Occurrence of the Enterotoxin Gene Cluster and the Toxic Shock Syndrome Toxin 1 Gene among Clinical Isolates of Methicillin-Resistant Staphy
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微生物临床杂志 2006年第5期
Department of Microbiology, School of Medicine, University of Patras, Patras, Greece
ABSTRACT
Analysis of 177 methicillin-resistant Staphylococcus aureus (MRSA) strains for the presence of egc and tst revealed that 60 strains carried at least one of the tested genes. MRSA strains were classified by pulsed-field gel electrophoresis into four clones belonging to agr groups I and III. Toxin genotypes were related by clonal type and agr group.
TEXT
The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) has posed a clinical threat worldwide. Staphylococcal enterotoxins, enterotoxin-like superantigens, and the toxic shock syndrome toxin 1 that belong to pyrogenic toxin superantigens (PTSAgs) are considered major virulence factors (5, 11). The enterotoxin gene cluster (egc) includes five enterotoxin-like superantigen genes (seo, sem, sei, sen, and seg) and two pseudogenes (ent1 and ent2) (6, 8, 11). Recently, a new toxin, SEU, encoded by the egc operon (egc2), was identified to result from sequence divergence in the region of the pseudogenes ent1/ent2 (9).
Expression of virulent genes in S. aureus is controlled by the accessory gene regulator (agr), with a polymorphism in the sequence of the autoinducing peptide and its receptor, according to which clinical strains can be divided into four agr groups (I to IV) (7, 10). S. aureus strains causing specific syndromes were linked to certain agr types (7). Thus, the purpose of the present study was to investigate the presence of the egc operon and tst in a set of 177 MRSA isolates collected from different patients admitted to the University Hospital of Patras, Greece, during 2001 to 2003, to determine the clones of isolates, and to correlate them with the agr groups and toxin gene profile.
(These results were partially presented as a poster at the 15th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 2 to 5 April 2005.)
All 177 MRSA isolates were tested by the disk diffusion method against various antistaphylococcal agents (12). Oxacillin MICs were determined by the agar dilution method (13), and the presence of the mecA gene was determined by PCR (14).
Clones were defined by pulsed-field gel electrophoresis of SmaI digests of chromosomal DNAs performed in a CHEFF DR III apparatus (Bio-Rad Laboratories, Richmond, California) according to published protocols (4, 15) and were compared to previously identified clones existing in our hospital (1).
agr typing was carried out by PCR as described previously (10) with modified thermal conditions as follows: predenaturation step for 5 min at 94°C, 25 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, extension for 1 min at 72°C, and a final extension step for 7 min at 72°C (J. Etienne and M. Bes, personal communication). Fri913 (agr class I), Fri137 (agr class II), ATCC 49775 (agr class III), and A920211 (agr class IV) were used as reference strains.
The sequences corresponding to seo, sem, sei, ent1/ent2 (ent), sen, and seg included in the egc1 operon and tst were detected by PCRs (6, 7). Fri137 and Fri913, carrying the egc2 operon and tst, respectively, were used as reference strains.
The presence of seu characterizing the egc2 operon (seo, sem, sei, seu, sen, and seg) was investigated by HindIII restriction fragment length polymorphism of the amplified ent region. The ent region of the seu-negative strain A900322 harboring egc1 (9) carries a unique restriction site for the HindIII enzyme at nucleotide 456, resulting in two restriction fragments of 456 and 181 bp. This region does not exist in the corresponding amplified part of the seu-positive strain Fri137 (GenBank accession no. AY205306) carrying egc2 (9). A restriction reaction in a final volume of 20 μl, containing 15 units HindIII (Promega, Madison, WI), 1x reaction buffer (Promega), and 7 μl of PCR product, was performed at 37°C with overnight incubation. Lamda () DNA was used as the restriction enzyme marker.
The 177 MRSA isolates with distinct antibiotic resistance phenotypes were classified into four clones, as previously described (1). All multiresistant isolates were characterized as clone B (48 strains or 27%). Most isolates (103 of 177 or 58%) belonged to clone C, expressing resistance to beta-lactams and fusidic acid (3). Three strains (2%) resistant to beta-lactams, erythromycin, and ciprofloxacin were characterized as clone F. Clone A included 23 isolates (13%) resistant only to beta-lactams.
Fifty-two isolates (29%) of clones B and F and a unique strain of clone C that did not possess the Panton-Valentine leukocidin (PVL) genes (3) belonged to agr class I. One hundred twenty-five (125 or 71%) isolates of clones A and C, with the exception of the aforementioned PVL-negative strain, belonged to agr class III (Table 1).
