Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in Enterococcus italicus Strains of Dairy Origin

Evaluation of Plasmid Content and Tetracycline ResistanceConjugative Transfer in Enterococcus italicus Strains of DairyOrigin

Francesca Borgo Æ Giovanni Ricci Æ Karsten Arends ÆKatarzyna Schiwon Æ Elisabeth Grohmann ÆMaria Grazia Fortina

Received: 31 March 2009 / Accepted: 7 May 2009 / Published online: 30 May 2009! Springer Science+Business Media, LLC 2009

Abstract Five Enterococcus italicus strains harbouringtet genes responsible for the tetracycline resistance were

subjected to plasmid profile determination studies. For four

strains tested the profiles showed between three and sixplasmid bands, the size of which ranged between 1.6 and

18.5 kb. Southern hybridization experiments associated

tetS and tetK genes with chromosomal DNA in all strainsand tetM gene with plasmids of around the same size

(18.5 kb) in two of the tested strains. The ability of the new

species to transfer tetM gene was studied by transferexperiments with the tetracycline-susceptible recipient

strains E. faecalis JH2-2 and OG1RF; mobilization

experiments were performed with E. faecalis JH 2-2 har-bouring the conjugative plasmid pIP501as helper plasmid.

The results obtained show that the new enterococcal spe-

cies was able to acquire antibiotic resistance by conjuga-tion, but not to transfer its plasmids to other bacteria.

Further PCR and hybridization experiments carried out to

assess the presence of mobilization sequences also suggestthat the tetM plasmid from E. italicus is a non-mobilizable

plasmid.

Introduction

Enterococci are one of the most controversial species of

lactic acid bacteria (LAB) found in food products. They arenaturally present in the gastrointestinal tract of mammals

and therefore in soil and polluted waters [17, 22]. Because

of their presence in these habitats, the enterococci enter thehuman food chain, where they can play different and in

many cases controversial roles [9]. The main question

related to the presence of enterococci in food is the highlevel of antibiotic resistance observed in these bacteria and

their potential role as reservoirs and vehicles for the dis-

semination of antibiotic resistance determinants via thefood chain [14, 26]. In enterococci, resistance factors are

often associated with mobile genetic elements, such as

transposons or conjugative plasmids: Tn916, discovered inE. faecalis, Tn5233 in E. faecium, and conjugative plas-

mids, such as pAMb1, pCF10, pIP501 and pRE25 [18, 24,

25]. These plasmids are also capable of mobilizing smallerplasmids and this fact may explain the dissemination of

smaller plasmids carrying antibiotic resistance genesamong Gram positive bacteria [12].

In previous studies, we isolated a new enterococcal spe-

cies E. italicus from typical Italian cheeses [8] and investi-gated its antibiotic resistance and virulence profile [7]. We

observed that E. italicus only possessed tetracycline resis-

tance determinants, particularly the RP (ribosomal protec-tion proteins) genes tetS and tetMand the tetKgene encoding

an EP (efflux pump protein). Interestingly, the strains tested

did not contain the Tn916 conjugative transposon. Similarresults have been reported for other strains ofE. italicus [20].In this study we investigated the potential of tetracycline

resistance transfer of this new species, with the aim to knowthe real relationship between the presence of E. italicus indairy products and food safety.

F. Borgo ! G. Ricci ! M. G. Fortina (&)Department of Food Science and Microbiology, IndustrialMicrobiology Section, University of Milan, Via Celoria 2,20133 Milan, Italye-mail: [email protected]

K. Arends ! K. Schiwon ! E. GrohmannEnvironmental Microbiology Genetics, University ofTechnology Berlin, Franklinstraße 29, 10587 Berlin, Germany

123

Curr Microbiol (2009) 59:261–266

DOI 10.1007/s00284-009-9428-5

Materials and Methods

Bacterial Strains and Culture Conditions

The strains used in this study and their characteristics areshown in Table 1. They were grown at 37"C in M17

medium (Difco, USA), supplemented with 1% glucose

(M17-G). When required, the cells were grown in M17-Gagar containing 25 lg/ml tetracycline 25 lg/ml fusidic

acid, 25 lg/ml chloramphenicol and/or 20 lg/ml

gentamicin.

