Plasmids in Listeria monocytogenes and other Listeria species PEARLI. PETERKIN,MARY-ANN GARDINER, AND NAEEMMALIK Bureau of Microbial Hazards, Food Directorate, Health Protection Branch, Ottawa, Ont., Canada KIA OL2 AND
EDMUND S. IDZIAK
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Department of Microbiology, Faculty of Agriculture, Macdonald College of McGill University, Ste-Anne-de-Bellevue, Que., Canada H9X 1CO Received August 17, 1991 Revision received October 3, 1991 Accepted October 8, 1991 M.-A., MALIK,N., and IDZIAK,E. S. 1992. Plasmids in Listeria monocytogenes and other PETERKIN, P. I., GARDINER, Listeria species. Can. J. Microbiol. 38: 161-164. One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance. Key words: plasmid, Listeria spp., Listeria monocytogenes. PETERKIN, P. I., GARDINER, M.-A., MALIK,N., et IDZIAK,E. S. 1992. Plasmids in Listeria monocytogenes and other Listeria species. Can. J. Microbiol. 38 : 161-164. La presence de plasmides a ete recherchee chez 122 souches de Listeria monocytogenes d'origine alimentaire, clinique et veterinaire. Vingt-cinq (20%) souches contenaient des plasmides dont la taille variait de 1,3 a 66 MDa. Chez 10 autres souches regroupant d'autres especes de Listeria (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi et L. murrayi), sept (70%) hebergeaient des plasmides dont la taille variait de 38 a 53 MDa. Aucune souche ne contenait plusieurs plasmides. Dans certains cas des plasmides de taille identique ont Cte isoles chez des souches apparentees. La presence d'un plasmide dans une souche n'etait pas reliee a des caracteres phenotypiques qui seraient transmis par des elements extrachromosomiques. Mots cfks : plasmide, Listeria spp., Listeria monocytogenes. [Traduit par la redaction]
The presence of plasmids in Listeria species was first reported by Perez-Diaz et al. (1982), who found a 38.5-MDa plasmid in 4 of 29 (14%) L. monocytogenes strains, both L. grayi strains and the single L. murrayi strain examined. When isolated from each of the seven strains, this plasmid had a similar pattern of restriction enzyme fragments. These authors found no plasmids in strains of clinical origin. In contrast, Flamm et al. (1984) found plasmids with a range of molecular sizes (32,34,44, and 53 MDa) in 7 of 29 (24%) L. monocytogenes strains, although the 34-MDa plasmid was found in four of these strains. DNA homology studies indicated that the plasmids shared common sequences. Recently, Fistrovici and Collins-Thompson (1990) reported the presence of plasmids (10 or 44 MDa) in 8 of 27 (30%) L. innocua strains, although they found none in 19 L. monocytogenes strains. No phenotypic character such as antibiotic resistance was associated with the presence of any of these plasmids. Incidences of plasmids at these levels in a food-borne pathogen such as L. monocytogenes are of little use in strain typing for epidemiological purposes. However, these studies were performed on a relatively small number of strains. We therefore undertook a more extensive study on a total of 122 (85 clinical, 10 food, 25 veterinary, and 2 environmental) strains of L. monocytogenes, as well as 10 strains of other Listeria species. These strains were also examined for phenotypic ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
characters, such as production of P-hemolysin, and antibiotic resistance. Bacterial strains used in this study (Table 1) were isolated in Canada, and were from the Health Protection Branch (HPB), Health and Welfare Canada, Ottawa, Ont., the Laboratoire de Sante de la Province de Quebec, Senneville, Que., and the Animal Diseases Research Institute, Agriculture Canada, Nepean, Ont. They were maintained at ambient temperature on Institut Pasteur maintenance medium (Peterkin et al. 1991) and grown overnight at 35°C in brain heart infusion (Difco Laboratories, Detroit, Mich.) broth as required. Molecular size markers were kindly provided by Dr. L.-K. Ng, Laboratory Centre for Disease Control, Ottawa, Ont . (60 and 86 MDa), Dr. C. Wyndham, Carleton University, Ottawa, Ont. (71 MDa), and Dr. F.L. Macrina, Virginia Commonwealth University, Richmond, Va. (1.4-35.8 MDa; Macrina et al. 1978). A modification of the method of Anderson and McKay (1983) was used in the plasmid isolation. To purify the preparation from residual contaminating RNA, a 10-min digestion on ice with DNase-free RNase (2 pg/mL) was added following the neutralization step. An L. innocua strain containing a 43-MDa plasmid was used as a positive control. Plasmid DNA was separated by gel electrophoresis at 7 V/cm in 0.7% agarose, both in the initial screening of strains and in determining the molecular size of identified plasmids. The hemolytic activity of strains was determined by stabbing the inoculum into 7% horse blood agar plates
162
CAN. J. MICROBIOL. VOL. 38, 1992
TABLE1 . Strains of Listeria spp. examined for plasmids Organism L. monocytogenes
Source Clinical
S e r ~ v a r No- of strains 1/2 4b 4d Others NK"
33 18 5
L. monocytogenes
Food
1/2 4b 6b
3 4 3
L. monocytogenes
Veterinary
1/2 4b
4 7
..