Among these 177 strains, 117 (66%) did not carry any PTSAg tested. Sixty MRSA isolates positive for at least one of the tested genes were classified into 11 genotypes (Table 1). The tst gene was always detected in combination with egc genes.
Twenty-three egc-positive isolates and six clinical isolates carrying the ent region (ent1/ent2-positive) were seu positive (egc2). The remaining four egc-positive isolates did not possess the seu gene (egc1) (Fig. 1). The Fri137 reference strain carried the seu gene, as expected (9).
The genotypes tst/egc2 and egc2 belonging to clone A were classified into agr group III. Four strains possessing egc1, classified to clones F (3 strains) and C (1 PVL-negative strain), belonged to agr group I (Table 1).
Figure 2 shows the distribution of specific genotypes for each year. Isolates harboring all tested genes reduced with time, whereas those which carried no genes remained high.
One-third (60/177) of the MRSA isolates tested carried a number of genes, ranging from one to seven, with an extensive variation between individual strains and a reducing tendency during the study period. Coexistence of tst with enterotoxin genes has been reported but not exclusively for all tested isolates (7), as shown in the present study. The prevalence of egc2 in our study is much higher than first reported (9). Discrepancies in the frequency of PTSAg genes from different countries are expected, since they are associated with mobile genetic elements and strains may gain a different number of genes.
The variable possession of egc genes suggests a more heterogeneous composition of this operon, leading to the emergence of the newly described seu gene (9) or the allelic variants of seg, sei, sen, and seu (2, 6). Identification of variable toxin gene profiles may be explained by horizontal transfer, since open reading frames for putative transposases are located nearby, although it is limited among lineages (7, 8).
Identification of agr groups classified the majority of our strains into group III and to a lesser extend into agr group I. In previous studies, most of the MRSA isolates were agr group I or II (7). Investigation of the relationship between agr groups, clones, and toxin gene distribution reveals that the presence of tested genes is clone related. Classification of tst-positive isolates in agr group III has been already reported among methicillin-susceptible S. aureus isolates (7).
The basic unit of bacterial pathogenicity may reside on the clone or lineage, since it expands, possessing particular combinations of virulence and regulatory genes in the appropriate genetic background (7). The sensitive clone A possessing all tested genes does not seem to be a threat to our patients, since it decreases with time.
In conclusion, one-third of 177 MRSA clinical isolates carried genes of the PTSAg family, with a reducing tendency during the study period. tst always coexisted in the same isolate with other genes of the egc operon. Clone A, which harbors all or the majority of tested toxin genes (tst/egc2 or egc2), expresses a sensitive phenotype; however, it could not correlate with a specific clinical syndrome. This is the first report of a representative collection of MRSA in Greece. A clonal relationship with agr group classification and toxin gene profile was identified, and it remains to be seen whether a specific lineage spread may occur. Identification of strains with variable toxin gene profiles belonging to different clones indicates that egc genes are not strictly linked and a multitude of combinations among themselves and other PTSAgs (tst) may occur in bacterial populations.
ACKNOWLEDGMENTS
This work was supported by "K. Karatheodori" grant no. B116 of the University of Patras awarded to I.S.
We thank Jerome Etienne from the Centre National de Reference des Staphylocoques, Lyon, France, for kindly providing the reference S. aureus strains and E. D. Anastassiou and E. Petinaki for editing the manuscript.
REFERENCES
Aires de Sousa, M., C. Bartzavali, I. Spiliopoulou, I. Santos Sanches, M. I. Crisostomo, and H. de Lencastre. 2003. Two international methicillin-resistant Staphylococcus aureus clones endemic in a university hospital in Patras, Greece. J. Clin. Microbiol. 41:2027-2032.
Becker, K., A. W. Friedrich, G. Peters, and C. von Eiff. 2004. Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SEIM, SEIO and SEIN. Mol. Nutr. Food Res. 48:488-495.
Chini, V., E. Petinaki, A. Foka, S. Paratiras, G. Dimitracopoulos, and I. Spiliopoulou. 2006. Spread of Staphylococcus aureus clinical isolates carrying Panton-Valentine leukocidin genes during a 3-year period in Greece. Clin. Microbiol. Infect. 12:29-34.
de Lencastre, H., I. Couto, I. Santos, J. Melo-Cristino, A. Torres-Pereira, and A. Tomasz. 1994. Methicillin-resistant Staphylococcus aureus disease in a Portuguese hospital: characterization of clonal types by a combination of DNA typing methods. Eur. J. Clin. Microbiol. Infect. Dis. 13:64-73.
Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13:16-34.
Jarraud, S., M. A. Peyrat, A. Lim, A. Tristan, M. Bes, C. Mougel, J. Etienne, F. Vandenesch, M. Bonneville, and G. Lina. 2001. egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166:669-677.
Jarraud, S., C. Mougel, J. Thioulouse, G. Lina, H. Meugnier, F. Forey, X. Nesme, J. Etienne, and F. Vandenesch. 2002. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 70:631-641.
Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N. K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Furuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, and K. Hiramatsu. 2001. Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225-1240.
Letertre, C., S. Perelle, F. Dilasser, and P. Fach. 2003. Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus. J. Appl. Microbiol. 95:38-43.
Lina, G., F. Boutite, A. Tristan, M. Bes, J. Etienne, and F. Vandenesch. 2003. Bacterial competition for human nasal cavity colonization: role of staphylococcal agr alleles. Appl. Environ. Microbiol. 69:18-23.
Lina, G., G. A. Bohach, S. P. Nair, K. Hiramatsu, E. Jouvin-Marche, and R. Mariuzza, for the International Nomenclature Committee for Staphylococcal Superantigens. 2004. Standard nomenclature for the superantigens expressed by Staphylococcus. J. Infect. Dis. 189:2334-2336.
National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial disk susceptibility tests, 8th ed. Approved standard M2-A8. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard M7-A6. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Petinaki, E., A. Arvaniti, G. Dimitracopoulos, and I. Spiliopoulou. 2001. Detection of mecA, mecR1 and mecI genes among clinical isolates of methicillin-resistant staphylococci by combined polymerase chain reactions. J. Antimicrob. Chemother. 47:297-304.
Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.(Vassiliki Chini, George D)
ABSTRACT
Analysis of 177 methicillin-resistant Staphylococcus aureus (MRSA) strains for the presence of egc and tst revealed that 60 strains carried at least one of the tested genes. MRSA strains were classified by pulsed-field gel electrophoresis into four clones belonging to agr groups I and III. Toxin genotypes were related by clonal type and agr group.
TEXT
The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) has posed a clinical threat worldwide. Staphylococcal enterotoxins, enterotoxin-like superantigens, and the toxic shock syndrome toxin 1 that belong to pyrogenic toxin superantigens (PTSAgs) are considered major virulence factors (5, 11). The enterotoxin gene cluster (egc) includes five enterotoxin-like superantigen genes (seo, sem, sei, sen, and seg) and two pseudogenes (ent1 and ent2) (6, 8, 11). Recently, a new toxin, SEU, encoded by the egc operon (egc2), was identified to result from sequence divergence in the region of the pseudogenes ent1/ent2 (9).
Expression of virulent genes in S. aureus is controlled by the accessory gene regulator (agr), with a polymorphism in the sequence of the autoinducing peptide and its receptor, according to which clinical strains can be divided into four agr groups (I to IV) (7, 10). S. aureus strains causing specific syndromes were linked to certain agr types (7). Thus, the purpose of the present study was to investigate the presence of the egc operon and tst in a set of 177 MRSA isolates collected from different patients admitted to the University Hospital of Patras, Greece, during 2001 to 2003, to determine the clones of isolates, and to correlate them with the agr groups and toxin gene profile.
(These results were partially presented as a poster at the 15th European Congress of Clinical Microbiology and Infectious Diseases, Copenhagen, Denmark, 2 to 5 April 2005.)
All 177 MRSA isolates were tested by the disk diffusion method against various antistaphylococcal agents (12). Oxacillin MICs were determined by the agar dilution method (13), and the presence of the mecA gene was determined by PCR (14).
Clones were defined by pulsed-field gel electrophoresis of SmaI digests of chromosomal DNAs performed in a CHEFF DR III apparatus (Bio-Rad Laboratories, Richmond, California) according to published protocols (4, 15) and were compared to previously identified clones existing in our hospital (1).
agr typing was carried out by PCR as described previously (10) with modified thermal conditions as follows: predenaturation step for 5 min at 94°C, 25 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 55°C, extension for 1 min at 72°C, and a final extension step for 7 min at 72°C (J. Etienne and M. Bes, personal communication). Fri913 (agr class I), Fri137 (agr class II), ATCC 49775 (agr class III), and A920211 (agr class IV) were used as reference strains.
