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Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage
International Journal of Food Microbiology 88 (2003) 325 – 329 www.elsevier.com/locate/ijfoodmicro
Mini-review
Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage Michael Teuber a,*, Franziska Schwarz a, Vincent Perreten b b
a Laboratory of Food Microbiology, ETH Zurich, Zurich CH-8092, Switzerland Institute for Veterinary Bacteriology, University of Berne, Bern CH-3012, Switzerland
Accepted 26 February 2003
Abstract Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies) , and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora. D 2003 Elsevier B.V. All rights reserved. Keywords: Antibiotic resistance; Enterococcus; Food; Multiresistance; Plasmid; Conjugation
1. Introduction The molecular characterisation of antibiotic resistance plasmids and transposons from food borne * Corresponding author. Tel.: +41-1-261-3417; fax: +41-1-2613441. E-mail address: [email protected] (M. Teuber). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1605(03)00195-8
entrococci, lactobacilli, staphylococci, and lactococci in our laboratory has provided evidence that such bacteria use the same resistance and transfer tools reported for bacterial isolates from human and animal pathogenic material (Teuber, 2001). Examples are pRE39, a pAMbeta-like, conjugative plasmid out of a meat isolate of Enterococcus sp. (Teuber et al., 1996), the Enterococcus faecalis transposon TnFO1,
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a Tn916-relative detected in a raw milk mountain cheese (Perreten et al., 1997a), the multiresistance plasmid pK214 found in Lactococcus lactis from a raw milk soft cheese (Perreten et al., 1997b), and several small resistance plasmids in coagulase-negative staphylococci out of raw fermented sausages and cheeses (Perreten et al., 1998). A strain of E. faecalis (RE25) isolated from a raw-fermented sausage was resistant to kanamycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, nourseothricin, and tetracycline. It contained a 50-kbp multiresistance plasmid which conjugated into E. faecalis JH2-2, L. lactis Bu2, and Listeria innocua
L19 (Schwarz et al., 2001). All described resistances were transferred with the exception of tetracycline.
2. Molecular structure of pRE25 The structure of pRE 25 was obtained by complete sequencing of the 50,237-bp DNA (GenBank/EMBL accession number X92945). The resulting gene organisation is shown in Fig. 1. Comparison with nucleotide sequences available in public databases yielded the similarities compiled in Table 1. It is immediately evident that a large part (module) of pRE25 is almost identical in amino acid sequences and gene organisa-
Fig. 1. Genomic structure of the 50 237 bp conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw meat sausage (GenBank accession number X92945). The basic structure comprising orf’s 6 to 40 is very similar to identical to the Inc18 family broad host range plasmid pIP501 from Streptococcus agalactiae and pSM19035 from Streptococcus pyogenes. The transfer region ranges from orf 24 (nickase) to orf 40 containing 10 genes for membrane spanning proteins. Resistance genes orf 10 and 14 code for a chloramphenicol acetyltransferase and a RNA-methylase, respectively, like in pIP501. This basic streptococcal plasmid has been obviously upgraded with the help of E. faecium IS elements (orf 41, 51, 54, 55, 56, 3, and 5) to contain another antibiotic resistance gene-assembly (orf 44, 45, 46) coding for an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglygoside phosphotransferase type III. In addition, pRE25 contains information for 3 replication proteins (orf 1, 6, and 11), two resolvases (orf 8, 53) and 2 ATPases (orf 16 and 48 with 98 to 100% amino acid identities with Clostridium perfringens/difficile proteins). The plasmid can be experimentally transferred by conjugation to Lactococcus lactis, Listeria innocua, and other enterococci. It is a demonstration that resistance genes are freely floating between commensal and pathogenic bacteria found in humans, animals and food (modified from F. Schwarz et al. 2001). Color code: dark red: aa identities (95 – 1000%) with Inc18 plasmid coded proteins (streptococcal module). Blue: enterococcal region (module). White: no homologies in data bases. Black: IS-elements. Blue bar: nucleotide sequences with a 98 – 100% identity to sequences in a vancomycin-resistant E. faecium whose genome has been completely sequenced in the USA (GenBank accession number NZ_AAAK01000000).