Total and Plasmid DNA Isolation and Hybridization

Analysis

Total DNA was obtained as described before [6]; for

plasmid DNA isolation, an alkaline extraction procedurewas used [1]. Plasmid purification was obtained by

ultracentrifugation to equilibrium in a caesium chloride–

ethidium bromide gradient (CsCl–EtBr). For each plasmid-bearing strain, a second-dimension electrophoresis of the

plasmid preparation for a further analysis of CCC and OC

forms was performed [13]. Total and plasmid DNAs,

undigested and digested with different restriction enzymes(Roche Diagnostics GmbH, Germany), were separated in a

1% w/v agarose gel and then transferred to a nylon mem-

brane (Roche) by Southern blotting, according to manu-facturer’s directions. The DIG DNA Labelling and

Detection kit (Roche) was used for digoxigenin labelling of

the probes tested. They included the 348, 656 and 667-bpfragments of the E. italicus tetK, tetM and tetS genes,

respectively, and the 413 and 396-bp fragments of thepMV158 and pSK41 relaxase genes, respectively. The

probes were obtained by PCR, as reported below. Prehy-

bridization and hybridization were performed in 50% (w/v)formamide at 42"C and stringency washes in 0.1 9 SSC at

65"C (0.1 9 SSC buffer contains 15 mM NaCl and

1.5 mM sodium citrate).

PCR Amplification Procedure

Genes responsible for resistance towards chloramphenicol

(cat) and tetracycline (tetK, tetM, tetS) were amplified by

PCR using primers and amplification conditions previously

Table 1 Enterococcal and control strains used in this study, plasmid profiling and antibiotic resistance determinants location

Strains Source/origin Relevant antibioticresistance/identifiedresistance determinantsa

Resistance determinantslocation

Plasmid profiling

No. of plasmids Molecular weight (kb)

E. italicus

TP1.5T b Toma cheese Tet (tetS, tetK) Chr Plasmid-free

TP2.3 Toma cheese Tet (tetS, tetK, tetM) P-located tetM 5 18.5; 12.8; 3.3; 2.2; 1.6

TP1.D Toma cheese Tet (tetS, tetK) Chr 6 6.0; 5.8; 5.0; 3.8; 3.5; 1.8

TP3.D Toma cheese Tet (tetS, tetK) Chr 4 13.5; 5.0; 3.3; 2.0

RP4 Robiola cheese Tet (tetS, tetK, tetM) P-located tetM 3 18.5; 14.0; 9.7

E. faecalis

JH2-2 LMG 19456 Rif, Fus Chr Plasmid-free

OG1RF DEMG/Bc Rif, Fus Chr Plasmid -free

JH2-2 pIP501 DEMG/B Rif, Fus, Cm (cat),Erm (ermB)

P-located cat, ermB 1 30.6

Staph.aureus SK982-pSK41

USd Gent (aacA), Kan(aphD), Neo (neo),Tob (aacA-aphD),Rif, Novo

P-located aacA, aphD,neo, aacA-aphD

1 46.4

Strept. pneumoniaeT4-600-pMV158

DMEG/B Tet (tetL) P-located tetL 1 5.5

Tet Tetracycline, Rif Rifampicin, Fus Fusidic acid, Cm Chloramphenicol, Gen Gentamicin, Kan Kanamycin, Neo Neomycin, Tob Tobramycin,Novo Novobiocin, Erm Erythromycin, Chr Chromosome, P Plasmida Antibiotic resistances were previously tested [7]; in the table only the resistance profile important for this study is indicatedb Strain TP1.5 = type strain = DSMZ15952T

c Department of Environmental Microbiology Genetics, University of Technology, Berlin (DEMG/B)d Kindly provided by Professor Ron Skurray from School of Biological Sciences, University of Sidney, NSW, Australia (US)

262 F. Borgo et al.: Evaluation of Plasmid Content and Tetracycline Resistance

123

described [7]. PCR assays with primers specific for E.faecalis and E. italicus were performed as described byDutka-Malen et al. [4] and Fortina et al. [6], respectively.

For detection and amplification of relaxase genes we

selected primer sets on the basis of the conserved regionsidentified by sequence comparison of selected relaxase

genes from the pMV158 superfamily. In detail, we designed

the primer pairs pMV158F 50-GAGTTACATGGTTGCAAGAAT-30 pMV158R 50-CTACTCCCATGTG

CATGTG-30and pSK41F 50-CTGGACTAAAAGGCATGCAA-30 pSK41R 50-GCAGTTTTCCATCACGCATA-30.