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 11/11/14 For personal use only.
5 24
6b
NK L. monocytogenes
Environmental
1/2
L. innocua
Food
L. welshimeri
Environmental
6b
L. seeligeri
Environmental
1 / 2 , NK
L. ivanovii
Food
L. grayi
Veterinary
L. murrayi
Environmental
4
5
'NK, not known.
(blood agar base; Oxoid Canada Inc., Nepean, Ont.), incubating the plates overnight at 37OC, and examining them by transmitted light for zones of clearing. The antibiotic resistance of the strains was determined by the disc assay method with the following antibiotics (Difco): streptomycin (10 pg), ampicillin (10 pg), erythromycin (15 pg), tetracycline (30 pg), and chloramphenicol (30 pg). Plasmids ranging in size from 1.3 to 66 MDa were found in 25 of the 122 L. monocytogenes strains examined (Table 2), an overall incidence of 20%. Plasmid incidence among the 85 clinical strains examined was 24% (20 strains). Examination of the plasmid profiles showed that the plasmids could be grouped according to size. The three strains (serovar 4b) containing small plasmids were isolated from stool specimens in a Halifax, N.S., hospital in 1982. Two of these plasmids were identical in size (1.5 MDa), and although the third was slightly smaller (1.3 MDa), these three form a group distinct from the much larger plasmids. Of the six strains containing plasmids of 37-MDa size, five were serovar 1/2, while the sixth had not been serotyped. Two of the 37-MDa plasmids, together with a slightly larger plasmid (40 MDa) also found in a serotype 1/2 strain, were found in strains isolated from pregnant women suffering from listeriosis, who were patients at a Halifax, N.S., hospital during 1984. Two of the remaining L. monocytogenes serovar 1/2 strains containing 37-MDa plasmids were isolated during 1985 from patients with septicaemia in hospitals in Ottawa, Ont., and Winnipeg, Man., respectively. Both the L. monocytogenes strains containing 66-MDa plasmids were serovar 1/2 strains isolated from blood samples at a hospital in MontrCal, Que., and the Provincial Laboratory of Health, Toronto, Ont. Too few plasmids were found among the food and veterinary strains to establish a pattern of plasmid profiles (Table 2). The strain isolated from the alfalfa tablets was
one of a food-clinical pair that had previously been demonstrated to be the same strain by DNA fingerprinting (P.I. Peterkin, unpublished results). Unexpectedly, although a plasmid of 44 MDa was found in the food isolate, no plasmid was isolated from its clinical pair. We conclude that a plasmid was lost, perhaps as a result of the length of storage. No plasmids were isolated from the two environmental strains examined. None of the strains examined contained more than one plasmid. There was a marked difference in the plasmid incidence between L. monocytogenes and the other Listeria species investigated. We found seven (70%) strains containing plasmids among the 10 strains that we examined (Table 3). The two L. ivanovii strains were isolated from raw cream samples, during an investigation of the 1985 listeriosis outbreak in California caused by contaminated Jalisco cheese. They may be the same strain, based on the presence of plasmids of identical size. It is interesting that one of the two L. seeligeri strains examined contained a plasmid of the same size as that found in the two L. ivanovii strains. The strain formerly known as L. denitrificans (ATCC 14870), but now reclassified as Jonesia denitrificans (Rocourt et al. 1987), did not contain any plasmids. The level of plasmid incidence among Listeria species, apart from L. monocytogenes, agrees with that reported recently by Pritchard and Donnelly (1992). In examining 111 environmental isolates of L. innocua, they found a 93% incidence of plasmids ranging in size from 3 to 54 MDa. Kolstad et al. (1990) also found a higher incidence (78%) of plasmids among 139 Listeria isolates. At these incidence levels, plasmid profiles would be of use in tracing sources of contamination. Recent reports on the presence of plasmids in L. monocytogenes strains freshly isolated from clinical specimens following outbreaks in .the United