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Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus
FEMS Microbiology Letters 202 (2001) 91^95
www.fems-microbiology.org
Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus Fernanda R. Buzzola a , Liliana S. Quelle a , Lynn Steele-Moore b , Donna Berg b , Graciela Denamiel c , Elida Gentilini c , Daniel O. Sordelli a; * a
Departamento de Microbiolog|¨a, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 p12, 1121 Buenos Aires, Argentina b Christiana Care Health Services, Wilmington, DE, USA c ¨ Departamento de Fisiolog|a y Etiopatogenia, Facultad de Veterinaria, Universidad de Buenos Aires, Buenos Aires, Argentina Received 28 March 2001 ; received in revised form 11 June 2001; accepted 13 June 2001 First published online 6 July 2001
Abstract Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Live-attenuated vaccine; Molecular typing; Bovine mastitis; Staphylococcus aureus
1. Introduction Mastitis due to Staphylococcus aureus is a disease of ruminants that produces substantial economic losses to the dairy industry reaching billions of dollars worldwide [1,2]. Bacterial cell extracts, capsular polysaccharides and/ or toxoids have been included in previously described vaccine preparations to prevent bovine mastitis [1]. Attempts at immunization using these preparations, however, have been largely ine¡ective and new approaches to vaccination are thus necessary to attempt prevention of mastitis in cows. In this regard, puri¢ed S. aureus components have been pointed out as targets for vaccine development and are currently under investigation [3,4]. Our laboratory is currently developing di¡erent live-attenuated mutants of S. aureus to be used as vaccines to promote adaptive immunity in the bovine mammary gland by local administration [5,6]. Before ¢eld trials are performed, a procedure for
* Corresponding author. Tel. : +54 (11) 4963 6669 ; Fax: +54 (11) 4508 3705. E-mail address : [email protected] (D.O. Sordelli).
discrimination of vaccine strains from clinical isolates needs to be established. Indeed the vaccine strain background has to be easily recognized from those of ¢eld isolates in the unlikely event that new cases of infection may be caused by putative derivatives of the vaccine strain. In the past decade, numerous molecular techniques with dissimilar discriminatory capacity were used for discrimination of S. aureus isolates in epidemiological studies [7]. Identi¢cation and comparison of S. aureus isolates from mastitic cow milk were established by several molecular methods [8,9]. The present study was designed to establish a procedure to di¡erentiate laboratory strains from S. aureus clinical isolates from bovines in Argentina. Molecular identi¢cation was performed by pulsed-¢eld gel electrophoresis (PFGE) band analysis after SmaI DNA macrorestriction and automated ribotyping. 2. Materials and methods 2.1. Bacterial strains and cultures Three sets of S. aureus isolates were included in the
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 9 4 - 4
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present study. Set 1 contained 21 epidemiologically unrelated isolates from bovine milk, collected from di¡erent farms located in one district in Argentina. These isolates were utilized to determine the index of discrimination (D) of the molecular methods used in this study. Set 2 comprised 55 bovine S. aureus isolates obtained from milk of cows with mastitis in herds placed in two districts located in eastern and western regions of the country. All isolates were identi¢ed as S. aureus using standard microbiological techniques [10]. Set 3 contained prototypic vaccine strains and their parental wild-types (wt). Vaccine strains included in this study were live-attenuated (la) mutants of S. aureus. Two temperature sensitive mutants isolated after nitrosoguanidine mutagenesis of S. aureus NCTC 8325-4 and an aromatic amino acid auxotrophic mutant obtained by insertional mutagenesis of parental strain RN6390 were included in the studies. 