The sequences corresponding to seo, sem, sei, ent1/ent2 (ent), sen, and seg included in the egc1 operon and tst were detected by PCRs (6, 7). Fri137 and Fri913, carrying the egc2 operon and tst, respectively, were used as reference strains.
The presence of seu characterizing the egc2 operon (seo, sem, sei, seu, sen, and seg) was investigated by HindIII restriction fragment length polymorphism of the amplified ent region. The ent region of the seu-negative strain A900322 harboring egc1 (9) carries a unique restriction site for the HindIII enzyme at nucleotide 456, resulting in two restriction fragments of 456 and 181 bp. This region does not exist in the corresponding amplified part of the seu-positive strain Fri137 (GenBank accession no. AY205306) carrying egc2 (9). A restriction reaction in a final volume of 20 μl, containing 15 units HindIII (Promega, Madison, WI), 1x reaction buffer (Promega), and 7 μl of PCR product, was performed at 37°C with overnight incubation. Lamda () DNA was used as the restriction enzyme marker.
The 177 MRSA isolates with distinct antibiotic resistance phenotypes were classified into four clones, as previously described (1). All multiresistant isolates were characterized as clone B (48 strains or 27%). Most isolates (103 of 177 or 58%) belonged to clone C, expressing resistance to beta-lactams and fusidic acid (3). Three strains (2%) resistant to beta-lactams, erythromycin, and ciprofloxacin were characterized as clone F. Clone A included 23 isolates (13%) resistant only to beta-lactams.
Fifty-two isolates (29%) of clones B and F and a unique strain of clone C that did not possess the Panton-Valentine leukocidin (PVL) genes (3) belonged to agr class I. One hundred twenty-five (125 or 71%) isolates of clones A and C, with the exception of the aforementioned PVL-negative strain, belonged to agr class III (Table 1).
Among these 177 strains, 117 (66%) did not carry any PTSAg tested. Sixty MRSA isolates positive for at least one of the tested genes were classified into 11 genotypes (Table 1). The tst gene was always detected in combination with egc genes.
Twenty-three egc-positive isolates and six clinical isolates carrying the ent region (ent1/ent2-positive) were seu positive (egc2). The remaining four egc-positive isolates did not possess the seu gene (egc1) (Fig. 1). The Fri137 reference strain carried the seu gene, as expected (9).
The genotypes tst/egc2 and egc2 belonging to clone A were classified into agr group III. Four strains possessing egc1, classified to clones F (3 strains) and C (1 PVL-negative strain), belonged to agr group I (Table 1).
Figure 2 shows the distribution of specific genotypes for each year. Isolates harboring all tested genes reduced with time, whereas those which carried no genes remained high.
One-third (60/177) of the MRSA isolates tested carried a number of genes, ranging from one to seven, with an extensive variation between individual strains and a reducing tendency during the study period. Coexistence of tst with enterotoxin genes has been reported but not exclusively for all tested isolates (7), as shown in the present study. The prevalence of egc2 in our study is much higher than first reported (9). Discrepancies in the frequency of PTSAg genes from different countries are expected, since they are associated with mobile genetic elements and strains may gain a different number of genes.
The variable possession of egc genes suggests a more heterogeneous composition of this operon, leading to the emergence of the newly described seu gene (9) or the allelic variants of seg, sei, sen, and seu (2, 6). Identification of variable toxin gene profiles may be explained by horizontal transfer, since open reading frames for putative transposases are located nearby, although it is limited among lineages (7, 8).
Identification of agr groups classified the majority of our strains into group III and to a lesser extend into agr group I. In previous studies, most of the MRSA isolates were agr group I or II (7). Investigation of the relationship between agr groups, clones, and toxin gene distribution reveals that the presence of tested genes is clone related. Classification of tst-positive isolates in agr group III has been already reported among methicillin-susceptible S. aureus isolates (7).
The basic unit of bacterial pathogenicity may reside on the clone or lineage, since it expands, possessing particular combinations of virulence and regulatory genes in the appropriate genetic background (7). The sensitive clone A possessing all tested genes does not seem to be a threat to our patients, since it decreases with time.
In conclusion, one-third of 177 MRSA clinical isolates carried genes of the PTSAg family, with a reducing tendency during the study period. tst always coexisted in the same isolate with other genes of the egc operon. Clone A, which harbors all or the majority of tested toxin genes (tst/egc2 or egc2), expresses a sensitive phenotype; however, it could not correlate with a specific clinical syndrome. This is the first report of a representative collection of MRSA in Greece. A clonal relationship with agr group classification and toxin gene profile was identified, and it remains to be seen whether a specific lineage spread may occur. Identification of strains with variable toxin gene profiles belonging to different clones indicates that egc genes are not strictly linked and a multitude of combinations among themselves and other PTSAgs (tst) may occur in bacterial populations.