States indicate a much higher plasmid incidence than previously described. Mottice et al. (1987) found a plasmid incidence of 59% among isolates from 42 patients. Bottone et al. (1990) found plasmids in 15 of 16 (94%) patient isolates, including 75 % of maternalneonatal pairs. It is not surprising that the incidence of plasmids is high in strains isolated during an outbreak. The clinical isolates are often from a common source where plasmids, if found, would be expected to be found frequently. In the present study, the incidence of plasmids was only 24% in clinical strains of L. monocytogenes that had been transferred many times in the laboratory. It may be that plasmid profiles would be of more use in the strain typing of fresh isolates than with strains that have been in the laboratory for some time. In addition to studying plasmid profiles for possible usefulness in strain typing, we also tested for the presence of two characters of known extrachromosomal inheritance, namely, toxin production and antibiotic resistance. All of the 122 L. monocytogenes strains tested showed production of the toxin P-hemolysin. When these L. monocytogenes strains, as well as the 10 strains of other Listeria species, were tested for antibiotic resistance, none of them showed resistance to streptomycin, ampicillin, erythromycin, tetracycline, or chloramphenicol. We conclude that the Phemolysin determinant is not inherited extrachromosomally and that none of the plasmids that we isolated carried resistance determinants for the antibiotics tested.
NOTES
TABLE2. Plasmids in L. monocytogenes Strain Strain source
Frequency"
Size ( M D ~ )
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 11/11/14 For personal use only.
Clinical
Location
Serovar
Halifax, N.S. Halifax, N.S. Halifax, N.S. Ottawa, Ont. Winnipeg, Man. Quebec Halifax, N.S. Fredericton, N.B Quebec Ottawa, Ont. St. John's, Nfld. Quebec Quebec Quebec Montreal, Que. Toronto, Ont.
Food Cheese Cheese Alfalfa tablets Veterinary Bovine Ovine Environmental
NK NK Lachute, Que. St. Bernardin, Ont. Smith's Falls, Ont.
'Frequency = (no. o f plasmids)/(no. o f strains tested). b ~ u m b e rin parentheses is the number of plasmids of that size found. 'NK. not known.
Conjugative transfer of plasmids bearing antibiotic resistance markers can occur in the laboratory between L. monocytogenes and strains of Streptococcus or Enterococcus; transfer rates vary from to (Flamm et al. 1984; Vicente et al. 1988; Leclercq et al. 1989). The resistance determinants were stably inherited. The conjugative plasmids used in these experiments varied from 17 to 22.4 MDa in size. In spite of their ability to effect conjugative transfer, there have only been three reports in the literature, all from Europe, of antibiotic-resistant strains of L. monocytogenes having been isolated from clinical specimens. A plasmid of 40 MDa was found in a streptomycinresistant strain (Facinelli et al. 1989), and curing of the plasmid led to loss of resistance. A plasmid of 24 MDa carrying multiple antibiotic-resistant determinants was isolated by Poyart-Salmeron et al. (1990) from a strain of the organism resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. This plasmid showed extensive sequence homology to pAM@l,the broad-host-range resistance plasmid found in enterococci and streptococci. Quentin et al. (1990) reported the isolation of a strain of L. monocytogenes resistant t o chloramphenicol, erythromycin, tetracycline, and minocycline. Although the authors did not attempt plasmid isolation, they noted that all resistance markers were lost simultaneously when the strain was treated with curing agents such as acridine orange, suggesting a plasmid location for these determinants. In view of the transfer of plasmids that can occur between L. monocytogenes and Enterococcus and Streptococcus species, the suggestion has been made by Slade and Collins-
TABLE 3. Plasmids in Listeria spp. other than L. monocytogenes Species
Serovar
Source
Plasmid size (MDa)
- -
L. L. L. L. L. L. L.
innocua innocua innocua welshimeri seeligeri ivanovii ivanovii
Meat Meat Meat NK NK Raw cream Raw cream
'NK, not known.