2.2. PFGE conditions and dendrogram generation Genomic DNA was digested with SmaI and DNA fragments were resolved by PFGE (CHEF DR-III, Bio-Rad, Hercules, CA, USA) using a standard protocol [11]. PFGE types and subtypes were identi¢ed with italicized small letters and arabic numerals, respectively. Similarity among PFGE types was evaluated by means of the Nei and Li coe¤cient [12]. Cluster analysis was performed by the unweighted pair group method average and data were analyzed with the TREECON program (version 2.1) for Windows. 2.3. Automated ribotyping Bacteria were also identi¢ed by EcoRI ribotyping using the automated RiboPrint system (Qualicon, Wilmington, DE, USA). Ribotyping was performed according to the standard protocol recommended by the vendor (Ribo-
Printer Microbial Characterization System, Chapter 2: Operations user's guide, Qualicon). Ribogroups were identi¢ed with roman numerals. 2.4. Discriminatory power, concordance analysis and statistical analysis To compare the discriminatory power of the typing methods, the Hunter and Gaston's [13] index (D) was applied. Concordance analysis of PFGE typing and ribotyping was performed as previously described [9]. Statistical signi¢cance was assessed by means of the M2 -test of independence (GraphPad, Prism 2.0 software). 3. Results 3.1. PFGE typing The discriminatory power of SmaI macrorestriction typing by PFGE was 0.91. The D index of EcoRI automated ribotyping was 0.69 (Set 1). PFGE typing discriminated the 55 isolates (Set 2) and laboratory strains (Set 3) into 17 ¢ngerprint groups consisting of 12^15 fragments in the size range from 6 48.5 to 485 kb (Fig. 1). At an 80% similarity level, Set 2 and 3 S. aureus strains were grouped into clusters A, B, C, and Q. SmaI PFGE band analysis revealed that 32 of 55 ¢eld isolates were indistinguishable from each other (cluster A), and the remainder were evenly distributed in 10 groups of one to four isolates. All prototypic vaccines and their parental wt strains exhibited a markedly di¡erent macrorestriction genotype (cluster Q) when compared with those of clinical isolates (clusters A, B and C) (Fig. 1). Band patterns from laboratory strains di¡ered in more than seven bands from those of bovine clinical isolates. The number of genetic di¡erences of the prototypic vaccine genome (8325 background) when com-
Fig. 1. SmaI PFGE types of S. aureus and percent of similarity dendrogram. Left panel: PFGE band patterns of laboratory strains with q1 background (wt NCTC 8325-4, live-attenuated A523 and live-attenuated B28, respectively, in the second, third and fourth lanes) and q2 (wt RN6390 and live-attenuated Sa306, respectively, in the ¢fth and sixth lanes). Lanes a, a1, a3, a9, a10, b1, b and c represent the most prevalent PFGE types and subtypes of S. aureus clinical bovine isolates. Lanes MW, molecular size marker (lambda concatemer). Right panel: dendrogram showing the levels of similarity between PFGE types of laboratory strains (cluster Q) and bovine clinical isolates of S. aureus (clusters A, B and C).
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pared with those of clinical isolates was three or more, which make them clonally di¡erent according to the criteria set by Tenover et al. [14]. Cluster Q shared only 49% of similarity with clusters A and B, and 35% of similarity with cluster C. Cluster Q strains were discriminated into two closely related clones (q1 and q2). Analysis of PFGE band patterns of these clones revealed di¡erences in three bands. These results show that prototypic vaccine strains were unequivocally discriminated from bovine milk S. aureus isolates using SmaI macrorestriction PFGE typing.
93
Table 1 Concordance analysis of ribotype with SmaI macrorestriction by PFGE typea Ribogroup I
Same Di¡erent
No. of isolate pairs (proportion of total comparisons) with PFGE typea Match
Mismatch
649 (0.366) 236 (0.133)
295 (0.166) 590 (0.333)
a Concordance equals the sum of the same ribotype-match and di¡erent ribotype-mismatch entries, expressed as a percentage of the total 1770 pairwise comparisons. For data presented here, concordance was 70%.
