APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1992, p. 3183-3186 0099-2240/92/093183-04$02.00/0 Copyright ©) 1992, American Society for Microbiology
Vol. 58, No. 9
Plasmids in Listeria monocytogenes in Relation to Cadmium Resistance MARYSE LEBRUN,"12 JOELLE
LOULERGUE,l ELISABETH CHASLUS-DANCLA,3
AND
ANDRE
AUDURIER'*
Laboratoire de Bacte6riologie, Faculte de Medecine, 37032 Tours, 1 and Laboratoire de Pathologie Infectieuse et Immunologie2 and Laboratoire de Pathologie Aviaire et Parasitologie,3 Institut National de la Recherche Agronomique, 37380 Nouzilly, France Received 27 March 1992/Accepted 30 June 1992
One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40%YV and 29%Yo, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. nwnocytogenes have been shown to be associated with cadmium resistance.
During the past decade, Listeria monocytogenes has been incriminated in a number of food-borne disease outbreaks and several sporadic episodes of listeric illness (8). In this context, typing of L. monocytogenes is a crucial step in incriminating a contaminated food source in human infection and in determining the relationship between L. monocytogenes in the final food product and the producing and processing environment. Routine typing of L. monocytogenes is based on the determination of serovar and lysovar. Sometimes these methods are unsuccessful, because of poor discrimination of serotyping (14) and high percentages of nontypable strains with the bacteriophages used (1). Other methods, including multilocus enzyme electrophoresis (17), restriction enzyme analysis of chromosomal DNA with highor low-frequency cleavage enzymes (5, 15), and analysis of restriction fragment length polymorphisms after hybridization with a deoxyribonucleotide probe (3, 23), are being investigated as possible improved typing systems. Although the isolation of plasmids from L. monocytogenes was reported in 1982 (16), there has been little research on their use as epidemiological markers. Plasmid typing has been used only once in conjunction with restriction chromosomal DNA analysis to confirm a case of cross-infection with L. monocytogenes (7). We have only minimal information on the prevalence and the functions of such plasmids. Antibiotic resistance has seldom been detected in L. monocytogenes. In 1990, Poyart-Salmeron et al. (18) isolated the first antibiotic-resistant strain, which carried a plasmid conferring resistance to chloramphenicol, erythromycin, streptomycin, and tetracycline. Although some strains from food or from sporadic cases of listeriosis have been reported as resistant to one or more antibiotics (6, 12, 19), in most cases plasmids of L. monocytogenes are cryptic. The purpose of this study was to evaluate the presence and diversity of plasmids of L. monocytogenes and their potential value as a basis for a typing method. We searched *
Corresponding author.
for strains that were highly resistant to antimicrobial agents in an effort to determine the genetic basis of this resistance. Prevalence and diversity of L. monocytogenes plasmids. We studied 173 strains of L. monocytogenes isolated in France from 1986 to 1990. All of the strains came from the faculte de Medecine de Tours collection, which includes more than 10,000 strains. The 173 strains were chosen according to their source, geographic origin, date of their isolation, serogroup, and phage type to eliminate replicates. Of the 173 strains, 33 were from meat and meat products, 49 were from milk and cheese, 21 were from the environment (silage, vegetables, and sewage farms), 41 were from human listeriosis cases, and 29 were from animal listeriosis cases. All of the isolates were biochemically confirmed as L. monocytogenes. Serotyping with specific antisera (Difco Laboratories, Detroit, Mich.) was performed and phage types were determined as previously described (2, 20). Plasmid DNA was extracted as described by Birnboim and Doly (4). The plasmid pIP112 (100.5 kb) (21) and the eight cryptic plasmids of the strain V517 (13) were used for molecular size markers. Of the strains tested, 28% were found to harbor plasmids. A striking feature of this study is the difference noted in the proportions of plasmid-carrying strains from different sources and serogroups (Table 1). Thirty-five percent of the serogroup 1 strains harbored plasmids, whereas 15% of the serogroup 4 strains harbored plasmids. Plasmids were carried by strains from all three sources; however, in serogroup 1 and 4 strains, plasmids were more frequently found in food and environmental isolates than in clinical strains (40, 29, and 13%, respectively). Almost half of the strains from meat harbored plasmids, as did one-third of the strains from milk and cheese. No significant difference (by chi-square analysis) in plasmid content between human and animal clinical isolates was observed (data not shown). Plasmids were more common among non-phage-typable strains (38.2%) than among phage-typable strains (22%) (data not shown). This significant difference (P < 0.05, chi square analysis) was confirmed by the successful extraction of plasmids from strains lysed by one or two phages but not from strains lysed 3183
APPL. ENVIRON. MICROBIOL.
