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International Journal of Food Microbiology 39 (1998) 231–236

Short communicationCharacterization of plasmids from Listeria monocytogenes and

Listeria innocua strains isolated from short-ripened cheeses

*´Abelardo Margolles, Clara G. de los Reyes-Gavilan

´ Instituto de Productos Lacteos de Asturias ( CSIC ). Crta. de Infiesto s / n, 33300 Villaviciosa, Asturias, Spain

Received 30 June 1997; received in revised form 2 October 1997; accepted 10 November 1997

Abstract

The plasmid content of 30 isolates of Listeria monocytogenes and 18 isolates of Listeria innocua obtained from

short-ripened cheeses was analysed. The isolates of L. monocytogenes serogroup 1 harboured a single plasmid, pLM33 (33.2

kbp), whereas the serogroup 4 isolates did not contain plasmids. One group of L. innocua strains harboured the plasmid

pLI71 (71 kbp) and another one contained two plasmids: pLI59 (59.5 kbp) and pLI56 (56.5 kbp). These plasmid groups

were in accordance with clusters previously defined by pulsed-field gel electrophoresis analysis of the chromosomal DNA of

Listeria isolates. Plasmids pLM33, pLI71 and pLI59 shared homology regions of at least 20 kbp. Plasmid pLI56 did not

encode genes for any known character (such as carbohydrate fermentation, resistance to antibiotics, heavy metals or

disinfectants, growth at low pH, NaCl tolerance or thermal inactivation by pasteurisation) and displayed different

characteristics to the other three plasmids. It was also the only one cured from the parent strain and the sole plasmid not

digested by the restriction enzyme Pst I. In addition, its lack of homology with pLM33, pLI71 and pLI59 enhanced the

possibility of a different origin for plasmid pLI56. © 1998 Elsevier Science B.V.

Keywords: Listeria monocytogenes; Listeria innocua; Plasmid; Cheeses

1. Introduction be frequently isolated from cheeses, dairy products

and other foods. The presence of plasmids in Listeria´ ´ Listeria monocytogenes is an opportunistic patho- was first reported by Perez-Dıaz et al. (1982) and

gen of humans and animals which in the last decade since this time, research has focused mainly on

has been implicated in several outbreaks and clinical isolates of L. monocytogenes. Most plasmids

sporadic cases of listeriosis traced to contaminated in Listeria are cryptic but several authors have

food (Farber and Peterkin, 1991). Listeria innocua, a recently shown the involvement of plasmids and

species closely related to L. monocytogenes, is transposons in cadmium and antibiotic resistance in

nonhemolytic and nonpathogenic. Both of them can Listeria (Poyart-Salmeron et al., 1990; Lebrun et al.,

1992; Poyart-Salmeron et al., 1992; Facinelli et al.,

* 1993; Hadorn et al., 1993; Lebrun et al., 1994a,b).Corresponding author. Tel.: 1 34 85892131; fax: 1 34

85892233; e-mail: [email protected] However, despite several studies on food and en-

0168-1605/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

P I I S 0 1 6 8 - 1 6 0 5 ( 9 7 ) 0 0 1 3 2 - 3

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´ 232 A. Margolles, C .G. de los Reyes-Gavilan / International Journal of Food Microbiology 39 (1998) 231 –236

vironmental strains (Fistrovici and Collins-Thomp- containing novobiocin at a subinhibitory concen-

son, 1990; Kolstad et al., 1990; Peterkin et al., 1992; tration (0.2 mg/ml). The culture was incubated for

Facinelli et al., 1993), there is still very little data 24 h at high temperature (408C), followed by 9

about plasmids in L. monocytogenes from nonclini- subcultures (1% inoculum) in the same medium

cal sources such as foods, as well as in other Listeria containing novobiocin. After growth under these

species. conditions, the cultures were plated on tryptone–

Recently, we reported the isolation of several L. soya agar (TSA) and individual colonies were sub-

monocytogenes and L. innocua strains from some sequently picked, purified and submitted to a plasmid

regional (Asturias, northern Spain) short-ripened extraction and endonuclease restriction analysis.

cheeses (Margolles et al., 1996). Analysis by pulsed- Parent and cured strains were phenotypically

field gel electrophoresis (PFGE) of ApaI and SmaI- characterised. In brief, lactose and melezitose fer-

digested chromosomal DNA defined five clusters in mentation ability were tested in an appropriate liquid

L. monocytogenes (m1 to m5 ) and two main clusters medium (peptone of meat 1%, sodium chloride

in L. innocua (i1 and i2 ). Clusters m1, m2 and m3 0.5%, bromocresol purple 0.02g/l, pH 6.8). Minimal

of L. monocytogenes harboured strains of serogroup inhibitory concentrations against several antibiotics