Thompson (1990) that resistance factor transfer may occur in the human bowel. Thus, although no antibiotic-resistant strains of this pathogen have yet been reported in Canada or the United States, the possible emergence of such strains must be kept in mind. Anderson, D.G., and McKay, L.L. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46: 549-552. Bottone, E.J., Namdari, H., and Sellers, G. 1990. Listeria monocytogenes: investigation of an unexpected increased incidence of infection by plasmid profiling. Abstr. Annu. Meet. Am. Soc. Microbiol. C-358. p. 403. Facinelli, B., Casolari, C., Montanari, M.P., et al. 1989. Plasmid DNA isolation from Listeria monocytogenes. Abstr. Annu. Meet. Ann. Soc. Microbiol. H-219. p. 206.
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CAN. J. MICROBIOL.
Fistrovici, E., and Collins-Thompson, D.L. 1990. Use of plasmid profiles and restriction endonuclease digest in environmental studies of Listeria spp. from raw milk. Int. J. Food Microbiol. 10: 43-50. Flamm, R.K., Hinrichs, D.J., and Thomashow, M.F. 1984. Introduction of pAMPl into Lkteria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44: 157-161. Kolstad, J., Rorvik, L.M., and Granum, P.E. 1990. Characterization of plasmids from Listeria sp. Int. J. Food Microbiol. 12: 123-132. Leclercq, R., Derlot, E., Weber, M., et al. 1989. Transferable vancomycin and teicoplanin in Enterococcus faecium. Antimicrob. Agents Chemother. 33: 10-1 5. Macrina, F.L., Kopecko, D. J., Jones, K.R., et al. 1978. A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid, 1: 417-420. Mottice, S., Robinson, W., and Reimer, L.G. 1987. Plasmid typing for the epidemiologic characterization of Lkteria monocytogenes. Abstr-. Annu. Meet. Am. Soc. Microbiol. C-206. p. 357. Perez-Diaz, J .C., Vicente, M.F., and Baquero, F. 1982. Plasmids in Listeria. Plasmid, 8: 112-1 18.
VOL. 38,
1992
Peterkin, P.I., Idziak, E.S., and Sharpe, A.N. 1991. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe. Appl. Environ. Microbiol. 57: 586-591. Poyart-Salmeron, C., Carlier, C., Trieu-Cuot, P., et al. 1990. Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes. Lancet, i: 1422- 1426. Pritchard, T.J., and Donnelly, C.W. 1992. Plasmid profile analysis of Listeria species to compare dairy plant environmental isolates. J. Food Prot. 55. In press. Quentin, C., Thibaut, M.C., Horovitz, J., and Bebear, C. 1990. Multiresistant strain of Listeria monocytogenes in septic abortion. Lancet, ii: 375. Rocourt, J., Wehmeyer, U., and Stackebrandt, E. 1987. Transfer of Listeria denitrificans to Jonesia gen. nov. as Jonesia denitrificans comb. nov. Int. J. Syst. Bacteriol. 37: 266-270. Slade, P.J., and Collins-Thompson, D.L. 1990. Listeria, plasmids, antibiotic resistance, and food. Lancet, ii: 1004. Vicente, M.F., Baquero, F., and Perez-Diaz, J.C. 1988. Conjugative acquisition and expression of antibiotic resistance determinants in Listeria spp. J. Antimicrobiol. Chemother. 21: 309-3 18.
Erratum: Bacterial culture history affects the attachment of Bradyrhizobium japonicum to host Glycine mar roots1 GEOFFREY B. SMITH^
AND
A. G. WOLLUM I1
Department of Soil Science, North Carolina State University, Box 7619, Raleigh, NC 27695-7619, U.S.A. (Ref. Can. J. Microbiol. 37: 730-736. 1991.)
On page 730, in line 9 of the English abstract, the phrase "and on new cell growth" should be deleted. On page 730, in line 11 of the French abstract, the phrase "suivie d'une nouvelle croissance des cellules" should be deleted. On page 734, in Table 2, the second line in the column entitled Treatment should read 5 mM PNS. '~eceivedat NRC December 10, 1991. 2~resentaddress: Department of Biology (Dept. 3AF), Box 30001, New Mexico State University, Las Cruces, NM 88003.