NOTES
3184
TABLE 1. Distribution of L. monocytogenes harboring plasmids by serogroup and source of the strains No. of strains harboring plasmids/total
Source
Serogroup 1
Clinical Food Environment Total
8/40 (20) 24/51 (47) 6/17 (35) 38/108 (35)d
(% harboring plasmids) Serogroup 4
1/30 (3) 9/31 (29) O/4C 10/65 (15)d
Total
9/70 (13)a"b 33/82 (40)6/21 (29)b 48/173 (28)
a Percentages of plasmid-bearing strains isolated in clinical samples versus those in strains from food were highly significantly different (P < 0.01) by chi-square analysis. b Percentages of plasmid-bearing strains isolated in clinical samples versus those in strains from the environment were not significantly different (P < 0.1) by chi-square analysis (r = 2.9). c Not representative. d Percentages of plasmid-bearing strains in serogroup 1 versus serogroup 4 were highly significantly different (P < 0.01) by chi-square analysis.
by all or nearly all of the phages used for phage typing. Nevertheless, the presence of plasmids was not related to a particular lysovar. Among the 48 plasmid-bearing strains, only one strain harbored two plasmids. On the basis of the number and sizes of the plasmids, 19 plasmid profiles were defined (Table 2). The sizes of the plasmids ranged from 14 to 106 kb; half of the plasmids were between 53 and 61 kb in size. No profile correlated with either a particular source or a specific lysovar (data not shown). Strains of serogroup 4 contained plasmids of only four different sizes, whereas serogroup 1 strains contained plasmids of 15 sizes (Table 2). Only one plasmid of 53 kb, isolated from a serogroup 4 strain, was identical in size to a plasmid found in serogroup 1 strains. Plasmids of common sizes (53 kb for serogroup 1 and 61 kb for serogroup 4) were analyzed after digestion by EcoRI (Appligene, Illkirch, France). Three different restriction patterns were observed (Fig. 1). Pattern A, observed in the 10 53-kb plasmids from strains of serogroup 1, was identical to that of the majority of plasmids (four of seven) isolated in TABLE 2. Distribution of plasmids by serogroup of L. monocytogenes strains Size(s) of plasmid(s) (kb)
14 29 32 36 38 41 43 47 52 53 55 59 61 64, 72 74 81 87 99 106
No. of strains (%) Serogroup 1
1 (2.6) 1 (2.6) 2 (5.3) 1 (2.6) 1 (2.6) 1 (2.6) 1 (2.6) 2 (5.3) 1 (2.6) 10 (26.3) 6 (15.8) 2 (5.3) 0 1 (2.6)
1(2.6) 5 (13.2) 0 0 2 (5.3)
Serogroup 4
0 0 0 0 0 0 0
0 0 1 (10) 0
0 6 (60)
0 0 0 2 (20) 1 (10)
0
Total
1 (2.1) 1 (2.1) 2 (4.1) 1 (2.1) 1 (2.1) 1 (2.1) 1 (2.1) 2 (4.1) 1 (2.1) 11 (23.0) 6 (12.5) 2 (4.1) 6 (12.5) 1 (2.1) 1 (2.1) 5 (10.4) 2 (4.1) 1 (2.1) 2 (4.1)
FIG. 1. EcoRl restriction profiles of L. monocytogenes plasmids. Lanes: 1 through 10, pattern A (53-kb plasmids isolated from serogroup 1 strains); 11, pattern C (53-kb plasmids isolated from a serogroup 4 strain); 12 through 17, pattern B (61-kb plasmids isolated from serogroup 4 strains). The lanes to the left and right of numbered lanes contain lambda phage DNA digested by EcoRI and
HindIlI (fragments of 21.2, 5.1, 4.9, 4.2, 3.5, 2.0, 1.9, 1.5, 1.3, 0.9, and 0.8 kbp).