1 whereas clusters m4 and m5 contained strains of (penicillin G, ampicillin, cephalotin, streptomycin,

serogroup 4 (Margolles et al., 1997). In the present gentamicin, kanamycin, neomycin, chloramphenicol,

work we report on the preliminary characterization tetracycline, erythromycin, rifampicin and phos-

of the plasmid content of these Listeria isolates. phomycin: 0.031 to 512 mg/ml) and heavy metals

salts (C H FeO ?H O, CuSO , HgCl , ZnSO ?6 5 7 2 4 2 4

7H O, Pb(NO ) , and CdSO ? 8H O: 2 to 20402 3 2 4 2

2. Material and methods mg / ml) were determined on Mueller–Hinton agar

and on TSA, respectively. Susceptibility to several

2.1. Plasmid profiles disinfectants (active agents: chlorine, peroxide, acid

or base) was evaluated by the Association of Official

Listeria strains were cultured on tryptone–soya Analytical Chemists (1984) method. Thermal in-

broth (TSB) (Adsa-Micro, Barcelona, Spain) at 308C. activation by HTST pasteurisation was tested on

Small-scale plasmid DNA preparations of Listeria overnight cultures washed and suspended in skimwere made as described by Simon et al. (1985) with milk. Heat treatment of milk was carried out in a

several modifications. Cells were harvested by cen- Linus Dualcycler (Linus, Madrid, Spain) and sur-

trifugation from 10 ml of exponential-growth-phase vivors were subsequently determined on TSA. The

cultures and protoplasts were prepared with lyso- ability to grow at low pH (3.5 to 5.5) produced by

zyme (final concentration: 10 mg/ ml) and incubated inorganic (HCl) or different organic acids (acetic,

for 1 h at 378C. In addition, after NaCl precipitation, citric and lactic acid: 50 and 150 mM) as well as

several phenol and chloroform extractions were NaCl tolerance (5 to 15%) were also evaluated using

made. DNA was examined on 0.6% horizontal microtiter plates with different concentrations of

agarose gels (Seakem, FMC Bioproducts, Rockland, acids or NaCl in TSB.

Maine) run at 3.5 V/cm during 5 h in TAE buffer

(40 mM Tris–acetate, pH 8.0, and 2 mM EDTA).Plasmids from Enterococcus faecalis BM 4100WT 2.3. Restriction endonuclease analysis and DNA–

(Courvalin et al., 1980) were used as molecular size DNA hybridisation

standards (70.1, 67.3, 49 and 2 kbp, respectively).

Restriction endonuclease digestions were per-

2.2. Plasmid curing experiments and phenotypic formed as recommended by the supplier (Boehringer

characterization of parent and cured derivative Mannheim, Germany). DNA was electrophoresed on

strains TAE buffer in 1% agarose gels at 1.2 V/cm during

16 h. DNA fragments from agarose gels were

An overnight culture of several Listeria strains in vacuum blotted to Hybond-N nylon membranes

TSB was inoculated (0.1 ml in 10 ml) into TSB- (Amersham, Buckinghamshire, UK) in a Bio-Rad

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´ A. Margolles, C .G. de los Reyes-Gavilan / International Journal of Food Microbiology 39 (1998) 231 –236 233

785 vacuum blotter (Bio-Rad, Richmond, CA) ac- al., 1992), our Listeria spp. isolates contained no

cording to the manufacturer instructions. more than two different plasmids per cell. In addi-

Southern hybridisations were done using as probes tion, no multiplasmic strains were found. On the

individual plasmids isolated from Listeria strains. other hand, 75.7% of our strains isolated from short-

Ten mg of EcoRI digests of the DNA probe were ripened cheeses contained plasmids. This was a

labelled with digoxigenin-dUTP (DIG DNA Label- similar level to that reported by Kolstad et al. (1990)

ling and Detection Kit, Boehringer). Hybridisation in Listeria spp. isolates from different origins,

and washing steps were performed at 658C. Sub- although the rather lower incidence of plasmid

sequent colorimetric detection with NBT (nitroblue positive strains (between 14 and 30%) has been´ ´tetrazolium salt) and X-Phosphate was performed as reported by other workers (Perez-Dıaz et al., 1982;

recommended by the supplier (Boehringer). Flamm et al., 1984; Fistrovici and Collins-Thomp-

son, 1990; Peterkin et al., 1992). These differences

can be explained if we take into account the different

3. Results and discussion origins of the isolates. Several studies indicate that

the percentage of plasmid positive strains in Listeria

Plasmid profiles of 30 isolates of L. monocyto- spp. is higher in strains of food and environmental