EDMUND S. IDZIAK
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Department of Microbiology, Faculty of Agriculture, Macdonald College of McGill University, Ste-Anne-de-Bellevue, Que., Canada H9X 1CO Received August 17, 1991 Revision received October 3, 1991 Accepted October 8, 1991 M.-A., MALIK,N., and IDZIAK,E. S. 1992. Plasmids in Listeria monocytogenes and other PETERKIN, P. I., GARDINER, Listeria species. Can. J. Microbiol. 38: 161-164. One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance. Key words: plasmid, Listeria spp., Listeria monocytogenes. PETERKIN, P. I., GARDINER, M.-A., MALIK,N., et IDZIAK,E. S. 1992. Plasmids in Listeria monocytogenes and other Listeria species. Can. J. Microbiol. 38 : 161-164. La presence de plasmides a ete recherchee chez 122 souches de Listeria monocytogenes d'origine alimentaire, clinique et veterinaire. Vingt-cinq (20%) souches contenaient des plasmides dont la taille variait de 1,3 a 66 MDa. Chez 10 autres souches regroupant d'autres especes de Listeria (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi et L. murrayi), sept (70%) hebergeaient des plasmides dont la taille variait de 38 a 53 MDa. Aucune souche ne contenait plusieurs plasmides. Dans certains cas des plasmides de taille identique ont Cte isoles chez des souches apparentees. La presence d'un plasmide dans une souche n'etait pas reliee a des caracteres phenotypiques qui seraient transmis par des elements extrachromosomiques. Mots cfks : plasmide, Listeria spp., Listeria monocytogenes. [Traduit par la redaction]
The presence of plasmids in Listeria species was first reported by Perez-Diaz et al. (1982), who found a 38.5-MDa plasmid in 4 of 29 (14%) L. monocytogenes strains, both L. grayi strains and the single L. murrayi strain examined. When isolated from each of the seven strains, this plasmid had a similar pattern of restriction enzyme fragments. These authors found no plasmids in strains of clinical origin. In contrast, Flamm et al. (1984) found plasmids with a range of molecular sizes (32,34,44, and 53 MDa) in 7 of 29 (24%) L. monocytogenes strains, although the 34-MDa plasmid was found in four of these strains. DNA homology studies indicated that the plasmids shared common sequences. Recently, Fistrovici and Collins-Thompson (1990) reported the presence of plasmids (10 or 44 MDa) in 8 of 27 (30%) L. innocua strains, although they found none in 19 L. monocytogenes strains. No phenotypic character such as antibiotic resistance was associated with the presence of any of these plasmids. Incidences of plasmids at these levels in a food-borne pathogen such as L. monocytogenes are of little use in strain typing for epidemiological purposes. However, these studies were performed on a relatively small number of strains. We therefore undertook a more extensive study on a total of 122 (85 clinical, 10 food, 25 veterinary, and 2 environmental) strains of L. monocytogenes, as well as 10 strains of other Listeria species. These strains were also examined for phenotypic ' ~ u t h o rto whom all correspondence should be addressed. Printed in Canada / Imprime au Canada
characters, such as production of P-hemolysin, and antibiotic resistance. Bacterial strains used in this study (Table 1) were isolated in Canada, and were from the Health Protection Branch (HPB), Health and Welfare Canada, Ottawa, Ont., the Laboratoire de Sante de la Province de Quebec, Senneville, Que., and the Animal Diseases Research Institute, Agriculture Canada, Nepean, Ont. They were maintained at ambient temperature on Institut Pasteur maintenance medium (Peterkin et al. 1991) and grown overnight at 35°C in brain heart infusion (Difco Laboratories, Detroit, Mich.) broth as required. Molecular size markers were kindly provided by Dr. L.-K. Ng, Laboratory Centre for Disease Control, Ottawa, Ont . (60 and 86 MDa), Dr. C. Wyndham, Carleton University, Ottawa, Ont. (71 MDa), and Dr. F.L. Macrina, Virginia Commonwealth University, Richmond, Va. (1.4-35.8 MDa; Macrina et al. 1978). A modification of the method of Anderson and McKay (1983) was used in the plasmid isolation. To purify the preparation from residual contaminating RNA, a 10-min digestion on ice with DNase-free RNase (2 pg/mL) was added following the neutralization step. An L. innocua strain containing a 43-MDa plasmid was used as a positive control. Plasmid DNA was separated by gel electrophoresis at 7 V/cm in 0.7% agarose, both in the initial screening of strains and in determining the molecular size of identified plasmids. The hemolytic activity of strains was determined by stabbing the inoculum into 7% horse blood agar plates
162
CAN. J. MICROBIOL. VOL. 38, 1992
TABLE1 . Strains of Listeria spp. examined for plasmids Organism L. monocytogenes
Source Clinical
S e r ~ v a r No- of strains 1/2 4b 4d Others NK"
33 18 5
L. monocytogenes
Food
1/2 4b 6b
3 4 3
L. monocytogenes
Veterinary
1/2 4b
4 7
..