the United States in 1984 by Flamm et al. (9). Pattern B was observed in the six 61-kb plasmids from strains of serogroup 4. The unique plasmid of 53 kb from L. monocytogenes of serogroup 4 had a distinct restriction pattern (pattern C). Thus, no identical plasmid was found in both serogroup 1 and 4 L. monocytogenes strains. The results suggest that the size diversity of the L. monocytogenes plasmids is important, especially for strains of serogroup 1, but molecular typing of plasmids should not be used for routine testing because only a small number of strains harbor plasmids. Many of the restriction fragment patterns of the plasmids were identical, which diminishes the usefulness of plasmids as epidemiological markers. The analysis of plasmids should nevertheless be undertaken when other typing methods are unsuccessful, particularly when strains are of serogroup 1 and not typable by the conventional phage typing method. Functions of L. monocytogenes plasmids. The susceptibilities of 90 strains to seven antibiotics and four heavy-metal salts were investigated. The agar dilution method (22) was used to determine the MICs of the following compounds: chloramphenicol, erythromycin, framycethin, nitrofurantoin, oxacillin, streptomycin, tetracycline, cadmium sulfate, mercuric chloride, silver nitrate, and nickel chloride (all from Sigma Chemical Co., St. Louis, Mo.). Staphylococcus aureus ATCC 25923 was included in each test as the control organism. The concentrations of inhibitors ranged from 0 to 128 ,ug of antibiotics per ml and from 0 to 1,024 ,ug of heavy-metal salts per ml (0 to 4,096 ,ug of nickel chloride per
ml). The patterns of susceptibility to each of the antibiotics were the same for all 90 strains tested. The results indicated that the acquisition of antibiotic resistance in L. monocytogenes strains is extremely rare and that the main functions of its plasmids are not related to antibiotic resistance. For
mercuric chloride, silver nitrate, and nickel chloride, all the
VOL. 58, 1992
NOTES
TABLE 3. In vitro susceptibilities of 90 L. monocytogenes strains to heavy-metal salts No. of isolates with salt MICs (,ug/ml) of:
Heavy-metal salt 4
8 16 32 64 128 256 512 1,024 2,048 4,096
3 23 21 3(CdSO4) * 8H20 41a 2 48a 42 HgCl2 3 55a 32 AgNO3 NiCl2
5a
81
4
a MIC is the same as that for S. aureus ATCC 25923.
strains tested also had the same pattern of susceptibility (Table 3), but two populations of L. monocytogenes showed susceptibility to cadmium. For 43 strains, the MICs of CdSO4 ranged from 4 to 8 p,g/ml; for 47 resistant strains, the MICs of CdSO4 were between 32 and 128 p,g/ml. Among the strains that were resistant to cadmium, 87.2% (41 of 47) harbored plasmids, whereas only 4.7% (2 of 43) of cadmiumsusceptible L. monocytogenes strains contained plasmids. This difference was significant (P < 0.01) by chi-square analysis. The two strains that were susceptible to cadmium and harbored plasmids were isolated from one human and one animal with listeriosis; the plasmids were 14 and 53 kb in size. All of the strains with pattern A or B were resistant to cadmium. The only strain with a distinct pattern C was cadmium susceptible. No plasmid DNA could be detected in 6 cadmium-resistant strains after three repeated extractions. To determine the genetics of cadmium resistance in L. monocytogenes, we examined the transfer of plasmids between strains of L. monocytogenes and plasmid curing. Conjugation between strains of L. monocytogenes was conducted by filter mating as described by Poyart-Salmeron et al. (18), and the transfer frequency was expressed as the number of transconjugants per donor CFU after mating. The recipient strain used in this experiment was L. monocytogenes EGD-SmR, provided by Gaillard et al. (10), which is a streptomycin-resistant mutant that contains no plasmids. L. monocytogenes Lm 106, which is resistant to cadmium, was chosen as donor of the 61-kb plasmid. The parental strains were of serogroup 4 for the donor strain and serogroup 1 for the recipient strain. The transconjugants were selected on Mueller-Hinton agar containing 100 p,g of streptomycin per ml and 32 ,ug of CdSO4 per ml. The 61-kb Lm 106 plasmid was transferred by conjugation to EGD-SmR at a frequency of 3.75 x 10'. Six colonies of the cadmium-resistant progeny of the mating were serotyped and screened to detection the plasmid. Each of the six colonies was of serogroup 1 (like the recipient strain) and harbored the 61-kb plasmid of the donor strain; when transferred to strain EGD-SmR, this plasmid conferred resistance to cadmium at the same level as that observed in the donor strain (data not shown). The loss of cadmium resistance in strains Lm 27, Lm 5, Lm 132, and Lm 119, respectively, carrying the 32-, 43-, 81-, and 106-kb plasmids was obtained by high-temperature treatment. After overnight culture at 43°C, 0.1-ml serial dilutions of each strain were streaked onto brain heart infusion agar and incubated at 37°C for 48 h; then 15 colonies were picked randomly, and the MICs of CdSO4 were determined. For each parental strain, the plasmid contents of two isogenic variants, one resistant and one susceptible to cadmium, were determined. All of the cadmium-susceptible isogenic variants were plasmid free, and all of the cadmiumresistant isogenic variants harbored plasmids.