3.2. Automated ribotyping Clinical isolates (Set 2) and laboratory strains (Set 3) were discriminated in 10 ribogroups by automated ribotyping. Only la-mutants and their parental wt strains were clustered into ribogroups IV (vendor code 135-50-4) and VIII (vendor code 135-55-4) (Fig. 2). Most clinical isolates were clustered into ribogroups II, III and IX of 30, eight and seven isolates, respectively. The remainder were evenly distributed in ¢ve ribogroups of one to four isolates (Fig. 2). Automated ribotyping identi¢ed as identical two la-S. aureus mutants (obtained by chemical mutagenesis) and discriminated the third (obtained by insertion mutagenesis) into a separate ribogroup. In this case, a single transposon insertion may have been the cause for the di¡erent pro¢le. In spite of the moderate discriminatory power observed, automated ribotyping permitted identi¢cation of laboratory strains that were assigned to ribogroups di¡erent from those of the ¢eld isolates. 3.3. Genotyping using both PFGE and ribotyping methods Because of the high number of clinical isolates identi¢ed as PFGE type a and ribogroup I, the concordance between both molecular typing methods in identifying genotypes was analyzed. The level of concordance of both methods was assessed by comparing all possible pairs from 55 isolates and laboratory strains (N total = 60).
Fig. 2. Digitalized pro¢les of ribotypes from laboratory strains and bovine clinical isolates of S. aureus. Arabic numbers represent the vendor database code whereas roman numerals were used to identify ribotypes easily.
For automated ribotyping and PFGE typing, each pair of isolates was classi¢ed according to: (a) the isolates possessed identical or di¡erent ribogroup I, and (b) whether they matched or mismatched PFGE type a (Table 1). From a total of 1770 possible pairwise combinations of the 60 strains under study, 36.6% of ribogroup I isolate pairs exhibited the PFGE type a pattern (match) and 33.3% of the isolate pairs with ribogroup di¡erent from I and PFGE type di¡erent from a (mismatch). The overall percentage of concordant results was 70%, which was highly signi¢cant (M2 -test of independence, M2 = 282, df = 1, P 6 1036 ), meaning that clonal identity association exists between type a and ribogroup I isolates. Combined use of SmaI PFGE typing and automated ribotyping permitted higher discrimination of laboratory strains. By means of automated ribotyping genotype q1 belonging to PFGE cluster Q was discriminated into two di¡erent ribogroups (IV and VIII). Thus, the two la-mutants obtained by chemical mutagenesis and their parental strains were classi¢ed within the q1/IV genotype whereas the insertional mutant of S. aureus was identi¢ed as the q1/VIII genotype. 4. Discussion SmaI PFGE typing analysis indicated that the prototypic vaccine strains described here were unequivocally discriminated from bovine S. aureus isolates from the geographical area under investigation. Previous data suggested that PFGE typing is the most discriminative of the currently available genotypic methods for S. aureus [15]. Although automated ribotyping exhibited low ability to di¡erentiate genealogically unrelated bacterial clones (D = 0.69), the prototypic vaccine strains were easily discriminated from the remaining bovine clinical isolates. By automated ribotyping, the band pattern from each isolate was compared with those in an existing database [16]. The discrimination criteria utilized by this method are less strict than those used for the analysis of SmaI macrorestriction PFGE pro¢les. High concordance between PFGE typing and ribotyping in identifying a similar genotype suggested the exis-
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tence of a prevalent clone (a/I) among S. aureus isolates from milk of bovines with mastitis in Argentina. This observation may represent a low gene £ow rate in the population of bovine S. aureus isolates under study, as suggested by Fitzgerald et al. [17]. Thus, it may not be necessary to repeat a study of similar characteristics before every ¢eld trial using la-vaccines constructed on the same background (S. aureus 8325) and performed within the same geographical region. Consequently, the results obtained by means of this typing and identi¢cation strategy may remain valid and be useful over several years. Moreover, preliminary studies from our laboratory have shown that no PFGE patterns similar to that of S. aureus NCTC 8325-4 have been found in a limited number of prevalent strains from the USA, Spain, Ireland, Iceland, Sweden, Finland, Norway and Denmark [18]. This study provides information on the procedure to be applied before la-S. aureus vaccine strains are introduced into the environment. One major concern when a liveattenuated vaccine is utilized is the possibility that new cases of the disease can be