The PCR conditions were: initial denaturation step at 94"Cfor 2 min followed by 35 cycles of denaturation at 94"C for1 min and annealing at 58"C for 1 min, with extension at

72"C for 1 min. The final cycle was followed by an addi-

tional 7 min elongation period at 72"C. PCR was performedin a 25-ll reaction mixture, containing 10 ng bacterial

DNA, 2.5 ll 10 9 reaction buffer (MBI-Fermentas),

200 lM of each dNTP, 2.5 mM MgCl2, 0.5 lM of eachprimer, and 0.5 U Taq polymerase (MBI-Fermentas). All

the amplification reactions were performed using a Gene

Amp PCR System 2400 (Perkin-Elmer, USA). Amplifica-tion products were separated on a 1.5% agarose gel stained

with ethidium bromide in 1 9 TAE buffer (40 mM Tris-

acetate, 1 mM EDTA, pH 8) and photographed in UV light.

Conjugation Assays, Transfer Frequency and Molecular

Typing of Transconjugants

Filter matings were performed according to Huys et al.

[15]. Donor and recipient strains were grown in selectiveM17 broth containing the appropriate antibiotics to the

mid-exponential phase of growth (approx. 4 h). Then,

different donor/recipient ratios were mixed and filteredthrough a sterile membrane filter (0.45 lm pore size, Sar-

torius, Germany). The filters were incubated overnight

either on non-selective M17 or on skim milk (Difco, 10%w/v) agar plates [21]. After mating, cells were washed from

the filters with 1 ml of 0.9% NaCl solution. Ten fold

dilutions of the mating mixtures were spread onto agarplates containing the appropriate antibiotic selection for

transconjugants (double selective M17 for biparental mat-

ing and triple selective M17 for triparental mating). Fol-lowing incubation for 24–48 h at 37"C, plates were

checked for the presence of growth. Colonies were picked

and inoculated into double and/or triple selective M17medium. Upon growth, the potential transconjugant cul-

tures were stored at -20"C. Donor and recipient cells were

mixed at ratios of 1:1 for biparental and 1:1:10 ofdonor1:donor2:recipient for triparental matings. Three

independent experiments of each mating pair were carried

out. A direct plate conjugation (DPC) mating experiment inskim milk medium was performed according to Broadbent

and Kondo [2]. Donor and recipient cells were incubated in

fresh M17 broth to the mid-exponential growth phase. Themating mixture consisted of 1 ml of donor and recipient

culture each and 3 ml of skim milk broth. They were

incubated overnight with gentle shaking, then aliquots ofthe mixture were spread onto double selective M17 plates.

The plates were screened for transconjugant colonies.

Transfer frequencies were expressed as the number oftransconjugants per recipient CFU. To determine the

transfer frequency, both the donor and the recipient werediluted in a 10-fold dilution series and enumerated by

plating onto appropriate selective medium agar. To verify

the genotypic properties of the potential transconjugants,PCR assays with primers specific for E. italicus, E. faecalisor targeting tetM and cat resistance genes were performed,

as specified above.

Results

Distribution of Plasmids and Location

of tet Determinants in the E. italicus Strains

We detected plasmid bands in four E. italicus strains: onlythe type strain of the species was plasmid-free. The profilesshowed between three and six plasmids in the CCC form

with molecular weights ranging from 1.6 to 18.5 kb

(Table 1). Figure 1a shows the electrophoretic pattern ofthe plasmid DNA isolated from the strains tested and

Fig. 1b shows an example of second dimension electro-

phoresis of plasmid DNA from strain TP1.D. It wasobserved that the isolates showed a distinctive plasmid

profile, so they could be easily distinguished by this tech-

nique. Interestingly, the two strains encoding tetM genesharboured a plasmid of the same molecular weight

(18.5 kb).

To identify the location of the tetK, tetM and tetS genesfound in the tested strains, undigested total DNA and

purified plasmid preparations were employed for Southern

blot hybridization experiments, using amplicons of the E.italicus tetK, tetM and tetS genes as probes. For tetS and

tetK genes, none of the plasmid samples showed a signal,

whereas in all the genomic DNA preparations a definiteband of high molecular weight was visualized (data not

shown), suggesting a chromosomal location of these

determinants. On the contrary, the tetM gene seemed to beassociated with extrachromosomal elements, particularly

with the 18.5 kb plasmid. Since this plasmid molecule has

approximately the same mobility as the chromosomalDNA, to analyse the presence of tetM determinants at

chromosomal level, we carried out Southern hybridization

experiments, employing total and plasmid DNA digested

F. Borgo et al.: Evaluation of Plasmid Content and Tetracycline Resistance 263

123

with the restriction enzymes EcoRI and HindIII. Hybrid-ization signals at the same position in digested total and

plasmid DNA suggest the tetM gene to be only present in

plasmid DNA and associated with the biggest plasmiddetected in the two strains harbouring this gene (Fig. 2).

