared the manuscript draft. JJJ, LG and CC participated in the microbiological and genetic analyses. RDC participated in the design and analysis of genotypic data. CH, LMC and PEH participated in the coordination and design of the study, analyzed the results and revised the manuscript. All authors read and approved the final version of the manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Official permission for collecting the griffon vulture feces was obtained from the Administrative and Official Control Section of the Parque Natural del Alto Tajo (Guadalajara, Spain).

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM)
(2)
Servicio de Microbiología, Hospital Universitario Ramón y Cajal, and Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS)

References

  1. O’Connor PM, Ross RP, Hill C, Cotter PD. Antimicrobial antagonists against food pathogens: a bacteriocin perspective. Curr Opin Food Sci. 2015;2:51–7.View ArticleGoogle Scholar
  2. Cotter PD, Ross RP, Hill C. Bacteriocins - a viable alternative to antibiotics? Nat Rev Microbiol. 2013;11:95–105.View ArticlePubMedGoogle Scholar
  3. Arbulu S, Jiménez JJ, Gútiez L, Cintas LM, Herranz C, Hernández PE. Cloning and expression of synthetic genes encoding the broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. BioMed Res Int. 2015;2015:767183.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Corr SC, Hill C, Gahan CGM. Understanding the mechanisms by which probiotics inhibit gastrointestinal pathogens. Adv Food Nutr Res. 2009;56:1–15.View ArticlePubMedGoogle Scholar
  5. Dobson A, Cotter PD, Ross RP, Hill C. Bacteriocin production: a probiotic trait? Appl Environ Microbiol. 2012;78:1–6.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Jiménez JJ, Diep DB, Borrero J, Gútiez L, Arbulu S, Nes IF, et al. Cloning strategies for heterologous expression of the bacteriocin enterocin A by Lactobacillus sakei Lb790, Lb. plantarum NC8 and Lb. casei CECT475. Microb Cell Factories. 2015;14:166.View ArticleGoogle Scholar
  7. Cotter PD, Hill C, Ross RP. Bacteriocins: developing innate immunity for food. Nat Rev Microbiol. 2005;3:777–88.View ArticlePubMedGoogle Scholar
  8. Nes IF, Diep DB, Ike Y. Enterococcal Bacteriocins and antimicrobial proteins that contribute to niche control. In: Gilmore MS, Clewell DB, Ike Y, Shankar N, editors. Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary; 2014.Google Scholar
  9. Kjos M, Borrero J, Opsata M, Birri DJ, Holo H, Cintas LM, et al. Target recognition, resistance, immunity and genome mining of class II bacteriocins from Gram-positive bacteria. Microbiology. 2011;157:3256–67.View ArticlePubMedGoogle Scholar
  10. Sánchez J, Basanta A, Gómez-Sala B, Herranz C, Cintas LM, Hernández PE. Antimicrobial and safety aspects, and biotechnological potential of bacteriocinogenic enterococci isolated from mallard ducks (Anas platyrhynchos). Int J Food Microbiol. 2007;117:295–305.View ArticlePubMedGoogle Scholar
  11. Muñoz-Atienza E, Gómez-Sala B, Araújo C, Campanero C, del Campo R, Hernández PE, et al. Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture. BMC Microbiol. 2013;13:15.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Gómez-Sala B, Muñoz-Atienza E, Sánchez J, Basanta A, Herranz C, Hernández PE, et al. Bacteriocin production by lactic acid bacteria isolated from fish, seafood and fish products. Eur Food Res Technol. 2015;241:341–56.View ArticleGoogle Scholar
  13. Margalida A, Colomer MA. Modelling the effects of sanitary policies on European vulture conservation. Sci Rep. 2012;2:753.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Ruiz-Rodríguez M, Soler JJ, Martín-Vivaldi M, Martín-Platero AM, Méndez M, Peralta-Sánchez JM, et al. Environmental factors shape the community of symbionts in the hoopoe uropygial gland more than genetic factors. Appl Environ Microbiol. 2014;80:6714–23.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Cintas LM, Casaus P, Fernández MF, Hernández PE. Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria. Food Microbiol. 1998;15:289–98.View ArticleGoogle Scholar
  16. Kullen MJ, Sanozky-Dawes RB, Crowell DC, Klaenhammer TR. Use of the DNA sequence of variable regions of the 16S rRNA gene for rapid and accurate identification of bacteria in the Lactobacillus acidophilus complex. J Appl Microbiol. 2000;89:511–6.View ArticlePubMedGoogle Scholar