M. Teuber et al. / International Journal of Food Microbiology 88 (2003) 325–329 Table 1 Homologies of open reading frames in plasmid pRE25 with open reading frames available in public databases orf Identification
aa Species identity (%)
Genetic element pK214
5
IS1216-V
100
8 10 13 14 15 17 24 25 26 27 28 29 30 31 32 33 34 38 40 41 42 46 47 48 51 6 55 45
resolvase cat MLS-leader protein ermB ORF6 ORF omega nickase hypoth. trsB hypoth. trsC hypoth. trsD hypoth. trsE hypoth. trsF hypoth. protein hypoth. protein hypoth. protein hypoth. protein hypoth. trsK ORF theta copR IS1216-V ORFX APH(3V)-III hypoth. protein ATPase IS1216-V repS IS1485 SAT4
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99.8 99.5 99.4
9 11 16 37 18 12 49 39 21 54 7 50 57 58 3 20
NH2-topoisomerase I hypoth. protein ATPase ORF eta hypoth. protein topoisomeraseCOOH I hypoth. protein ORF iota hypoth. protein transposase ORF alpha ORF zeta hypoth. prot. PrgO hypoth. prot. PrgP IS1062 hypoth. protein
96.7 96.3 95.7 94.9 94.6 81.4 81.3 79.2 76.5 61.9
44
aad
58.2
98.7 98.4 98 98 97.8 97
Lactococcus lactis S. pyogenes S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. pyogenes S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. pyogenes S. agalactiae L. lactis S. aureus S. aureus S. aureus C. difficile L. lactis S. pyogenes E. faecium Campylobacter coli S. pyogenes S. agalactiae C. perfringens S. pyogenes S. agalactiae S. pyogenes S. agalactiae S. agalactiae E. faecium E. faecium S. pyogenes S. pyogenes E. faecalis E. faecalis E. faecalis Lactococcus lactis B. subtilis
pSM19035 pIP501 pIP501 pIP501 pIP501 pSM19035 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pSM19035 pIP501 pK214 Tn5405 Tn5405 Tn5405 Genome pK214 pSM19035 pEME19
pSM19035 pIP501 genome pSM19035 pIP501 pSM19035 pIP501 pIP501 pEF1 pSM19035 pSM19035 pCF10 pCF10 pPD1
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Table 1 (continued) orf Identification
52 22 1 2 53 30 33 36 4 23 43
cell filamentation protein hypoth. protein repR hypothet. prot. PrgN resolvase ORF14 trsK trsL hypoth. protein hypoth. protein hypoth. protein
aa Species identity (%) 57.5 52.2 50.6 49.5 49.7 43.1 39.7 23 0 0 0
Neisseria meningitidis L. lactis S. agalactiae E. faecalis L. lactis E. faecalis L. lactis L. lactis
Genetic element
pIP501 pCF10 pK214 Tn916 pMRC01 pMRC01
The homologies are listed in descending amino acid similarities. It is obvious that many of the detected open reading frames and the coded proteins have been described on conjugative, plasmids and transposons. Most genes seem to originate from Gram-positive bacteria especially Streptococcus pyogenes, S. agalactiae, Clostridium difficile/perfringens, Lactococcus, and enterococci (modified from Schwarz et al., 2001).