caused by revertants derived from the vaccine strain. The appearance of cows with mastitis in the vaccinated herd cannot be rejected because 100% protection from infection cannot be assumed. Indeed, the e¤cacy of vaccination with la-S. aureus to prevent bovine mastitis has to be determined in clinical trials. The procedure described in this report can safely be used to assess stability of vaccine strains in cows as well it may provide information on spreading of a vaccine strain spontaneous derivative, should this unlikely event occur. In order to be able to identify a putative derivative of the vaccine strain in humans in contact with vaccinated cows, the use of a marker to identify the vaccine strain may be prudent. Addition to the genome of a marker gene to identify vaccine strains has been suggested in the past. Indeed, antibiotic resistance genes were used to identify such laboratory strains [19]. This strategy, however, may not be acceptable to identify revertants of the vaccine strain in the environment because it may not be appropriate to construct attenuated vaccine strains bearing antibiotic resistance. In this report we demonstrate the value of molecular typing as screening method for a vaccine genotype. The S. aureus 8325 background is widely used in genetic studies [20]. In addition, most readily available information on the S. aureus genome to the general scienti¢c community was obtained from the 8325 background [21]. Therefore, our study provides information to researchers using the S. aureus 8325 strain or its derivatives in studies in which the research subject, e.g. an attenuated mutant of S. aureus, may be released to the environment. We are currently screening strains from di¡erent sources worldwide in order to extend the geographical validity of our observations. In conclusion, SmaI macrorestriction PFGE typing and automated ribotyping demonstrated to be useful methods
for discrimination of our prototypic vaccine strains from S. aureus bovine isolates in controlled studies performed on isolated and small size herds. The signi¢cant concordance between the most prevalent types by PFGE and ribotyping supports these ¢ndings. Acknowledgements This work was supported in part by Grants from UBACYT Argentina (integrado IM-05 ; trienal TM-55), CONICET Argentina (PIP 0944/98), ANPCyT (PICT 047460) and the `A.J. Roemmers Foundation'. The authors thank Scott Frischell and Eileen Cole (Qualicon, Wilmington, DE, USA) for providing the RiboPrint ribotyping kits and for her help with data analysis, respectively.
References [1] Yancey Jr., R.J. (1999) Vaccines and diagnostic methods for bovine mastitis: fact and ¢ction. Adv. Vet. Med. 41, 257^273. [2] Calzolari, A., Giraudo, J.E., Rampone, H., Odierno, L., Giraudo, A.T., Frigerio, C., Bettera, S., Raspanti, C., Herna¨ndez, J., Wehbe, M., Mattea, M., Ferrari, M., Larriestra, A. and Nagel, R. (1997) Field trials of a vaccine against bovine mastitis 2. Evaluation in two commercial dairy herds. J. Dairy Sci. 80, 854^858. [3] O'Brien, C., Guidry, A., Fattom, A., Shepherd, S., Douglass, L. and Westho¡, D. (2000) Production of antibodies to Staphylococcus aureus serotypes 5, 8 and 336 using poly(DL-lactide-CO-glycolide) microspheres. J. Dairy Sci. 83, 1758^1766. [4] McKeenney, D., Pouliot, K., Wang, Y., Murthy, V., Ulrich, M., Doring, G., Lee, J.C., Goldmann, D. and Pier, G. (2000) Vaccine potential of poly-1,6 L-DN-succinylglucosamine, an immunoprotective surface polysaccharide of Staphylococcus aureus and Staphylococcus epidermidis. J. Biotechnol. 29, 37^44. [5] Go¨mez, M.I., Garc|¨a, V.E., Gherardi, M.M., Cerquetti, M.C. and Sordelli, D.O. (1998) Intramammary immunization with live-attenuated Staphylococcus aureus protects mice from experimental mastitis. FEMS Immunol. Med. Microbiol. 20, 21^27. [6] Sordelli, D.O., Cerquetti, M.C., Buzzola, F.R., Garc|¨a, V.E., Go¨mez, M.I. and Morris Hooke, A. (1998) Attenuated mutants of Staphylococcus aureus with two temperature-sensitive lesions: isolation and characterization. Microbios 94, 95^102. [7] Tenover, F.C., Arbeit, R.D., Archer, G., Biddle, J., Byrne, R., Goering, R., Hancock, G., Herbert, A., Hill, B., Hollis, R., Jarvis, W., Kreiswirth, B., Eisner, W., Maslow, J., McDougal, L., Miller, M., Mulligan, M. and Pfaller, M. (1994) Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus. J. Clin. Microbiol. 32, 407^415. [8] Fiztgerald, J.R., Hartigan, P.J., Meaney, W.J. and Smyth, C.J. (2000) Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection. J. Appl. Microbiol. 88, 1028^1037. [9] Mullarky, I.K., Su, C., Frieze, N., Park, Y.H. and Sordillo, L.M. (2001) Staphylococcus aureus agr genotypes with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect. Immun. 69, 45^51. [10] Kloos, W.E. and Bannerman, T.L. (1999) Staphylococcus and Micrococcus. In: Manual of Clinical Microbiology (Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., Eds.), pp. 264^ 282. ASM Press, Washington, DC. [11] Maslow, J.N., Mulligan, M.E. and Arbeit, R.D. (1993) Molecular