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5 24
6b
NK L. monocytogenes
Environmental
1/2
L. innocua
Food
L. welshimeri
Environmental
6b
L. seeligeri
Environmental
1 / 2 , NK
L. ivanovii
Food
L. grayi
Veterinary
L. murrayi
Environmental
4
5
'NK, not known.
(blood agar base; Oxoid Canada Inc., Nepean, Ont.), incubating the plates overnight at 37OC, and examining them by transmitted light for zones of clearing. The antibiotic resistance of the strains was determined by the disc assay method with the following antibiotics (Difco): streptomycin (10 pg), ampicillin (10 pg), erythromycin (15 pg), tetracycline (30 pg), and chloramphenicol (30 pg). Plasmids ranging in size from 1.3 to 66 MDa were found in 25 of the 122 L. monocytogenes strains examined (Table 2), an overall incidence of 20%. Plasmid incidence among the 85 clinical strains examined was 24% (20 strains). Examination of the plasmid profiles showed that the plasmids could be grouped according to size. The three strains (serovar 4b) containing small plasmids were isolated from stool specimens in a Halifax, N.S., hospital in 1982. Two of these plasmids were identical in size (1.5 MDa), and although the third was slightly smaller (1.3 MDa), these three form a group distinct from the much larger plasmids. Of the six strains containing plasmids of 37-MDa size, five were serovar 1/2, while the sixth had not been serotyped. Two of the 37-MDa plasmids, together with a slightly larger plasmid (40 MDa) also found in a serotype 1/2 strain, were found in strains isolated from pregnant women suffering from listeriosis, who were patients at a Halifax, N.S., hospital during 1984. Two of the remaining L. monocytogenes serovar 1/2 strains containing 37-MDa plasmids were isolated during 1985 from patients with septicaemia in hospitals in Ottawa, Ont., and Winnipeg, Man., respectively. Both the L. monocytogenes strains containing 66-MDa plasmids were serovar 1/2 strains isolated from blood samples at a hospital in MontrCal, Que., and the Provincial Laboratory of Health, Toronto, Ont. Too few plasmids were found among the food and veterinary strains to establish a pattern of plasmid profiles (Table 2). The strain isolated from the alfalfa tablets was
one of a food-clinical pair that had previously been demonstrated to be the same strain by DNA fingerprinting (P.I. Peterkin, unpublished results). Unexpectedly, although a plasmid of 44 MDa was found in the food isolate, no plasmid was isolated from its clinical pair. We conclude that a plasmid was lost, perhaps as a result of the length of storage. No plasmids were isolated from the two environmental strains examined. None of the strains examined contained more than one plasmid. There was a marked difference in the plasmid incidence between L. monocytogenes and the other Listeria species investigated. We found seven (70%) strains containing plasmids among the 10 strains that we examined (Table 3). The two L. ivanovii strains were isolated from raw cream samples, during an investigation of the 1985 listeriosis outbreak in California caused by contaminated Jalisco cheese. They may be the same strain, based on the presence of plasmids of identical size. It is interesting that one of the two L. seeligeri strains examined contained a plasmid of the same size as that found in the two L. ivanovii strains. The strain formerly known as L. denitrificans (ATCC 14870), but now reclassified as Jonesia denitrificans (Rocourt et al. 1987), did not contain any plasmids. The level of plasmid incidence among Listeria species, apart from L. monocytogenes, agrees with that reported recently by Pritchard and Donnelly (1992). In examining 111 environmental isolates of L. innocua, they found a 93% incidence of plasmids ranging in size from 3 to 54 MDa. Kolstad et al. (1990) also found a higher incidence (78%) of plasmids among 139 Listeria isolates. At these incidence levels, plasmid profiles would be of use in tracing sources of contamination. Recent reports on the presence of plasmids in L. monocytogenes strains freshly isolated from clinical specimens following outbreaks in .the United States indicate a much