´ ´genes and 18 of L. innocua were analysed. All L. origin than in clinical isolates (Perez-Dıaz et al.,

monocytogenes isolates serogroup 1 harboured a 1982; Nocera et al., 1990). Several authors have

single plasmid (pLM33) whereas serogroup 4 iso- found higher levels of strains harbouring plasmids in

lates did not contain plasmids (Fig. 1 ). However, L. innocua (93%) than in L. monocytogenes (20%)

plasmids were present in all L. innocua strains: (Peterkin et al., 1992), as was also the case with our

isolates of one group (PFGE cluster i1 ) harboured Listeria isolates.

one plasmid (pLI71) and the isolates of the other Curing experiments were carried out with novo-

group (PFGE cluster i2 ) contained two plasmids biocin and at a high temperature as curing agents on

(pLI59 and pLI56). As previously reported by other isolates representative of all plasmid groups. After

´ ´authors (Perez-Dıaz et al., 1982; Flamm et al., 1984; several attempts, only pLI56 was cured from strain

Fistrovici and Collins-Thompson, 1990; Peterkin et Li16, giving rise to the cured derivative Li16c which

only harboured the plasmid pLI59 (Fig. 1). Nodifferences were found between Li16 and Li16c

based on the phenotypic characters tested in this

work (lactose and melezitose fermentation, suscep-

tibility to antibiotics, heavy metals and disinfectants,

growth at low pH and with different organic acids,

tolerance to NaCl and thermal inactivation by pas-

teurisation). This indicates that pLI56 did not encode

genes for the above mentioned properties. On the

other hand, as it was reported above, all our serog-

roup 1 L. monocytogenes isolates contained a unique

plasmid (pLM33), whereas serogroup 4 isolates didnot contain plasmids. This suggests a possible corre-

lation between the serogroup and the presence of

plasmid in this species. Since no cured variants

lacking plasmid were obtained from serogroup 1

strains, this hypothesis could not be proved. To this

respect, Lebrun et al. (1992) indicated that the

conjugative introduction of plasmids in L. monocyto-Fig. 1. Plasmids of Listeria strains. Lanes: 1, pLM33 ( L. mono-

genes strains did not cause a change of its serogroupcytogenes Lm1 strain); 2, pLI71 ( L. innocua Li17 strain); 3, pLI59

and Kolstad et al. (1990) did not find a relationand pLI56 ( L. innocua Li16 strain); 4, pLI59 ( L. innocua Li16c

strain). between serotype and plasmid content in their work.

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It is also interesting to note that Nocera et al. (1990) Li16 strain and its derivative Li16c was quite lower

reported an absence of extrachromosomal DNA in than that expected from the electrophoretic migration

L. monocytogenes serotype 4b strains related to of the undigested plasmid. Because of that, the

the Swiss epidemic of listeriosis (1983–1987) and enzyme HindIII was used for this purpose giving a´ ´Perez-Dıaz et al. (1982) did not find plasmids in size of 56.5 kbp for pLI56 (data not shown). On the

clinical serogroup 4 strains. It is also necessary to other hand, restriction analysis with EcoRI and Pst I

take into account that the plasmid content in Listeria proved that the plasmid content of all isolates shared

strains isolated from foods could be underestimated numerous bands with identical molecular size, sug-

because isolates are usually obtained by enrichment gesting that homology regions could exist between

in selective media containing acriflavin, a potential plasmids. Furthermore, comparison of Li16 and

curing agent (Slade et al., 1988). Li16c Pst I restriction profiles indicated that this

Total plasmid content of Lm1 (pLM33), Li17 enzyme did not cut the DNA of pLI 56 probably by

(pLI71) Li16 (pLI59 and pLI56) and Li16c (pLI59) absence of recognising sequences for Pst I.

strains was analysed by endonuclease restriction. For The EcoRI digests of the plasmid content of

plasmids pLM33, pLI71 and pLI59, the molecular strains from each plasmid group were hybridised to

sizes calculated from EcoRI digestions were 33.2, 71 digoxigenin-labelled probes prepared from plasmids

and 59.5 kbp, respectively (Fig. 2) which were pLM33, pLI59, and pLI71. Using the EcoRI digest

similar to those of other Listeria strains (Kolstad et of pLI71 as a probe, homology was found with all

al., 1990). However, the molecular size of pLI56 fragments of pLM33 and pLI59 [Fig. 2B(a)]. When

estimated by comparing EcoRI digestion profiles of the probe was prepared from pLI59, all fragments of

Fig. 2. (A) EcoRI and Pst I restriction profiles of the different plasmid groups. Lanes 1 and 6, l DNA digested with Pst I (1) and HindIII (6)

(molecular sizes in kbp). Plasmids pLM33, pLI71, pLI591 pLI56, and pLI59 digested with EcoRI (lanes 2 to 5) and with Pst I (lanes 7 to

10). (B) Respective hybridisations of lines 1 to 6 obtained using the EcoRI digests of pLI71 (a), pLI59 (b), and pLM33 (c) as a probe.