tion with the streptococcal plasmids pSM19035 (S. pyogenes) and pIP501 (S. agalactiae). Unfortunately, the complete 30.5-kbp sequence of pIP501 has not been reported. We propose that the pRE25-segment consisting of open reading frames 6– 40 (about 30 kbp) represents a direct derivative of pIP501. The evidence in favour of this hypothesis is obvious: the structure and sequence of the resistance part containing cat and ermB is identical. Specifically, the insertion of the cat region into a topoisomerase I-sequence is convincing. In addition, the region responsible for conjugative transfer is identical as far as the sequence of pIP501 is known (Wang and Macrina, 1995). It also includes the recently reported pIP501 sequences corresponding to the pRE25 orf’s 30 to 34 (Kurenbach et al., 2002). The tra-operon of pIP501 (orf 1 to orf 11; orf 24 to orf 34 in pRE25) was shown to be transcribed as a single messenger RNA. The remaining section of pRE25 (orf 41 to orf 58, and orf 1 to orf 5) seems to be a Enterococcus-derived module. It has been assembled with the aid of enterococcal IS-elements: two copies of IS1216V (orf 5 and orf 41) border the pIP501-derived regions. Another IS1216V-element (orf 49) may have been responsible for the incorporation of the second antibiotic resist-
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ance region (orf 44 to orf 46). It consists of genes for an aminoglycoside 6-adenyltransferase (aadK, orf 44), a streptothricin acetyltransferase (sat 4, orf 45), and a type III aminoglycoside phosphotransferase (aph(3‘)-III, orf 46). Identical packages (aadE-sat4aphA-3) have been previously observed in S. aureus and E. faecalis (Derbise et al., 1996; Trieu-Cuot and Courvalin, 1983). Recently, it was also detected in E. faecium (disseminated!) and S. intermedius (Boerlin et al., 2001; Werner et al., 2001). Two more complete IS-elements (IS1485, orf 55, and IS1062, orf 3) are present in the enterococcal part. IS1485 is inserted into still another IS-element (transposase of E. faecium 6T1a). The significance of these IS-elements in the evolution of pRE25 is not clear. The replication region of the enterococcal part is represented by orf 1. A streptococcal origin of replication identical to pIP501 is present between orf 6 and orf 7 (see Fig. 1). It is not known which replication system is at work. The origin of transfer (oriT) is in its proper place, in front of orf 24 (nickase). The function of the two genes for ATPases (orf 16 and orf 48) with 98 – 100% identities to Clostridium perfringens/difficile genes and enzymes is unknown. It demonstrates, however, the close genetic ties probably via horizontal transfer between Gram-positive bacteria of human and animal intestinal habitats.
3. Discussion Screening of E. faecalis RE25 for known virulence factors (Eaton and Gasson, 2001) was negative for gelatinase, haemolysis, cytolysin, and aggregation proteins (phenotypic and genotypic). However, the enterococcal surface protein (esp) was clearly demonstrated by PCR using specific probes (Franziska Rossi et al., manuscript in preparation). An interesting and important observation is the fact that large stretches of the enterococcal module show 98– 100% identities on the nucleotide level with sequences of the E. faecium complete genome sequencing project in the United States (GenBank accession number NZ_AAAK01000000; see blue inset bars in Fig. 1). After publication of the pRE25 sequence in the GenBank/EMBL database, we were informed by colleagues in France (P. Courvalin, personal communication), Denmark (P. Jensen, per-
sonal communication), and Switzerland (H. Ha¨chler, personal communication) that they discovered identical sequences in plasmids from enterococci and listerias. The broad host range tools of Gram-positive bacteria and particularly enterococci seem to be used and present all over the world. The presence of enterococci in food which carry such mobile genetic elements including resistance and possibly virulence genes gives the enterococci a new dimension regarding their potential pathogenicity for immunocompromised persons. Since it has been demonstrated that food borne vancomycin-resistant enterococci reach the intestine and colonise transiently (Sorensen et al., 2001), it is just a matter of time when transfer of resistance genes from ingested enterococci to the intestinal microflora of a consumer will be experimentally demonstrated. As a consequence, the presence of enterococci in fermented food (where they may be numerous, Teuber et al., 1996, 1999) and their use as starter cultures or probiotics needs a careful reevaluation. In the sake of a good food microbiological practice, it is proposed not to feed the consumer with high levels of resistant and virulence factor-loaded enterococci because food borne enterococci with mobile genetic elements like conjugative plasmids and transposons contribute to the dissemination of antibiotic resistance genes in the human population. Since it is known that animal enterococci enter fermented food from the raw material (meat or milk) and do multiply during food processing, such food items (raw milk cheeses and raw meat sausages, and the raw milk and meat itself) are a direct link between the animal and human microflora (Teuber et al., 1999). If food technologies developed before the age of antibiotic resistance are used, resistant bacteria find their way into the traditional products. It seems necessary to discuss this new dimension in food microbiology and at the same time to develop new technologies to avoid this new threat. References Boerlin, P., Burnens, A.P., Frey, J., Kuhnert, P., Nicolet, J., 2001. Molecular epidemiology and genetic linkage of macrolide and aminoglycoside resistance in Staphylococcus intermedius of canine origin. Veterinary Microbiology 79, 155 – 169. Derbise, S., Dyke, K.G.H., El Solh, N., 1996. Characterization of a
M. Teuber et al. / International Journal of Food Microbiology 88 (2003) 325–329 Staphylococcus aureus transposon, Tn5405, located within Tn5404 and carrying the aminoglycoside resistance genes, aphA-3 and aadE. Plasmid 35, 174 – 188. Eaton, T.J., Gasson, M.J., 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Applied Environmental Microbiology 67, 1628 – 1635. Kurenbach, B., Grothe, D., Farias, M.E., Szewzyk, U., Grohmann, E., 2002. The tra-region of the conjugative plasmid pIP501 is organized in an operon with the first gene encoding the relaxase. Journal Bacteriology 184, 1801 – 1805. Perreten, V., Kollo¨ffel, B., Teuber, M., 1997a. Conjugal transfer of the Tn916-like transposon TnFO1 from Enterococcus faecalis isolated from cheese to other Gram-positive bacteria. Systematic Applied Microbiology 20, 27 – 38. Perreten, V., Schwarz, F., Cresta, L., Boeglin, M., Dasen, G., Teuber, M., 1997b. Antibiotic resistance spread in food. Nature 389, 801 – 802. Perreten, V., Giampa, N., Schuler-Schmid, U., Teuber, M., 1998. Antibiotic resistance genes in coagulase-negative staphylococci from food. Systematic Applied Microbiology 21, 113 – 120. Schwarz, F.V., Perreten, V., Teuber, M., 2001. Sequence of the 50kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25. Plasmid 46, 170 – 187.
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Sorensen, T.L., Blom, N., Monnet, D.L., Frimodt-Moller, N., Poulsen, R.L., Espersen, F., 2001. Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork. New England Journal of Medicine 345, 1161 – 1166. Teuber, M., 2001. Veterinary use and antibiotic resistance. Current Opinion in Microbiology 4, 493 – 499. Teuber, M., Perreten, V., Wirsching, F., 1996. Antibiotikumresistente Bakterien: eine neue Dimension in der Lebensmittelmikrobiologie. Lebensmittel-Technologie 29, 182 – 199 (URL http:// e-collection.ethbib.ethz.ch/ecol-pool/bericht/bericht_50.pdf). Teuber, M., Meile, L., Schwarz, F., 1999. Antibiotic resistance of lactic acid bacteria from food. Antonie van Leeuwenhoek 76, 115 – 137. Trieu-Cuot, P., Courvalin, P., 1983. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3V5V-aminoglycoside phosphotransferase type III. Gene 23, 331 – 341. Wang, A., Macrina, F.L., 1995. Streptococcal plasmid pIP501 has a functional oriT site. Journal Bacteriology 177, 4199 – 4206. Werner, G., Hildebrandt, B., Witte, W., 2001. Aminoglycosidestreptothricin resistance gene cluster aadE-sat4-aphA-3 disseminated among multiresistant isolates of Enterococcus faecium. Antimicrobial Agents Chemotherapy 45, 3267 – 3269.