higher plasmid incidence than previously described. Mottice et al. (1987) found a plasmid incidence of 59% among isolates from 42 patients. Bottone et al. (1990) found plasmids in 15 of 16 (94%) patient isolates, including 75 % of maternalneonatal pairs. It is not surprising that the incidence of plasmids is high in strains isolated during an outbreak. The clinical isolates are often from a common source where plasmids, if found, would be expected to be found frequently. In the present study, the incidence of plasmids was only 24% in clinical strains of L. monocytogenes that had been transferred many times in the laboratory. It may be that plasmid profiles would be of more use in the strain typing of fresh isolates than with strains that have been in the laboratory for some time. In addition to studying plasmid profiles for possible usefulness in strain typing, we also tested for the presence of two characters of known extrachromosomal inheritance, namely, toxin production and antibiotic resistance. All of the 122 L. monocytogenes strains tested showed production of the toxin P-hemolysin. When these L. monocytogenes strains, as well as the 10 strains of other Listeria species, were tested for antibiotic resistance, none of them showed resistance to streptomycin, ampicillin, erythromycin, tetracycline, or chloramphenicol. We conclude that the Phemolysin determinant is not inherited extrachromosomally and that none of the plasmids that we isolated carried resistance determinants for the antibiotics tested.
NOTES
TABLE2. Plasmids in L. monocytogenes Strain Strain source
Frequency"
Size ( M D ~ )
Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 11/11/14 For personal use only.
Clinical
Location
Serovar
Halifax, N.S. Halifax, N.S. Halifax, N.S. Ottawa, Ont. Winnipeg, Man. Quebec Halifax, N.S. Fredericton, N.B Quebec Ottawa, Ont. St. John's, Nfld. Quebec Quebec Quebec Montreal, Que. Toronto, Ont.
Food Cheese Cheese Alfalfa tablets Veterinary Bovine Ovine Environmental
NK NK Lachute, Que. St. Bernardin, Ont. Smith's Falls, Ont.
'Frequency = (no. o f plasmids)/(no. o f strains tested). b ~ u m b e rin parentheses is the number of plasmids of that size found. 'NK. not known.
Conjugative transfer of plasmids bearing antibiotic resistance markers can occur in the laboratory between L. monocytogenes and strains of Streptococcus or Enterococcus; transfer rates vary from to (Flamm et al. 1984; Vicente et al. 1988; Leclercq et al. 1989). The resistance determinants were stably inherited. The conjugative plasmids used in these experiments varied from 17 to 22.4 MDa in size. In spite of their ability to effect conjugative transfer, there have only been three reports in the literature, all from Europe, of antibiotic-resistant strains of L. monocytogenes having been isolated from clinical specimens. A plasmid of 40 MDa was found in a streptomycinresistant strain (Facinelli et al. 1989), and curing of the plasmid led to loss of resistance. A plasmid of 24 MDa carrying multiple antibiotic-resistant determinants was isolated by Poyart-Salmeron et al. (1990) from a strain of the organism resistant to chloramphenicol, erythromycin, streptomycin, and tetracycline. This plasmid showed extensive sequence homology to pAM@l,the broad-host-range resistance plasmid found in enterococci and streptococci. Quentin et al. (1990) reported the isolation of a strain of L. monocytogenes resistant t o chloramphenicol, erythromycin, tetracycline, and minocycline. Although the authors did not attempt plasmid isolation, they noted that all resistance markers were lost simultaneously when the strain was treated with curing agents such as acridine orange, suggesting a plasmid location for these determinants. In view of the transfer of plasmids that can occur between L. monocytogenes and Enterococcus and Streptococcus species, the suggestion has been made by Slade and Collins-
TABLE 3. Plasmids in Listeria spp. other than L. monocytogenes Species
Serovar
Source
Plasmid size (MDa)
- -
L. L. L. L. L. L. L.
innocua innocua innocua welshimeri seeligeri ivanovii ivanovii
Meat Meat Meat NK NK Raw cream Raw cream
'NK, not known.