3185
We have shown that 95% of the plasmids of L. monocytogenes confer cadmium resistance and that the plasmids can be transmitted between strains of this species. Resistance to cadmium is widespread among S. aureus strains (11) and is described almost exclusively in this species, where it is mainly conferred by extrachromosomal DNA. Six of the studied strains did not have plasmids but grew in the presence of high concentrations of cadmium. We can put forward two hypotheses to explain this result. The first is that the plasmid extraction method used in this study does not permit the detection of plasmids present in these strains. The second is that cadmium resistance can be chromosomal, as has been shown in S. aureus (24). We are currently investigating the genetic and molecular basis of cadmium resistance. Cadmium is highly toxic to most life forms and often present in the environment. It can act as an efficient selective agent in the environment, and this could explain the high percentage of plasmids in strains isolated from food and the environment. Cadmium resistance is a very selective advantage for the persistence of L. monocytogenes in the environment; thus, plasmids could contribute to the survival of such strains in the environment. We are grateful to P. Berche for providing the EGD-SmR strain used in this study, A. Fenneteau for the serotyping and phage typing, M. C. Lesage for information on the extraction of plasmids, and P. Pardon for helpful discussions. We are indebted to A. G. Taylor (London) for reading the manuscript. This work was supported by a grant from the Conseil R6gional de la Region Centre, France. REFERENCES 1. Audurier, A., and C. Martin. 1989. Phage typing of Listeria monocytogenes. Int. J. Food. Microbiol. 8:251-257. 2. Audurier, A., J. Rocourt, and A. Courtieu. 1977. Isolement et caracterisation de bacteriophages de Listena monocytogenes. Ann. Microbiol. (Paris) 128A:185-198. 3. Baloga, A. O., and S. K. Harlander. 1991. Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol. 57:2324-2331. 4. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 5. Carriere, C., A. Allardet-Servent, G. Bourg, A. Audurier, and M. Ramuz. 1991. DNA polymorphism in strains of Listeria monocytogenes. J. Clin. Microbiol. 29:1351-1355. 6. Facinelli, B., E. Giovanetti, P. E. Varaldo, C. Casolari, and U. Fabio. 1991. Antibiotic resistance in foodborne Listeria. Lancet 338:1272. 7. Facinelli, B., P. E. Varaldo, C. Casolari, and U. Fabio. 1988. Cross-infection with Listeria monocytogenes confirmed by DNA fingerprinting. Lancet ii:1247-1248. 8. Farber, J. M., and P. 1. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476-511. 9. Flamm, R. K., D. J. Hinrichs, and M. F. Thomashow. 1984. Introduction of pAM 1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44:157-161. 10. Gaillard, J. L., P. Berche, and P. Sansonetti. 1986. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 52:50-55. 11. Lyon, B. R., and R. Skurray. 1987. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiol. Rev. 51:88134. 12. MacGowan, A. P., D. S. Reeves, and J. McLauchlin. 1990. Antibiotic resistance of Listeria monocytogenes. Lancet 336: 513-514.
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13. Macrina, F. L., D. J. Kopecko, K. R. Jones, D. J. Ayers, and S. M. McCowen. 1978. A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid 1:417-420. 14. McLauchlin, J., A. Audurier, and A. L. Courtieu. 1987. Listeria monocytogenes, recent advances in taxonomy and epidemiology of listeriosis in humans. J. Appl. Bacteriol. 63:1-11. 15. Nocera, D., E. Bannerman, J. Rocourt, K. Jaton-Ogay, and J. Bille. 1990. Characterization by DNA restriction endonuclease analysis of Listeria monocytogenes strains related to Swiss epidemic of listeriosis. J. Clin. Microbiol. 28:2259-2263. 16. Perez-Diaz, J. C., M. F. Vicente, and F. Baquero. 1982. Plasmids in Listeria. Plasmid 8:112-118. 17. Piffaretti, J. C., H. Kresseburch, M. Aeschbacher, J. Bille, E. Bannerman, J. M. Musser, R. K. Selander, and J. Rocourt. 1989. Genetic characterization of clones of bacterium Listeria monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. USA 86:3818-3822. 18. Poyart-Salmeron, C., C. Carlier, P. Trieu-Cuot, A. L. Courtieu, and P. Courvalin. 1990. Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes. Lancet 335:14221426. 19. Quentin, C., M. C. Thibaut, J. Horovitz, and C. Bebear. 1990. Multiresistant strain of Listeria monocytogenes in septic abor-
APPL. ENVIRON. MICROBIOL.