Mini-review
Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage Michael Teuber a,*, Franziska Schwarz a, Vincent Perreten b b
a Laboratory of Food Microbiology, ETH Zurich, Zurich CH-8092, Switzerland Institute for Veterinary Bacteriology, University of Berne, Bern CH-3012, Switzerland
Accepted 26 February 2003
Abstract Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies) , and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora. D 2003 Elsevier B.V. All rights reserved. Keywords: Antibiotic resistance; Enterococcus; Food; Multiresistance; Plasmid; Conjugation
1. Introduction The molecular characterisation of antibiotic resistance plasmids and transposons from food borne * Corresponding author. Tel.: +41-1-261-3417; fax: +41-1-2613441. E-mail address: [email protected] (M. Teuber). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-1605(03)00195-8
entrococci, lactobacilli, staphylococci, and lactococci in our laboratory has provided evidence that such bacteria use the same resistance and transfer tools reported for bacterial isolates from human and animal pathogenic material (Teuber, 2001). Examples are pRE39, a pAMbeta-like, conjugative plasmid out of a meat isolate of Enterococcus sp. (Teuber et al., 1996), the Enterococcus faecalis transposon TnFO1,
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a Tn916-relative detected in a raw milk mountain cheese (Perreten et al., 1997a), the multiresistance plasmid pK214 found in Lactococcus lactis from a raw milk soft cheese (Perreten et al., 1997b), and several small resistance plasmids in coagulase-negative staphylococci out of raw fermented sausages and cheeses (Perreten et al., 1998). A strain of E. faecalis (RE25) isolated from a raw-fermented sausage was resistant to kanamycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, nourseothricin, and tetracycline. It contained a 50-kbp multiresistance plasmid which conjugated into E. faecalis JH2-2, L. lactis Bu2, and Listeria innocua
L19 (Schwarz et al., 2001). All described resistances were transferred with the exception of tetracycline.
2. Molecular structure of pRE25 The structure of pRE 25 was obtained by complete sequencing of the 50,237-bp DNA (GenBank/EMBL accession number X92945). The resulting gene organisation is shown in Fig. 1. Comparison with nucleotide sequences available in public databases yielded the similarities compiled in Table 1. It is immediately evident that a large part (module) of pRE25 is almost identical in amino acid sequences and gene organisa-
Fig. 1. Genomic structure of the 50 237 bp conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw meat sausage (GenBank accession number X92945). The basic structure comprising orf’s 6 to 40 is very similar to identical to the Inc18 family broad host range plasmid pIP501 from Streptococcus agalactiae and pSM19035 from Streptococcus pyogenes. The transfer region ranges from orf 24 (nickase) to orf 40 containing 10 genes for membrane spanning proteins. Resistance genes orf 10 and 14 code for a chloramphenicol acetyltransferase and a RNA-methylase, respectively, like in pIP501. This basic streptococcal plasmid has been obviously upgraded with the help of E. faecium IS elements (orf 41, 51, 54, 55, 56, 3, and 5) to contain another antibiotic resistance gene-assembly (orf 44, 45, 46) coding for an aminoglycoside 6-adenylyltransferase, a streptothricin acetyltransferase, and an aminoglygoside phosphotransferase type III. In addition, pRE25 contains information for 3 replication proteins (orf 1, 6, and 11), two resolvases (orf 8, 53) and 2 ATPases (orf 16 and 48 with 98 to 100% amino acid identities with Clostridium perfringens/difficile proteins). The plasmid can be experimentally transferred by conjugation to Lactococcus lactis, Listeria innocua, and other enterococci. It is a demonstration that resistance genes are freely floating between commensal and pathogenic bacteria found in humans, animals and food (modified from F. Schwarz et al. 2001). Color code: dark red: aa identities (95 – 1000%) with Inc18 plasmid coded proteins (streptococcal module). Blue: enterococcal region (module). White: no homologies in data bases. Black: IS-elements. Blue bar: nucleotide sequences with a 98 – 100% identity to sequences in a vancomycin-resistant E. faecium whose genome has been completely sequenced in the USA (GenBank accession number NZ_AAAK01000000).