Thompson (1990) that resistance factor transfer may occur in the human bowel. Thus, although no antibiotic-resistant strains of this pathogen have yet been reported in Canada or the United States, the possible emergence of such strains must be kept in mind. Anderson, D.G., and McKay, L.L. 1983. Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol. 46: 549-552. Bottone, E.J., Namdari, H., and Sellers, G. 1990. Listeria monocytogenes: investigation of an unexpected increased incidence of infection by plasmid profiling. Abstr. Annu. Meet. Am. Soc. Microbiol. C-358. p. 403. Facinelli, B., Casolari, C., Montanari, M.P., et al. 1989. Plasmid DNA isolation from Listeria monocytogenes. Abstr. Annu. Meet. Ann. Soc. Microbiol. H-219. p. 206.
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Fistrovici, E., and Collins-Thompson, D.L. 1990. Use of plasmid profiles and restriction endonuclease digest in environmental studies of Listeria spp. from raw milk. Int. J. Food Microbiol. 10: 43-50. Flamm, R.K., Hinrichs, D.J., and Thomashow, M.F. 1984. Introduction of pAMPl into Lkteria monocytogenes by conjugation and homology between native L. monocytogenes plasmids. Infect. Immun. 44: 157-161. Kolstad, J., Rorvik, L.M., and Granum, P.E. 1990. Characterization of plasmids from Listeria sp. Int. J. Food Microbiol. 12: 123-132. Leclercq, R., Derlot, E., Weber, M., et al. 1989. Transferable vancomycin and teicoplanin in Enterococcus faecium. Antimicrob. Agents Chemother. 33: 10-1 5. Macrina, F.L., Kopecko, D. J., Jones, K.R., et al. 1978. A multiple plasmid-containing Escherichia coli strain: convenient source of size reference plasmid molecules. Plasmid, 1: 417-420. Mottice, S., Robinson, W., and Reimer, L.G. 1987. Plasmid typing for the epidemiologic characterization of Lkteria monocytogenes. Abstr-. Annu. Meet. Am. Soc. Microbiol. C-206. p. 357. Perez-Diaz, J .C., Vicente, M.F., and Baquero, F. 1982. Plasmids in Listeria. Plasmid, 8: 112-1 18.
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Peterkin, P.I., Idziak, E.S., and Sharpe, A.N. 1991. Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe. Appl. Environ. Microbiol. 57: 586-591. Poyart-Salmeron, C., Carlier, C., Trieu-Cuot, P., et al. 1990. Transferable plasmid-mediated antibiotic resistance in Listeria monocytogenes. Lancet, i: 1422- 1426. Pritchard, T.J., and Donnelly, C.W. 1992. Plasmid profile analysis of Listeria species to compare dairy plant environmental isolates. J. Food Prot. 55. In press. Quentin, C., Thibaut, M.C., Horovitz, J., and Bebear, C. 1990. Multiresistant strain of Listeria monocytogenes in septic abortion. Lancet, ii: 375. Rocourt, J., Wehmeyer, U., and Stackebrandt, E. 1987. Transfer of Listeria denitrificans to Jonesia gen. nov. as Jonesia denitrificans comb. nov. Int. J. Syst. Bacteriol. 37: 266-270. Slade, P.J., and Collins-Thompson, D.L. 1990. Listeria, plasmids, antibiotic resistance, and food. Lancet, ii: 1004. Vicente, M.F., Baquero, F., and Perez-Diaz, J.C. 1988. Conjugative acquisition and expression of antibiotic resistance determinants in Listeria spp. J. Antimicrobiol. Chemother. 21: 309-3 18.
Erratum: Bacterial culture history affects the attachment of Bradyrhizobium japonicum to host Glycine mar roots1 GEOFFREY B. SMITH^
AND
A. G. WOLLUM I1
Department of Soil Science, North Carolina State University, Box 7619, Raleigh, NC 27695-7619, U.S.A. (Ref. Can. J. Microbiol. 37: 730-736. 1991.)
On page 730, in line 9 of the English abstract, the phrase "and on new cell growth" should be deleted. On page 730, in line 11 of the French abstract, the phrase "suivie d'une nouvelle croissance des cellules" should be deleted. On page 734, in Table 2, the second line in the column entitled Treatment should read 5 mM PNS. '~eceivedat NRC December 10, 1991. 2~resentaddress: Department of Biology (Dept. 3AF), Box 30001, New Mexico State University, Las Cruces, NM 88003.