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´ A. Margolles, C .G. de los Reyes-Gavilan / International Journal of Food Microbiology 39 (1998) 231 –236 235

pLM33 and most of pLI71 fragments showed hybrid- Spain (Grant ALI93-0114). A. Margolles was the

isation [Fig. 2B(b)]. Also, the probe from pLM33 recipient of a predoctoral fellowship from the Fun-´ ´ ´ ´displayed homology with most but not all fragments dacion para la Investigacion Cientıfica y Tecnica

of pLI59 and pLI71 [Fig. 2B(c)]. Using each one of (Asturias, Spain). We thank Ian Bytheway for his

these three plasmids as a probe, five common helpful comments about the English usage on this

hybridisation bands to pLM33, pLI59 and pLI71 manuscript.

were always obtained whose molecular sizes were

9.0, 8.3, 1.5, 0.8 and 0.5 kbp. This data corroborated

the genetic proximity between plasmids pLM33, ReferencespLI59 and pLI71 which have homology regions of at

least 20 kbp. The presence of homology zonesAssociation of Official Analytical Chemists, 1984. Disinfectants.

between native plasmids in Listeria has already been In: Williams, S. (Ed.), Official Methods of Analysis of the

shown by other authors (Kolstad et al., 1990; Flamm Association of Official Analytical Chemists. Arlington, VA, p.

70.et al., 1984). On the other hand, using each one of Charpentier, E., Gerbaud, G., Courvalin, P., 1994. Presence of thethe above three plasmids as a probe, hybridisation

Listeria tetracycline resistance gene tet ( S ) in Enterococcusprofiles of the strain Li16 and its curing derivative

faecalis. Antimicrob. Agents Chemother. 38, 2330–2335.Li16c were identical, indicating the lack of homol- Courvalin, P., Carlier, C., Collatz, E., 1980. Plasmid-mediatedogy between pLI56 and the other three plasmids of resistance to aminocyclitol antibiotics in group D streptococci.

J. Bacteriol. 143, 541–551. L. monocytogenes and L. innocua. Different charac-Facinelli, B., Roberts, M.C., Giovanetti, E., Casolari, C., Fabio,teristics of pLI56 to the other plasmids (pLM33,

U., Varaldo, P.E., 1993. Genetic basis of tetracycline resistancepLI71 and pLI59) strengthened the hypothesis of a

in food-borne isolates of Listeria innocua. Appl. Environ.different origin for this plasmid. It has been sug- Microbiol. 59, 614–616.gested that Listeria spp. can acquire plasmids in Farber, J.M., Peterkin, P.I., 1991. Listeria monocytogenes, a food-

borne pathogen. Microbiol. Rev. 55, 476–511.certain environments such as the fecal tract (Fis-Fistrovici, E., Collins-Thompson, D.L., 1990. Use of plasmidtrovici and Collins-Thompson, 1990). In addition,

profiles and restriction endonuclease digest in environmentalseveral authors have effectively demonstrated thatstudies of Listeria spp. from raw milk. Int. J. Food Microbiol.

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duction of pAMb1 into Listeria monocytogenes by conjuga-biotic resistance in L. monocytogenes and L. innocuation and homology between native Listeria monocytogenes(Flamm et al., 1984; Vicente et al., 1988; Poyart-plasmids. Infect. Immun. 44, 157–161.Salmeron et al., 1990, 1992; Facinelli et al., 1993;

¨Hadorn, K., Hachler, H., Schaffner, A., Kayser, F.H., 1993.Charpentier et al., 1994). Due to its ubiquitous

Genetic characterization of plasmid-encoded multiple anti-nature, Listeria as well as the genetically related biotic resistance in a strain of Listeria monocytogenes causing

enterococci and Streptococcus spp. can be present in endocarditis. Eur. J. Clin. Microbiol. Infect. Dis. 12, 928–937.

Kolstad, J., Rørvik, L.M., Granum, P.E., 1990. Characterization of many different environments. Thus, a genetic trans-plasmics from Listeria sp. Int. J. Food Microbiol. 12, 123–fer from other microorganisms could explain the132.

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Finally, hybridisation of the different probes with cadmium resistance genes in Listeria monocytogenes are

the total DNA of the strains studied did not show similar to cad A and cad C of Staphylococcus aureus and areinduced by cadmium. J. Bacteriol. 176, 3040–3048.additional hybridisation bands different from those of

Lebrun, M., Audurier, A., Cossart, P., 1994. Plasmid-bornethe owner plasmid indicating that no homologycadmium resistance genes in Listeria monocytogenes are

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´ ´Margolles, A., Rodrıguez, A., de los Reyes-Gavilan, C., 1996.This work was financially supported by the Comi- Some chemical and bacteriological characteristics of regional

´sion Interministerial de Ciencia y Tecnologıa of cheeses from Asturias. Spain. J. Food Prot. 59, 509–515.

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