tion. Lancet 336:375. 20. Rocourt, J., A. Audurier, A. L. Courtieu, J. Durst, S. Ortel, A. Schrettenbrunner, and A. G. Taylor. 1985. A multi-center study on the phage typing of Listeria monocytogenes. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 259:489-497. 21. Roussel, A., C. Carlier, G. Gerbaud, Y. A. Chabbert, 0. Croissant, and D. Blangy. 1979. Reversible translocation of antibiotic resistance determinants in Salmonella ordonez. Mol. Gen. Genet. 169:13-25. 22. Sahm, D. F., and J. A. Washington II. 1991. Antimicrobial susceptibility tests: dilution methods, p. 1105-1116. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. 23. Wesley, I. V., R. D. Wesley, J. Heisick, F. Harrell, and D. Wagner. 1990. Characterization of Listeria monocytogenes isolates by southern blot hybridization. Vet. Microbiol. 24:341353. 24. Witte, W., L. Green, T. K. Misra, and S. Silver. 1986. Resistance to mercury and cadmium in chromosomally resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 29: 663-669.
Vol. 58, No. 9
Plasmids in Listeria monocytogenes in Relation to Cadmium Resistance MARYSE LEBRUN,"12 JOELLE
LOULERGUE,l ELISABETH CHASLUS-DANCLA,3
AND
ANDRE
AUDURIER'*
Laboratoire de Bacte6riologie, Faculte de Medecine, 37032 Tours, 1 and Laboratoire de Pathologie Infectieuse et Immunologie2 and Laboratoire de Pathologie Aviaire et Parasitologie,3 Institut National de la Recherche Agronomique, 37380 Nouzilly, France Received 27 March 1992/Accepted 30 June 1992
One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40%YV and 29%Yo, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. nwnocytogenes have been shown to be associated with cadmium resistance.
During the past decade, Listeria monocytogenes has been incriminated in a number of food-borne disease outbreaks and several sporadic episodes of listeric illness (8). In this context, typing of L. monocytogenes is a crucial step in incriminating a contaminated food source in human infection and in determining the relationship between L. monocytogenes in the final food product and the producing and processing environment. Routine typing of L. monocytogenes is based on the determination of serovar and lysovar. Sometimes these methods are unsuccessful, because of poor discrimination of serotyping (14) and high percentages of nontypable strains with the bacteriophages used (1). Other methods, including multilocus enzyme electrophoresis (17), restriction enzyme analysis of chromosomal DNA with highor low-frequency cleavage enzymes (5, 15), and analysis of restriction fragment length polymorphisms after hybridization with a deoxyribonucleotide probe (3, 23), are being investigated as possible improved typing systems. Although the isolation of plasmids from L. monocytogenes was reported in 1982 (16), there has been little research on their use as epidemiological markers. Plasmid typing has been used only once in conjunction with restriction chromosomal DNA analysis to confirm a case of cross-infection with L. monocytogenes (7). We have only minimal information on the prevalence and the functions of such plasmids. Antibiotic resistance has seldom been detected in L. monocytogenes. In 1990, Poyart-Salmeron et al. (18) isolated the first antibiotic-resistant strain, which carried a plasmid conferring resistance to chloramphenicol, erythromycin, streptomycin, and tetracycline. Although some strains from food or from sporadic cases of listeriosis have been reported as resistant to one or more antibiotics (6, 12, 19), in most cases plasmids of L. monocytogenes are cryptic. The purpose of this study was to evaluate the presence and diversity of plasmids of L. monocytogenes and their potential value as a basis for a typing method. We searched *
Corresponding author.