  17. Poyart C, Quesne G, Boumaila C, Trieu-Cuot P. Rapid and accurate species-level identification of coagulase-negative staphylococci by using the sodA gene as a target. J Clin Microbiol. 2001;39:4296–301.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Eaton TJ, Gasson MJ. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol. 2001;67:1628–35.View ArticlePubMedPubMed CentralGoogle Scholar
  19. EFSA Panel on Additives and Products or Substances Used in Animal Feed (FEEDAP). Guidance on the safety assessment of Enterococcus faecium in animal nutrition. EFSA J. 2012;10(5):2682.View ArticleGoogle Scholar
  20. Muñoz-Atienza E, Araújo C, del Campo R, Hernández PE, Herranz C, Cintas LM. Safety assessment and molecular genetic profiling by pulsed-field gel electrophoresis (PFGE) and PCR-based techniques of Enterococcus faecium strains of food origin. LWT. Food Sci Technol. 2016;65:357–62.Google Scholar
  21. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. Wayne: CLSI; 2014.Google Scholar
  22. Ruiz-Garbajosa P, Bonten MJM, Robinson DA, Top J, Nallapareddy SR, Torres C, et al. Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination. J Clin Microbiol. 2006;44:2220–8.Google Scholar
  23. Martín M, Gutiérrez J, Criado R, Herranz C, Cintas LM, Hernández PE. Cloning, production and expression of the bacteriocin enterocin A produced by Enterococcus faecium PLBC21 in Lactococcus lactis. Appl Microbiol Biotechnol. 2007;76:667–75.View ArticlePubMedGoogle Scholar
  24. Borrero J, Brede DA, Skaugen M, Diep DB, Herranz C, Nes IF, et al. Characterization of garvicin ML, a novel circular bacteriocin produced by Lactococcus garvieae DCC43, Isolated from mallard ducks (Anas platyrhynchos). Appl Environ Microbiol. 2011;77:369–73.View ArticlePubMedGoogle Scholar
  25. Soler JJ, Martin-Vivaldi M, Peralta-Sanchez JM, Ruiz-Rodriguez M. Antibiotic-producing bacteria as a possible defence of birds against pathogenic microorganisms. Open Ornithol J. 2010;3:93–100.View ArticleGoogle Scholar
  26. Vela AI, Casas-Díaz E, Fernández-Garayzábal JF, Serrano E, Agustí S, Porrero MC, et al. Estimation of cultivable bacterial diversity in the cloacae and pharynx in Eurasian griffon vultures (Gyps fulvus). Microb Ecol. 2015;69:597–607.View ArticlePubMedGoogle Scholar
  27. Rodríguez-Ruano SM, Martín-Vivaldi M, Martín-Platero AM, López-López JP, Peralta-Sánchez JM, Ruiz-Rodríguez M, et al. The hoopoe’s uropygial gland hosts a bacterial community influenced by the living conditions of the bird. PLoS One. 2015;10(10):e0139734.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Marin C, Palomeque MD, Marco-Jiménez F, Vega S. Wild griffon vultures (Gyps fulvus) as a source of Salmonella and Campylobacter in Eastern Spain. PLoS One. 2014;9:e94191.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Lohans CT, van Belkum MJ, Li J, Vederas JC. Characterization of bacterial antimicrobial peptides active against Campylobacter jejuni. Can J Chem. 2014;93:381–8.View ArticleGoogle Scholar
  30. Lee M-H, Lee J, Nam Y-D, Lee JS, Seo M-J, Yi S-H. Characterization of antimicrobial lipopeptides produced by Bacillus sp. LM7 isolated from chungkookjang, a Korean traditional fermented soybean food. Int J Food Microbiol. 2016;221:12–8.View ArticlePubMedGoogle Scholar
  31. Martín M, Gutiérrez J, Criado R, Herranz C, Cintas LM, Hernández PE. Genes encoding bacteriocins and their expression and potential virulence factors of enterococci isolated from wood pigeons (Columba palumbus). J Food Prot. 2006;69:520–31.PubMedGoogle Scholar
  32. Criado R, Gutiérrez J, Martín M, Herranz C, Hernández PE, Cintas LM. Immunochemical characterization of temperature-regulated production of enterocin L50 (EntL50A and EntL50B), enterocin P, and enterocin Q by Enterococcus faecium L50. Appl Environ Microbiol. 2006;72:7634–43.View ArticlePubMedPubMed CentralGoogle Scholar
  33. Franz CMAP, Huch M, Abriouel H, Holzapfel W, Gálvez A. Enterococci as probiotics and their implications in food safety. Int J Food Microbiol. 2011;151:125–40.View ArticlePubMedGoogle Scholar
  34. Willems RJL, Top J, van Schaik W, Leavis H, Bonten M, Sirén J, et al. Restricted gene flow among hospital subpopulations of Enterococcus faecium. mBio. 2012;3:e00151–00112.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Pessione A, Lamberti C, Cocolin L, Campolongo S, Grunau A, Giubergia S, et al. Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis. Proteomics. 2012;12:431–47.View ArticlePubMedGoogle Scholar