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[12]
[13]
[14]
[15]
[16]
epidemiology: application of contemporary techniques to the typing of microorganisms. Clin. Infect. Dis. 17, 153^164. Nei, M. and Li, W.H. (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76, 5269^5273. Hunter, P.R. and Gaston, M.A. (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26, 2465^2466. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H. and Swaminathan, B. (1995) Interpreting chromosomal DNA restriction patterns produced by pulse-¢eld gel electrophoresis : criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233^2239. Galdbart, J.O., Morvan, A. and El Sohl, N. (2000) Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in France from 1995 to 1997. J. Clin. Microbiol. 38, 185^190. Bruce, J. (1996) Automated system rapidly identi¢es and characterizes microorganisms in food. Food Technol. (Chicago) 50, 77^81.
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[17] Fitzgerald, J.R., Meaney, W.J., Hartigan, P.J., Smyth, C.J. and Kapur, V. (1997) Fine-structure molecular epidemiological analysis of Staphylococcus aureus recovered from cows. Epidemiol. Infect. 119, 261^269. [18] Quelle, L.S., Buzzola, F.R. and Sordelli, D.O. (2001) Phylogenetic relationship of bovine mastitis Staphylococcus aureus from America and Europe. 101th General Meeting of the American Society for Microbiology, Abstract C-407, p. 246, Orlando, FL. [19] Hooke, A.M., Bellanti, J.A. and Oeschger, M.P. (1985) Live-attenuated vaccines: new approaches for safety and e¤cacy. Lancet 8444, 1472^1474. [20] Novick, P.R. (1989) The Staphylococcus as a molecular genetic system. In: Molecular Biology of the Staphylococci (Novick, R.P., Ed.), pp. 1^37. VCH Publishers. [21] Iandolo, J.J. (2000) Genetic and physical map of the chromosome of the Staphylococcus aureus 8325. In: Gram-Positive Pathogens (Fischetti, V.A., Novick, R.P., Ferretti, J.J., Portnoy, D.A. and Rood, J.I., Eds.), pp. 317^325. ASM Press, Washington, DC.
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Molecular diversity of live-attenuated prototypic vaccine strains and clinical isolates of Staphylococcus aureus Fernanda R. Buzzola a , Liliana S. Quelle a , Lynn Steele-Moore b , Donna Berg b , Graciela Denamiel c , Elida Gentilini c , Daniel O. Sordelli a; * a
Departamento de Microbiolog|¨a, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 p12, 1121 Buenos Aires, Argentina b Christiana Care Health Services, Wilmington, DE, USA c ¨ Departamento de Fisiolog|a y Etiopatogenia, Facultad de Veterinaria, Universidad de Buenos Aires, Buenos Aires, Argentina Received 28 March 2001 ; received in revised form 11 June 2001; accepted 13 June 2001 First published online 6 July 2001
Abstract Bovine mastitis Staphylococcus aureus isolates and prototypic live-attenuated vaccine strains were analyzed by SmaI pulsed-field gel electrophoresis (PFGE) typing and automated ribotyping. The discriminatory index of these methods was 0.91 and 0.69, respectively. SmaI PFGE typing assigned all laboratory strains into cluster Q, which shared 49% similarity with clusters A and B, and 35% similarity with cluster C. Automated ribotyping placed laboratory strains within ribogroups different from those of bovine isolates. These methods have 70% concordance and permitted identification of the prototypic vaccine background from those of clinical isolates. This information is required before conducting field trials with the vaccine. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Live-attenuated vaccine; Molecular typing; Bovine mastitis; Staphylococcus aureus
1. Introduction Mastitis due to Staphylococcus aureus is a disease of ruminants that produces substantial economic losses to the dairy industry reaching billions of dollars worldwide [1,2]. Bacterial cell extracts, capsular polysaccharides and/ or toxoids have been included in previously described vaccine preparations to prevent bovine mastitis [1]. Attempts at immunization using these preparations, however, have been largely ine¡ective and new approaches to vaccination are thus necessary to attempt prevention of mastitis in cows. In this regard, puri¢ed S. aureus components have been pointed out as targets for vaccine development and are currently under investigation [3,4]. Our laboratory is currently developing di¡erent live-attenuated mutants of S. aureus to be used as vaccines to promote adaptive immunity in the bovine mammary gland by local administration [5,6]. Before ¢eld trials are performed, a procedure for
* Corresponding author. Tel. : +54 (11) 4963 6669 ; Fax: +54 (11) 4508 3705. E-mail address : [email protected] (D.O. Sordelli).