for strains that were highly resistant to antimicrobial agents in an effort to determine the genetic basis of this resistance. Prevalence and diversity of L. monocytogenes plasmids. We studied 173 strains of L. monocytogenes isolated in France from 1986 to 1990. All of the strains came from the faculte de Medecine de Tours collection, which includes more than 10,000 strains. The 173 strains were chosen according to their source, geographic origin, date of their isolation, serogroup, and phage type to eliminate replicates. Of the 173 strains, 33 were from meat and meat products, 49 were from milk and cheese, 21 were from the environment (silage, vegetables, and sewage farms), 41 were from human listeriosis cases, and 29 were from animal listeriosis cases. All of the isolates were biochemically confirmed as L. monocytogenes. Serotyping with specific antisera (Difco Laboratories, Detroit, Mich.) was performed and phage types were determined as previously described (2, 20). Plasmid DNA was extracted as described by Birnboim and Doly (4). The plasmid pIP112 (100.5 kb) (21) and the eight cryptic plasmids of the strain V517 (13) were used for molecular size markers. Of the strains tested, 28% were found to harbor plasmids. A striking feature of this study is the difference noted in the proportions of plasmid-carrying strains from different sources and serogroups (Table 1). Thirty-five percent of the serogroup 1 strains harbored plasmids, whereas 15% of the serogroup 4 strains harbored plasmids. Plasmids were carried by strains from all three sources; however, in serogroup 1 and 4 strains, plasmids were more frequently found in food and environmental isolates than in clinical strains (40, 29, and 13%, respectively). Almost half of the strains from meat harbored plasmids, as did one-third of the strains from milk and cheese. No significant difference (by chi-square analysis) in plasmid content between human and animal clinical isolates was observed (data not shown). Plasmids were more common among non-phage-typable strains (38.2%) than among phage-typable strains (22%) (data not shown). This significant difference (P < 0.05, chi square analysis) was confirmed by the successful extraction of plasmids from strains lysed by one or two phages but not from strains lysed 3183
APPL. ENVIRON. MICROBIOL.
NOTES
3184
TABLE 1. Distribution of L. monocytogenes harboring plasmids by serogroup and source of the strains No. of strains harboring plasmids/total
Source
Serogroup 1
Clinical Food Environment Total
8/40 (20) 24/51 (47) 6/17 (35) 38/108 (35)d
(% harboring plasmids) Serogroup 4
1/30 (3) 9/31 (29) O/4C 10/65 (15)d
Total
9/70 (13)a"b 33/82 (40)6/21 (29)b 48/173 (28)
a Percentages of plasmid-bearing strains isolated in clinical samples versus those in strains from food were highly significantly different (P < 0.01) by chi-square analysis. b Percentages of plasmid-bearing strains isolated in clinical samples versus those in strains from the environment were not significantly different (P < 0.1) by chi-square analysis (r = 2.9). c Not representative. d Percentages of plasmid-bearing strains in serogroup 1 versus serogroup 4 were highly significantly different (P < 0.01) by chi-square analysis.
by all or nearly all of the phages used for phage typing. Nevertheless, the presence of plasmids was not related to a particular lysovar. Among the 48 plasmid-bearing strains, only one strain harbored two plasmids. On the basis of the number and sizes of the plasmids, 19 plasmid profiles were defined (Table 2). The sizes of the plasmids ranged from 14 to 106 kb; half of the plasmids were between 53 and 61 kb in size. No profile correlated with either a particular source or a specific lysovar (data not shown). Strains of serogroup 4 contained plasmids of only four different sizes, whereas serogroup 1 strains contained plasmids of 15 sizes (Table 2). Only one plasmid of 53 kb, isolated from a serogroup 4 strain, was identical in size to a plasmid found in serogroup 1 strains. Plasmids of common sizes (53 kb for serogroup 1 and 61 kb for serogroup 4) were analyzed after digestion by EcoRI (Appligene, Illkirch, France). Three different restriction patterns were observed (Fig. 1). Pattern A, observed in the 10 53-kb plasmids from strains of serogroup 1, was identical to that of the majority of plasmids (four of seven) isolated in TABLE 2. Distribution of plasmids by serogroup of L. monocytogenes strains Size(s) of plasmid(s) (kb)
14 29 32 36 38 41 43 47 52 53 55 59 61 64, 72 74 81 87 99 106
No. of strains (%) Serogroup 1
1 (2.6) 1 (2.6) 2 (5.3) 1 (2.6) 1 (2.6) 1 (2.6) 1 (2.6) 2 (5.3) 1 (2.6) 10 (26.3) 6 (15.8) 2 (5.3) 0 1 (2.6)
1(2.6) 5 (13.2) 0 0 2 (5.3)
Serogroup 4
0 0 0 0 0 0 0
0 0 1 (10) 0
0 6 (60)
0 0 0 2 (20) 1 (10)
0
Total
1 (2.1) 1 (2.1) 2 (4.1) 1 (2.1) 1 (2.1) 1 (2.1) 1 (2.1) 2 (4.1) 1 (2.1) 11 (23.0) 6 (12.5) 2 (4.1) 6 (12.5) 1 (2.1) 1 (2.1) 5 (10.4) 2 (4.1) 1 (2.1) 2 (4.1)
FIG. 1. EcoRl restriction profiles of L. monocytogenes plasmids. Lanes: 1 through 10, pattern A (53-kb plasmids isolated from serogroup 1 strains); 11, pattern C (53-kb plasmids isolated from a serogroup 4 strain); 12 through 17, pattern B (61-kb plasmids isolated from serogroup 4 strains). The lanes to the left and right of numbered lanes contain lambda phage DNA digested by EcoRI and
HindIlI (fragments of 21.2, 5.1, 4.9, 4.2, 3.5, 2.0, 1.9, 1.5, 1.3, 0.9, and 0.8 kbp).