  36. Hollenbeck BL, Rice LB. Intrinsic and acquired resistance mechanisms in Enterococcus. Virulence. 2012;3:421–33.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Arias CA, Murray BE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol. 2012;10:266–78.View ArticlePubMedPubMed CentralGoogle Scholar
  38. Leavis HL, Bonten MJM, Willems RJL. Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance. Curr Opin Microbiol. 2006;9:454–60.View ArticlePubMedGoogle Scholar
  39. Galloway-Peña JR, Rice LB, Murray BE. Analysis of PBP5 of early U.S. isolates of Enterococcus faecium: sequence variation alone does not explain increasing ampicillin resistance over time. Antimicrob Agents Chemother. 2011;55:3272–7.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Gútiez L, Borrero J, Jiménez JJ, Gómez-Sala B, Recio I, Cintas LM, et al. Genetic and biochemical evidence that recombinant Enterococcus spp. strains expressing gelatinase (GelE) produce bovine milk-derived hydrolysates with high angiotensin converting enzyme-inhibitory activity (ACE-IA). J Agric Food Chem. 2014;62:5555–64.View ArticlePubMedGoogle Scholar
  41. Gútiez L, Gómez-Sala B, Recio I, del Campo R, Cintas LM, Herranz C, et al. Enterococcus faecalis strains from food, environmental, and clinical origin produce ACE-inhibitory peptides and other bioactive peptides during growth in bovine skim milk. Int J Food Microbiol. 2013;166:93–101.View ArticlePubMedGoogle Scholar
  42. Casaus P, Nilsen T, Cintas LM, Nes IF, Hernández PE, Holo H. Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology. 1997;143:2287–94.View ArticlePubMedGoogle Scholar
  43. Kawamoto S, Shima J, Sato R, Eguchi T, Ohmomo S, Shibato J, et al. Biochemical and genetic characterization of mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393. Appl Environ Microbiol. 2002;68:3830–40.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Saavedra L, Minahk C, de Ruiz Holgado AP, Sesma F. Enhancement of the enterocin CRL35 activity by a synthetic peptide derived from the NH2-terminal sequence. Antimicrob Agents Chemother. 2004;48:2778–81.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Feng G, Guron GKP, Churey JJ, Worobo RW. Characterization of mundticin L, a class IIa anti-Listeria bacteriocin from Enterococcus mundtii CUGF08. Appl Environ Microbiol. 2009;75:5708–13.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Birri DJ, Brede DA, Forberg T, Holo H, Nes IF. Molecular and genetic characterization of a novel bacteriocin locus in Enterococcus avium isolates from infants. Appl Environ Microbiol. 2010;76:483–92.View ArticlePubMedGoogle Scholar
  47. Salvucci E, Saavedra L, Hebert EM, Haro C, Sesma F. Enterocin CRL35 inhibits Listeria monocytogenes in a murine model. Foodborne Pathog Dis. 2012;9:68–74.View ArticlePubMedGoogle Scholar
  48. Ennahar S, Sashihara T, Sonomoto K, Ishizaki A. Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol Rev. 2000;24:85–106.View ArticlePubMedGoogle Scholar
  49. Arbulu S, Lohans CT, van Belkum M, Cintas LM, Herranz C, Vederas JC, Hernández PE. Solution structure of enterocin HF, an antilisterial bacteriocin produced by Enterococcus faecium M3K31. J Agric Food Chem. 2015;63:10689–95.View ArticlePubMedGoogle Scholar
  50. Diep DB, Skaugen M, Salehian Z, Holo H, Nes IF. Common mechanisms of target cell recognition and immunity for class II bacteriocins. Proc Natl Acad Sci U S A. 2007;104:2384–9.View ArticlePubMedPubMed CentralGoogle Scholar
  51. Kjos M, Salehian Z, Nes IF, Diep DB. An extracellular loop of the mannose phosphotransferase system component IIC is responsible for specific targeting by class IIa bacteriocins. J Bacteriol. 2010;192:5906–13.View ArticlePubMedPubMed CentralGoogle Scholar
  52. Riboulet-Bisson E, Sturme MHJ, Jeffery IB, O’Donnell MM, Neville BA, Forde BM, et al. Effect of Lactobacillus salivarius bacteriocin Abp118 on the mouse and pig intestinal microbiota. PLoS One. 2012;7:e31113.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Arbulu S, Frantzen C, Lohans CT, Cintas LM, Herranz C, Holo H, et al. Draft genome sequence of the bacteriocin-producing strain Enterococcus faecium M3K31, isolated from griffon vultures (Gyps fulvus subsp. fulvus). Genome Announc. 2016;4(2):e00055–16.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Balla E, Dicks LM, Du Toit M, Van Der Merwe MJ, Holzapfel WH. Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071. Appl Environ Microbiol. 2000;66:1298–304.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© The Author(s). 2016

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