ACKNOWLEDGMENTS
This work was supported by "K. Karatheodori" grant no. B116 of the University of Patras awarded to I.S.
We thank Jerome Etienne from the Centre National de Reference des Staphylocoques, Lyon, France, for kindly providing the reference S. aureus strains and E. D. Anastassiou and E. Petinaki for editing the manuscript.
REFERENCES
Aires de Sousa, M., C. Bartzavali, I. Spiliopoulou, I. Santos Sanches, M. I. Crisostomo, and H. de Lencastre. 2003. Two international methicillin-resistant Staphylococcus aureus clones endemic in a university hospital in Patras, Greece. J. Clin. Microbiol. 41:2027-2032.
Becker, K., A. W. Friedrich, G. Peters, and C. von Eiff. 2004. Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SEIM, SEIO and SEIN. Mol. Nutr. Food Res. 48:488-495.
Chini, V., E. Petinaki, A. Foka, S. Paratiras, G. Dimitracopoulos, and I. Spiliopoulou. 2006. Spread of Staphylococcus aureus clinical isolates carrying Panton-Valentine leukocidin genes during a 3-year period in Greece. Clin. Microbiol. Infect. 12:29-34.
de Lencastre, H., I. Couto, I. Santos, J. Melo-Cristino, A. Torres-Pereira, and A. Tomasz. 1994. Methicillin-resistant Staphylococcus aureus disease in a Portuguese hospital: characterization of clonal types by a combination of DNA typing methods. Eur. J. Clin. Microbiol. Infect. Dis. 13:64-73.
Dinges, M. M., P. M. Orwin, and P. M. Schlievert. 2000. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13:16-34.
Jarraud, S., M. A. Peyrat, A. Lim, A. Tristan, M. Bes, C. Mougel, J. Etienne, F. Vandenesch, M. Bonneville, and G. Lina. 2001. egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J. Immunol. 166:669-677.
Jarraud, S., C. Mougel, J. Thioulouse, G. Lina, H. Meugnier, F. Forey, X. Nesme, J. Etienne, and F. Vandenesch. 2002. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 70:631-641.
Kuroda, M., T. Ohta, I. Uchiyama, T. Baba, H. Yuzawa, I. Kobayashi, L. Cui, A. Oguchi, K. Aoki, Y. Nagai, J. Lian, T. Ito, M. Kanamori, H. Matsumaru, A. Maruyama, H. Murakami, A. Hosoyama, Y. Mizutani-Ui, N. K. Takahashi, T. Sawano, R. Inoue, C. Kaito, K. Sekimizu, H. Hirakawa, S. Kuhara, S. Goto, J. Yabuzaki, M. Kanehisa, A. Yamashita, K. Oshima, K. Furuya, C. Yoshino, T. Shiba, M. Hattori, N. Ogasawara, H. Hayashi, and K. Hiramatsu. 2001. Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet 357:1225-1240.
Letertre, C., S. Perelle, F. Dilasser, and P. Fach. 2003. Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus. J. Appl. Microbiol. 95:38-43.
Lina, G., F. Boutite, A. Tristan, M. Bes, J. Etienne, and F. Vandenesch. 2003. Bacterial competition for human nasal cavity colonization: role of staphylococcal agr alleles. Appl. Environ. Microbiol. 69:18-23.
Lina, G., G. A. Bohach, S. P. Nair, K. Hiramatsu, E. Jouvin-Marche, and R. Mariuzza, for the International Nomenclature Committee for Staphylococcal Superantigens. 2004. Standard nomenclature for the superantigens expressed by Staphylococcus. J. Infect. Dis. 189:2334-2336.
National Committee for Clinical Laboratory Standards. 2003. Performance standards for antimicrobial disk susceptibility tests, 8th ed. Approved standard M2-A8. National Committee for Clinical Laboratory Standards, Wayne, Pa.
National Committee for Clinical Laboratory Standards. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Approved standard M7-A6. National Committee for Clinical Laboratory Standards, Wayne, Pa.
Petinaki, E., A. Arvaniti, G. Dimitracopoulos, and I. Spiliopoulou. 2001. Detection of mecA, mecR1 and mecI genes among clinical isolates of methicillin-resistant staphylococci by combined polymerase chain reactions. J. Antimicrob. Chemother. 47:297-304.
Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233-2239.(Vassiliki Chini, George D)