discrimination of vaccine strains from clinical isolates needs to be established. Indeed the vaccine strain background has to be easily recognized from those of ¢eld isolates in the unlikely event that new cases of infection may be caused by putative derivatives of the vaccine strain. In the past decade, numerous molecular techniques with dissimilar discriminatory capacity were used for discrimination of S. aureus isolates in epidemiological studies [7]. Identi¢cation and comparison of S. aureus isolates from mastitic cow milk were established by several molecular methods [8,9]. The present study was designed to establish a procedure to di¡erentiate laboratory strains from S. aureus clinical isolates from bovines in Argentina. Molecular identi¢cation was performed by pulsed-¢eld gel electrophoresis (PFGE) band analysis after SmaI DNA macrorestriction and automated ribotyping. 2. Materials and methods 2.1. Bacterial strains and cultures Three sets of S. aureus isolates were included in the
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 9 4 - 4
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present study. Set 1 contained 21 epidemiologically unrelated isolates from bovine milk, collected from di¡erent farms located in one district in Argentina. These isolates were utilized to determine the index of discrimination (D) of the molecular methods used in this study. Set 2 comprised 55 bovine S. aureus isolates obtained from milk of cows with mastitis in herds placed in two districts located in eastern and western regions of the country. All isolates were identi¢ed as S. aureus using standard microbiological techniques [10]. Set 3 contained prototypic vaccine strains and their parental wild-types (wt). Vaccine strains included in this study were live-attenuated (la) mutants of S. aureus. Two temperature sensitive mutants isolated after nitrosoguanidine mutagenesis of S. aureus NCTC 8325-4 and an aromatic amino acid auxotrophic mutant obtained by insertional mutagenesis of parental strain RN6390 were included in the studies. 2.2. PFGE conditions and dendrogram generation Genomic DNA was digested with SmaI and DNA fragments were resolved by PFGE (CHEF DR-III, Bio-Rad, Hercules, CA, USA) using a standard protocol [11]. PFGE types and subtypes were identi¢ed with italicized small letters and arabic numerals, respectively. Similarity among PFGE types was evaluated by means of the Nei and Li coe¤cient [12]. Cluster analysis was performed by the unweighted pair group method average and data were analyzed with the TREECON program (version 2.1) for Windows. 2.3. Automated ribotyping Bacteria were also identi¢ed by EcoRI ribotyping using the automated RiboPrint system (Qualicon, Wilmington, DE, USA). Ribotyping was performed according to the standard protocol recommended by the vendor (Ribo-
Printer Microbial Characterization System, Chapter 2: Operations user's guide, Qualicon). Ribogroups were identi¢ed with roman numerals. 2.4. Discriminatory power, concordance analysis and statistical analysis To compare the discriminatory power of the typing methods, the Hunter and Gaston's [13] index (D) was applied. Concordance analysis of PFGE typing and ribotyping was performed as previously described [9]. Statistical signi¢cance was assessed by means of the M2 -test of independence (GraphPad, Prism 2.0 software). 