the United States in 1984 by Flamm et al. (9). Pattern B was observed in the six 61-kb plasmids from strains of serogroup 4. The unique plasmid of 53 kb from L. monocytogenes of serogroup 4 had a distinct restriction pattern (pattern C). Thus, no identical plasmid was found in both serogroup 1 and 4 L. monocytogenes strains. The results suggest that the size diversity of the L. monocytogenes plasmids is important, especially for strains of serogroup 1, but molecular typing of plasmids should not be used for routine testing because only a small number of strains harbor plasmids. Many of the restriction fragment patterns of the plasmids were identical, which diminishes the usefulness of plasmids as epidemiological markers. The analysis of plasmids should nevertheless be undertaken when other typing methods are unsuccessful, particularly when strains are of serogroup 1 and not typable by the conventional phage typing method. Functions of L. monocytogenes plasmids. The susceptibilities of 90 strains to seven antibiotics and four heavy-metal salts were investigated. The agar dilution method (22) was used to determine the MICs of the following compounds: chloramphenicol, erythromycin, framycethin, nitrofurantoin, oxacillin, streptomycin, tetracycline, cadmium sulfate, mercuric chloride, silver nitrate, and nickel chloride (all from Sigma Chemical Co., St. Louis, Mo.). Staphylococcus aureus ATCC 25923 was included in each test as the control organism. The concentrations of inhibitors ranged from 0 to 128 ,ug of antibiotics per ml and from 0 to 1,024 ,ug of heavy-metal salts per ml (0 to 4,096 ,ug of nickel chloride per
ml). The patterns of susceptibility to each of the antibiotics were the same for all 90 strains tested. The results indicated that the acquisition of antibiotic resistance in L. monocytogenes strains is extremely rare and that the main functions of its plasmids are not related to antibiotic resistance. For
mercuric chloride, silver nitrate, and nickel chloride, all the
VOL. 58, 1992
NOTES
TABLE 3. In vitro susceptibilities of 90 L. monocytogenes strains to heavy-metal salts No. of isolates with salt MICs (,ug/ml) of:
Heavy-metal salt 4
8 16 32 64 128 256 512 1,024 2,048 4,096
3 23 21 3(CdSO4) * 8H20 41a 2 48a 42 HgCl2 3 55a 32 AgNO3 NiCl2
5a
81
4
a MIC is the same as that for S. aureus ATCC 25923.
strains tested also had the same pattern of susceptibility (Table 3), but two populations of L. monocytogenes showed susceptibility to cadmium. For 43 strains, the MICs of CdSO4 ranged from 4 to 8 p,g/ml; for 47 resistant strains, the MICs of CdSO4 were between 32 and 128 p,g/ml. Among the strains that were resistant to cadmium, 87.2% (41 of 47) harbored plasmids, whereas only 4.7% (2 of 43) of cadmiumsusceptible L. monocytogenes strains contained plasmids. This difference was significant (P < 0.01) by chi-square analysis. The two strains that were susceptible to cadmium and harbored plasmids were isolated from one human and one animal with listeriosis; the plasmids were 14 and 53 kb in size. All of the strains with pattern A or B were resistant to cadmium. The only strain with a distinct pattern C was cadmium susceptible. No plasmid DNA could be detected in 6 cadmium-resistant strains after three repeated extractions. To determine the genetics of cadmium resistance in L. monocytogenes, we examined the transfer of plasmids between strains of L. monocytogenes and plasmid curing. Conjugation between strains of L. monocytogenes was conducted by filter mating as described by Poyart-Salmeron et al. (18), and the transfer frequency was expressed as the number of transconjugants per donor CFU after mating. The recipient strain used in this experiment was L. monocytogenes EGD-SmR, provided by Gaillard et al. (10), which is a streptomycin-resistant mutant that contains no plasmids. L. monocytogenes Lm 106, which is resistant to cadmium, was chosen as donor of the 61-kb plasmid. The parental strains were of serogroup 4 for the donor strain and serogroup 1 for the recipient strain. The transconjugants were selected on Mueller-Hinton agar containing 100 p,g of streptomycin per ml and 32 ,ug of CdSO4 per ml. The 61-kb Lm 106 plasmid was transferred by conjugation to EGD-SmR at a frequency of 3.75 x 10'. Six colonies of the cadmium-resistant progeny of the mating were