The segregational stability of tetM in these strains was

high, since the strains were always subcultured in antibi-otic-free media, from isolation to maintenance in labora-

tory collection.

Tetracycline Resistance Transfer

Filter mating experiments with E. faecalis JH2-2 (RifR,FusR) harbouring the broad-host range pIP501 plasmid

(CmR) as donor and E. italicus RP4 strain (TetR) as recipient(1:1 ratio donor/recipient) showed that the chloramphenicolresistance phenotype was transferred to E. italicus at fre-

quencies ranging between 10-4 and 10-5 transconjugants

per recipient. Similar results were obtained in direct plate

p6

p1

p3 p4

p5

BFirst dimension

Second dimension

6.0

A

2.0

3.9

8.0

16.2

1 2 3 4 M

kb

p2

Fig. 1 Gel electrophoresis ofpurified plasmid DNA (panel a)from E. italicus strains. Lanes 1through 4, plasmid DNA fromTP2.3, TP1.D, TP3.D, RP4strains, respectively. M,Supercoiled DNA Ladder(Invitrogen-Life Technologies,USA). Panel b, banding patternof plasmid DNA from E.italicus strain TP1.D after two-dimensional agarose gelelectrophoresis. Symbols: p1–p6 refer to the six plasmids inthe CCC form; O1–O5 to theOC form of the correspondingplasmids originally present inthe plasmid DNA preparation;O1*–O6* to the OC formsnewly originated from CCCforms (C) after UV irradiation

BA

1 2 3 4 5 M1 M2 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

Fig. 2 Gel electrophoresis (panel a) and Southern blot analysis(panel b) of total and plasmid DNA from two E. italicus tetracyclineresistant strains. As a probe, an internal segment of tetM obtained byspecific PCR was used. Lane 1, undigested plasmid DNA from strainRP4; lane 2, RP4 plasmid DNA digested with EcoRI; lane 3, RP4total DNA digested with EcoRI; lane 4, RP4 plasmid DNA digestedwith HindIII; lane 5, RP4 total DNA digested with HindIII; lane 6,

undigested plasmid DNA from strain TP2.3; lane 7, TP2.3 plasmidDNA digested with EcoRI; lane 8, TP2.3 total DNA digested withEcoRI; lane 9, TP2.3 plasmid DNA digested with HindIII; lane 10,TP2.3 total DNA digested with HindIII. M1, Supercoiled DNA Ladder(Invitrogen); M2, linear Gene Ruler DNA Ladder Mix (MBI-Fermentas, Lithuania)

264 F. Borgo et al.: Evaluation of Plasmid Content and Tetracycline Resistance

123

conjugation mating in milk. Potential transconjugant colo-

nies isolated from the double selective medium (containingboth 25 lg/ml chloramphenicol and 25 lg/ml tetracycline)

displayed the TetR/CmR phenotype. Further confirmation of

the transconjugant’s identity was obtained by cat and tetM-specific PCR and by using species-specific probe for E. ita-licus and E. faecalis. Genotypic verification of the trans-

ferred plasmid was also carried out. All transconjugantsexhibited the same plasmid profile as theE. italicus recipientand additionally harboured the R-plasmid of the donor strain(data not shown). After 100 generations in the absence of

chloramphenicol, the transconjugants retained the CmR

phenotype and the conjugative plasmid pIP501.On the basis of the data obtained, we investigated the

mobility of the plasmid-located tetM gene in E. italicusstrain RP4 (TetR) in filter mating experiments with tetra-cycline-susceptible (TetS) recipient strain E. faecalis JH2-2(plasmid-free, RifR FusR). Several attempts to obtain

transconjugants from the double selective medium (con-taining both 25 lg/ml fusidic acid and 25 lg/ml tetracy-

cline) by using different donor-recipient ratios failed: no

transfer of tetracycline resistance from E. italicus to E.faecalis was found. The same results were obtained with

direct plate conjugation mating experiments. No trans-

conjugants were obtained with another recipient strain E.faecalis OG1RF (plasmid-free, TetS, RifR FusR).