M. Teuber et al. / International Journal of Food Microbiology 88 (2003) 325–329 Table 1 Homologies of open reading frames in plasmid pRE25 with open reading frames available in public databases orf Identification
aa Species identity (%)
Genetic element pK214
5
IS1216-V
100
8 10 13 14 15 17 24 25 26 27 28 29 30 31 32 33 34 38 40 41 42 46 47 48 51 6 55 45
resolvase cat MLS-leader protein ermB ORF6 ORF omega nickase hypoth. trsB hypoth. trsC hypoth. trsD hypoth. trsE hypoth. trsF hypoth. protein hypoth. protein hypoth. protein hypoth. protein hypoth. trsK ORF theta copR IS1216-V ORFX APH(3V)-III hypoth. protein ATPase IS1216-V repS IS1485 SAT4
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 99.8 99.5 99.4
9 11 16 37 18 12 49 39 21 54 7 50 57 58 3 20
NH2-topoisomerase I hypoth. protein ATPase ORF eta hypoth. protein topoisomeraseCOOH I hypoth. protein ORF iota hypoth. protein transposase ORF alpha ORF zeta hypoth. prot. PrgO hypoth. prot. PrgP IS1062 hypoth. protein
96.7 96.3 95.7 94.9 94.6 81.4 81.3 79.2 76.5 61.9
44
aad
58.2
98.7 98.4 98 98 97.8 97
Lactococcus lactis S. pyogenes S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. pyogenes S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. agalactiae S. pyogenes S. agalactiae L. lactis S. aureus S. aureus S. aureus C. difficile L. lactis S. pyogenes E. faecium Campylobacter coli S. pyogenes S. agalactiae C. perfringens S. pyogenes S. agalactiae S. pyogenes S. agalactiae S. agalactiae E. faecium E. faecium S. pyogenes S. pyogenes E. faecalis E. faecalis E. faecalis Lactococcus lactis B. subtilis
pSM19035 pIP501 pIP501 pIP501 pIP501 pSM19035 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pIP501 pSM19035 pIP501 pK214 Tn5405 Tn5405 Tn5405 Genome pK214 pSM19035 pEME19
pSM19035 pIP501 genome pSM19035 pIP501 pSM19035 pIP501 pIP501 pEF1 pSM19035 pSM19035 pCF10 pCF10 pPD1
327
Table 1 (continued) orf Identification
52 22 1 2 53 30 33 36 4 23 43
cell filamentation protein hypoth. protein repR hypothet. prot. PrgN resolvase ORF14 trsK trsL hypoth. protein hypoth. protein hypoth. protein
aa Species identity (%) 57.5 52.2 50.6 49.5 49.7 43.1 39.7 23 0 0 0
Neisseria meningitidis L. lactis S. agalactiae E. faecalis L. lactis E. faecalis L. lactis L. lactis
Genetic element
pIP501 pCF10 pK214 Tn916 pMRC01 pMRC01
The homologies are listed in descending amino acid similarities. It is obvious that many of the detected open reading frames and the coded proteins have been described on conjugative, plasmids and transposons. Most genes seem to originate from Gram-positive bacteria especially Streptococcus pyogenes, S. agalactiae, Clostridium difficile/perfringens, Lactococcus, and enterococci (modified from Schwarz et al., 2001).