3. Results 3.1. PFGE typing The discriminatory power of SmaI macrorestriction typing by PFGE was 0.91. The D index of EcoRI automated ribotyping was 0.69 (Set 1). PFGE typing discriminated the 55 isolates (Set 2) and laboratory strains (Set 3) into 17 ¢ngerprint groups consisting of 12^15 fragments in the size range from 6 48.5 to 485 kb (Fig. 1). At an 80% similarity level, Set 2 and 3 S. aureus strains were grouped into clusters A, B, C, and Q. SmaI PFGE band analysis revealed that 32 of 55 ¢eld isolates were indistinguishable from each other (cluster A), and the remainder were evenly distributed in 10 groups of one to four isolates. All prototypic vaccines and their parental wt strains exhibited a markedly di¡erent macrorestriction genotype (cluster Q) when compared with those of clinical isolates (clusters A, B and C) (Fig. 1). Band patterns from laboratory strains di¡ered in more than seven bands from those of bovine clinical isolates. The number of genetic di¡erences of the prototypic vaccine genome (8325 background) when com-
Fig. 1. SmaI PFGE types of S. aureus and percent of similarity dendrogram. Left panel: PFGE band patterns of laboratory strains with q1 background (wt NCTC 8325-4, live-attenuated A523 and live-attenuated B28, respectively, in the second, third and fourth lanes) and q2 (wt RN6390 and live-attenuated Sa306, respectively, in the ¢fth and sixth lanes). Lanes a, a1, a3, a9, a10, b1, b and c represent the most prevalent PFGE types and subtypes of S. aureus clinical bovine isolates. Lanes MW, molecular size marker (lambda concatemer). Right panel: dendrogram showing the levels of similarity between PFGE types of laboratory strains (cluster Q) and bovine clinical isolates of S. aureus (clusters A, B and C).
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pared with those of clinical isolates was three or more, which make them clonally di¡erent according to the criteria set by Tenover et al. [14]. Cluster Q shared only 49% of similarity with clusters A and B, and 35% of similarity with cluster C. Cluster Q strains were discriminated into two closely related clones (q1 and q2). Analysis of PFGE band patterns of these clones revealed di¡erences in three bands. These results show that prototypic vaccine strains were unequivocally discriminated from bovine milk S. aureus isolates using SmaI macrorestriction PFGE typing.
93
Table 1 Concordance analysis of ribotype with SmaI macrorestriction by PFGE typea Ribogroup I
Same Di¡erent
No. of isolate pairs (proportion of total comparisons) with PFGE typea Match
Mismatch
649 (0.366) 236 (0.133)
295 (0.166) 590 (0.333)
a Concordance equals the sum of the same ribotype-match and di¡erent ribotype-mismatch entries, expressed as a percentage of the total 1770 pairwise comparisons. For data presented here, concordance was 70%.
3.2. Automated ribotyping Clinical isolates (Set 2) and laboratory strains (Set 3) were discriminated in 10 ribogroups by automated ribotyping. Only la-mutants and their parental wt strains were clustered into ribogroups IV (vendor code 135-50-4) and VIII (vendor code 135-55-4) (Fig. 2). Most clinical isolates were clustered into ribogroups II, III and IX of 30, eight and seven isolates, respectively. The remainder were evenly distributed in ¢ve ribogroups of one to four isolates (Fig. 2). Automated ribotyping identi¢ed as identical two la-S. aureus mutants (obtained by chemical mutagenesis) and discriminated the third (obtained by insertion mutagenesis) into a separate ribogroup. In this case, a single transposon insertion may have been the cause for the di¡erent pro¢le. In spite of the moderate discriminatory power observed, automated ribotyping permitted identi¢cation of laboratory strains that were assigned to ribogroups di¡erent from those of the ¢eld isolates. 3.3. Genotyping using both PFGE and ribotyping methods Because of the high number of clinical isolates identi¢ed as PFGE type a and ribogroup I, the concordance between both molecular typing methods in identifying genotypes was analyzed. The level of concordance of both methods was assessed by comparing all possible pairs from 55 isolates and laboratory strains (N total = 60).