Search for a Mobilization Region in the tetM Plasmid

To verify if the non-conjugative tetM plasmid of E. italicusRP4 could bemobilized in the presence of additional transferfunctions, we used the conjugative plasmid pIP501 in tri-

parentalmating experiments, withE. italicusRP4 as donor 1,E. faecalis JH 2-2 harbouring the conjugative plasmidpIP501 as donor 2 and the tetracycline-susceptible (TetS)

recipient strain E. faecalis JH2-2 (plasmid-free). However,

also in these experiments no transfer of tetracycline resis-tance from E. italicus to E. faecaliswas detected in the tripleselective M17 medium (containing 25 lg/ml fusidic acid,

25 lg/ml tetracycline and 25 lg/ml chloramphenicol).To assess the presence of a mobilization region on the

tetM plasmid of E. italicus RP4, we designed a primer pair

on basis of the reported nucleotide sequences of selectedrelaxases of the pMV158 superfamily [11]. The primer pair

matched to the relaxases encoded by the E. faecalis plas-mid pAMa1 [10], the Staphylococcus plasmids pUB110,pSK41, and pIP1714, the Bacillus cereus plasmid pBC16

and the Geobacillus plasmids pTB19 and pTB53. Addi-

tionally, we designed a primer pair matching the relaxasesencoded by the Streptococcus plasmids pMV158,

pSMQ172, pER13, and pSSU1 [11]. Positive signals were

only obtained either in PCR or in hybridization experi-ments for the positive controls pSK41 and pMV158.

Discussion

A lot of research has focused on the potential role of food

enterococci as reservoirs and/or vehicles of antibiotic-

resistance [5, 15]. As antibiotic resistant strains ofEnterococcus have been isolated from raw foods and some

believe that water and foods are possible vectors of strain

transmission to human intestinal microflora [16, 27], thecontrol of enterococci in foods has assumed a new level of

importance in food processing and food microbiology.

From several studies it appears that tetracycline resistance(TetR) is one of the most common phenotypes of acquired

antibiotic resistance in enterococcal food isolates [23, 26].

Since this antimicrobial agent is still used in human therapyand it is also in use in veterinary and aquaculture settings,

detailed characterization of new TetR food-associated en-

terococcal strains is of great importance.Previously, we carried out a phenotypic characterization

and a preliminary genotypic study for the new enterococcal

species E. italicus [7]. From these studies we deduced thatthe tested strains of dairy origin are associated with a low

virulence profile, with the exception of a TetR phenotype,

characterized at the molecular level by the presence of twoor more tetracycline determinants. For this reason, and for

the recent detection of an E italicus isolate from a clinically

significant human source [3], in this survey, the E. italicusstrains were investigated to evaluate their potential of

horizontal tetracycline resistance transfer. Since the strainswere negative by PCR for the integrase element (int),indicating that they do not contain a conjugative transposon

[7], we evaluated the presence of extrachromosomal ele-ments and the location of tet determinants. The majority of

the tested strains showed the presence of several plasmid

molecules; one of them, of 18.5 kb, seemed to be associ-ated with the presence of the tetM determinant in two E.italicus strains. However, according to its relatively low

molecular weight and the results of filter mating experi-ments, this plasmid should be considered a non-conjugative

plasmid. The other tetracycline determinants found in E.italicus seem to be chromosomally located.

To completely evaluate E. italicus food safety, we car-

ried out further experiments with the aim to verify if the

18.5 kb plasmid of E. italicus could be classified as amobilizable plasmid. Naturally occurring mobilizable

plasmids lack the functions required for mating pair for-

mation, but can be transmissible in the presence of addi-tional conjugative function, since they carry the genetic

information necessary for relaxosome formation and pro-

cessing [11, 12, 19]. Firstly we exploited the naturalmobilization capacity of plasmid pIP501. pIP501 is a

30.6 kb Inc18 broad-host range conjugative plasmid that

shows two important characteristics: it is able to self-transfer to a wide variety of Gram positive genera and to

F. Borgo et al.: Evaluation of Plasmid Content and Tetracycline Resistance 265

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mobilize non-self-transmissible plasmids, such as pMV158

[18]. The results of the triparental filter mating experimentsshowed that the 18.5 kb tetM plasmid from E. italicuscould not be mobilized by pIP501. Since the 18.5 kb

plasmid may possess mobilization functions not recognizedby the pIP501 transfer machinery, we tested for the pres-

ence of the relaxase functions encoded by a variety of

plasmids originating from Gram positive bacteria, all ofthem belonging to the pMV158 relaxase superfamily [11]

Additionally, we tested the 18.5 kb plasmid for the pres-ence of the Streptococcus plasmids pMV158, pSMQ172,

pER13 and pSSU1-encoded relaxase gene. In both cases,

no positive signals were obtained.Our data suggest that the new enterococcal species

neither uses transposition nor possesses transmissible or

mobilizable plasmids as mechanism for the disseminationof tet genes. For this reason we argue that the presence of

E. italicus in the food chain could be associated with low

health risk. The determination of the complete nucleotidesequence of the 18.5 kb plasmid and the collection and

study of new E. italicus isolates from different origin will

be a challenging task for future work.

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