tion with the streptococcal plasmids pSM19035 (S. pyogenes) and pIP501 (S. agalactiae). Unfortunately, the complete 30.5-kbp sequence of pIP501 has not been reported. We propose that the pRE25-segment consisting of open reading frames 6– 40 (about 30 kbp) represents a direct derivative of pIP501. The evidence in favour of this hypothesis is obvious: the structure and sequence of the resistance part containing cat and ermB is identical. Specifically, the insertion of the cat region into a topoisomerase I-sequence is convincing. In addition, the region responsible for conjugative transfer is identical as far as the sequence of pIP501 is known (Wang and Macrina, 1995). It also includes the recently reported pIP501 sequences corresponding to the pRE25 orf’s 30 to 34 (Kurenbach et al., 2002). The tra-operon of pIP501 (orf 1 to orf 11; orf 24 to orf 34 in pRE25) was shown to be transcribed as a single messenger RNA. The remaining section of pRE25 (orf 41 to orf 58, and orf 1 to orf 5) seems to be a Enterococcus-derived module. It has been assembled with the aid of enterococcal IS-elements: two copies of IS1216V (orf 5 and orf 41) border the pIP501-derived regions. Another IS1216V-element (orf 49) may have been responsible for the incorporation of the second antibiotic resist-
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ance region (orf 44 to orf 46). It consists of genes for an aminoglycoside 6-adenyltransferase (aadK, orf 44), a streptothricin acetyltransferase (sat 4, orf 45), and a type III aminoglycoside phosphotransferase (aph(3‘)-III, orf 46). Identical packages (aadE-sat4aphA-3) have been previously observed in S. aureus and E. faecalis (Derbise et al., 1996; Trieu-Cuot and Courvalin, 1983). Recently, it was also detected in E. faecium (disseminated!) and S. intermedius (Boerlin et al., 2001; Werner et al., 2001). Two more complete IS-elements (IS1485, orf 55, and IS1062, orf 3) are present in the enterococcal part. IS1485 is inserted into still another IS-element (transposase of E. faecium 6T1a). The significance of these IS-elements in the evolution of pRE25 is not clear. The replication region of the enterococcal part is represented by orf 1. A streptococcal origin of replication identical to pIP501 is present between orf 6 and orf 7 (see Fig. 1). It is not known which replication system is at work. The origin of transfer (oriT) is in its proper place, in front of orf 24 (nickase). The function of the two genes for ATPases (orf 16 and orf 48) with 98 – 100% identities to Clostridium perfringens/difficile genes and enzymes is unknown. It demonstrates, however, the close genetic ties probably via horizontal transfer between Gram-positive bacteria of human and animal intestinal habitats.
3. Discussion Screening of E. faecalis RE25 for known virulence factors (Eaton and Gasson, 2001) was negative for gelatinase, haemolysis, cytolysin, and aggregation proteins (phenotypic and genotypic). However, the enterococcal surface protein (esp) was clearly demonstrated by PCR using specific probes (Franziska Rossi et al., manuscript in preparation). An interesting and important observation is the fact that large stretches of the enterococcal module show 98– 100% identities on the nucleotide level with sequences of the E. faecium complete genome sequencing project in the United States (GenBank accession number NZ_AAAK01000000; see blue inset bars in Fig. 1). After publication of the pRE25 sequence in the GenBank/EMBL database, we were informed by colleagues in France (P. Courvalin, personal communication), Denmark (P. Jensen, per-
sonal communication), and Switzerland (H. Ha¨chler, personal communication) that they discovered identical sequences in plasmids from enterococci and listerias. The broad host range tools of Gram-positive bacteria and particularly enterococci seem to be used and present all over the world. The presence of enterococci in food which carry such mobile genetic elements including resistance and possibly virulence genes gives the enterococci a new dimension regarding their potential pathogenicity for immunocompromised persons. Since it has been demonstrated that food borne vancomycin-resistant enterococci reach the intestine and colonise transiently (Sorensen et al., 2001), it