serotyped and screened to detection the plasmid. Each of the six colonies was of serogroup 1 (like the recipient strain) and harbored the 61-kb plasmid of the donor strain; when transferred to strain EGD-SmR, this plasmid conferred resistance to cadmium at the same level as that observed in the donor strain (data not shown). The loss of cadmium resistance in strains Lm 27, Lm 5, Lm 132, and Lm 119, respectively, carrying the 32-, 43-, 81-, and 106-kb plasmids was obtained by high-temperature treatment. After overnight culture at 43°C, 0.1-ml serial dilutions of each strain were streaked onto brain heart infusion agar and incubated at 37°C for 48 h; then 15 colonies were picked randomly, and the MICs of CdSO4 were determined. For each parental strain, the plasmid contents of two isogenic variants, one resistant and one susceptible to cadmium, were determined. All of the cadmium-susceptible isogenic variants were plasmid free, and all of the cadmiumresistant isogenic variants harbored plasmids.
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We have shown that 95% of the plasmids of L. monocytogenes confer cadmium resistance and that the plasmids can be transmitted between strains of this species. Resistance to cadmium is widespread among S. aureus strains (11) and is described almost exclusively in this species, where it is mainly conferred by extrachromosomal DNA. Six of the studied strains did not have plasmids but grew in the presence of high concentrations of cadmium. We can put forward two hypotheses to explain this result. The first is that the plasmid extraction method used in this study does not permit the detection of plasmids present in these strains. The second is that cadmium resistance can be chromosomal, as has been shown in S. aureus (24). We are currently investigating the genetic and molecular basis of cadmium resistance. Cadmium is highly toxic to most life forms and often present in the environment. It can act as an efficient selective agent in the environment, and this could explain the high percentage of plasmids in strains isolated from food and the environment. Cadmium resistance is a very selective advantage for the persistence of L. monocytogenes in the environment; thus, plasmids could contribute to the survival of such strains in the environment. We are grateful to P. Berche for providing the EGD-SmR strain used in this study, A. Fenneteau for the serotyping and phage typing, M. C. Lesage for information on the extraction of plasmids, and P. Pardon for helpful discussions. We are indebted to A. G. Taylor (London) for reading the manuscript. This work was supported by a grant from the Conseil R6gional de la Region Centre, France. REFERENCES 1. Audurier, A., and C. Martin. 1989. Phage typing of Listeria monocytogenes. Int. J. Food. Microbiol. 8:251-257. 2. Audurier, A., J. Rocourt, and A. Courtieu. 1977. Isolement et caracterisation de bacteriophages de Listena monocytogenes. Ann. Microbiol. (Paris) 128A:185-198. 3. Baloga, A. O., and S. K. Harlander. 1991. Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol. 57:2324-2331. 4. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 5. Carriere, C., A. Allardet-Servent, G. Bourg, A. Audurier, and M. Ramuz. 1991. DNA polymorphism in strains of Listeria monocytogenes. J. Clin. Microbiol. 29:1351-1355. 6. Facinelli, B., E. Giovanetti, P. E. Varaldo, C. Casolari, and U. Fabio. 1991. Antibiotic resistance in foodborne Listeria. Lancet 338:1272. 7. Facinelli, B., P. E. Varaldo, C. Casolari, and U. Fabio. 1988. Cross-infection with Listeria monocytogenes confirmed by DNA fingerprinting. Lancet ii:1247-1248. 8. Farber, J. M., and P. 1. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476-511. 9. Flamm, R. K., D. J. Hinrichs, and M. F. Thomashow. 1984. Introduction of pAM 1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44:157-161. 10. Gaillard, J. L., P. Berche, and P. Sansonetti. 1986. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence of Listeria monocytogenes. Infect. Immun. 52:50-55. 11. Lyon, B. R., and R. Skurray. 1987. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiol. Rev. 51:88134. 12. MacGowan, A. P., D. S. Reeves, and J. McLauchlin. 1990. Antibiotic resistance of Listeria monocytogenes. Lancet 336: 513-514.
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