is just a matter of time when transfer of resistance genes from ingested enterococci to the intestinal microflora of a consumer will be experimentally demonstrated. As a consequence, the presence of enterococci in fermented food (where they may be numerous, Teuber et al., 1996, 1999) and their use as starter cultures or probiotics needs a careful reevaluation. In the sake of a good food microbiological practice, it is proposed not to feed the consumer with high levels of resistant and virulence factor-loaded enterococci because food borne enterococci with mobile genetic elements like conjugative plasmids and transposons contribute to the dissemination of antibiotic resistance genes in the human population. Since it is known that animal enterococci enter fermented food from the raw material (meat or milk) and do multiply during food processing, such food items (raw milk cheeses and raw meat sausages, and the raw milk and meat itself) are a direct link between the animal and human microflora (Teuber et al., 1999). If food technologies developed before the age of antibiotic resistance are used, resistant bacteria find their way into the traditional products. It seems necessary to discuss this new dimension in food microbiology and at the same time to develop new technologies to avoid this new threat. References Boerlin, P., Burnens, A.P., Frey, J., Kuhnert, P., Nicolet, J., 2001. Molecular epidemiology and genetic linkage of macrolide and aminoglycoside resistance in Staphylococcus intermedius of canine origin. Veterinary Microbiology 79, 155 – 169. Derbise, S., Dyke, K.G.H., El Solh, N., 1996. Characterization of a
M. Teuber et al. / International Journal of Food Microbiology 88 (2003) 325–329 Staphylococcus aureus transposon, Tn5405, located within Tn5404 and carrying the aminoglycoside resistance genes, aphA-3 and aadE. Plasmid 35, 174 – 188. Eaton, T.J., Gasson, M.J., 2001. Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Applied Environmental Microbiology 67, 1628 – 1635. Kurenbach, B., Grothe, D., Farias, M.E., Szewzyk, U., Grohmann, E., 2002. The tra-region of the conjugative plasmid pIP501 is organized in an operon with the first gene encoding the relaxase. Journal Bacteriology 184, 1801 – 1805. Perreten, V., Kollo¨ffel, B., Teuber, M., 1997a. Conjugal transfer of the Tn916-like transposon TnFO1 from Enterococcus faecalis isolated from cheese to other Gram-positive bacteria. Systematic Applied Microbiology 20, 27 – 38. Perreten, V., Schwarz, F., Cresta, L., Boeglin, M., Dasen, G., Teuber, M., 1997b. Antibiotic resistance spread in food. Nature 389, 801 – 802. Perreten, V., Giampa, N., Schuler-Schmid, U., Teuber, M., 1998. Antibiotic resistance genes in coagulase-negative staphylococci from food. Systematic Applied Microbiology 21, 113 – 120. Schwarz, F.V., Perreten, V., Teuber, M., 2001. Sequence of the 50kb conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25. Plasmid 46, 170 – 187.
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Sorensen, T.L., Blom, N., Monnet, D.L., Frimodt-Moller, N., Poulsen, R.L., Espersen, F., 2001. Transient intestinal carriage after ingestion of antibiotic-resistant Enterococcus faecium from chicken and pork. New England Journal of Medicine 345, 1161 – 1166. Teuber, M., 2001. Veterinary use and antibiotic resistance. Current Opinion in Microbiology 4, 493 – 499. Teuber, M., Perreten, V., Wirsching, F., 1996. Antibiotikumresistente Bakterien: eine neue Dimension in der Lebensmittelmikrobiologie. Lebensmittel-Technologie 29, 182 – 199 (URL http:// e-collection.ethbib.ethz.ch/ecol-pool/bericht/bericht_50.pdf). Teuber, M., Meile, L., Schwarz, F., 1999. Antibiotic resistance of lactic acid bacteria from food. Antonie van Leeuwenhoek 76, 115 – 137. Trieu-Cuot, P., Courvalin, P., 1983. Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3V5V-aminoglycoside phosphotransferase type III. Gene 23, 331 – 341. Wang, A., Macrina, F.L., 1995. Streptococcal plasmid pIP501 has a functional oriT site. Journal Bacteriology 177, 4199 – 4206. Werner, G., Hildebrandt, B., Witte, W., 2001. Aminoglycosidestreptothricin resistance gene cluster aadE-sat4-aphA-3 disseminated among multiresistant isolates of Enterococcus faecium. Antimicrobial Agents Chemotherapy 45, 3267 – 3269.