Fig. 2. Digitalized pro¢les of ribotypes from laboratory strains and bovine clinical isolates of S. aureus. Arabic numbers represent the vendor database code whereas roman numerals were used to identify ribotypes easily.
For automated ribotyping and PFGE typing, each pair of isolates was classi¢ed according to: (a) the isolates possessed identical or di¡erent ribogroup I, and (b) whether they matched or mismatched PFGE type a (Table 1). From a total of 1770 possible pairwise combinations of the 60 strains under study, 36.6% of ribogroup I isolate pairs exhibited the PFGE type a pattern (match) and 33.3% of the isolate pairs with ribogroup di¡erent from I and PFGE type di¡erent from a (mismatch). The overall percentage of concordant results was 70%, which was highly signi¢cant (M2 -test of independence, M2 = 282, df = 1, P 6 1036 ), meaning that clonal identity association exists between type a and ribogroup I isolates. Combined use of SmaI PFGE typing and automated ribotyping permitted higher discrimination of laboratory strains. By means of automated ribotyping genotype q1 belonging to PFGE cluster Q was discriminated into two di¡erent ribogroups (IV and VIII). Thus, the two la-mutants obtained by chemical mutagenesis and their parental strains were classi¢ed within the q1/IV genotype whereas the insertional mutant of S. aureus was identi¢ed as the q1/VIII genotype. 4. Discussion SmaI PFGE typing analysis indicated that the prototypic vaccine strains described here were unequivocally discriminated from bovine S. aureus isolates from the geographical area under investigation. Previous data suggested that PFGE typing is the most discriminative of the currently available genotypic methods for S. aureus [15]. Although automated ribotyping exhibited low ability to di¡erentiate genealogically unrelated bacterial clones (D = 0.69), the prototypic vaccine strains were easily discriminated from the remaining bovine clinical isolates. By automated ribotyping, the band pattern from each isolate was compared with those in an existing database [16]. The discrimination criteria utilized by this method are less strict than those used for the analysis of SmaI macrorestriction PFGE pro¢les. High concordance between PFGE typing and ribotyping in identifying a similar genotype suggested the exis-
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tence of a prevalent clone (a/I) among S. aureus isolates from milk of bovines with mastitis in Argentina. This observation may represent a low gene £ow rate in the population of bovine S. aureus isolates under study, as suggested by Fitzgerald et al. [17]. Thus, it may not be necessary to repeat a study of similar characteristics before every ¢eld trial using la-vaccines constructed on the same background (S. aureus 8325) and performed within the same geographical region. Consequently, the results obtained by means of this typing and identi¢cation strategy may remain valid and be useful over several years. Moreover, preliminary studies from our laboratory have shown that no PFGE patterns similar to that of S. aureus NCTC 8325-4 have been found in a limited number of prevalent strains from the USA, Spain, Ireland, Iceland, Sweden, Finland, Norway and Denmark [18]. This study provides information on the procedure to be applied before la-S. aureus vaccine strains are introduced into the environment. One major concern when a liveattenuated vaccine is utilized is the possibility that new cases of the disease can be caused by revertants derived from the vaccine strain. The appearance of cows with mastitis in the vaccinated herd cannot be rejected because 100% protection from infection cannot be assumed. Indeed, the e¤cacy of vaccination with la-S. aureus to prevent bovine mastitis has to be determined in clinical trials. The procedure described in this report can safely be used to assess stability of vaccine strains in cows as well it may provide information on spreading of a vaccine strain spontaneous derivative, should this unlikely event occur. In order to be able to identify a putative derivative of the vaccine strain in humans in contact with vaccinated cows, the use of a marker to identify the vaccine strain may be prudent. Addition to the genome of a marker gene to identify vaccine strains has been suggested in the past. Indeed, antibiotic resistance genes were used to identify such laboratory strains [19]. This strategy, however, may not be acceptable to identify revertants of the vaccine strain in the environment because it may not be appropriate to construct attenuated vaccine strains bearing antibiotic resistance. In this report we demonstrate the value of molecular typing as screening method for a vaccine genotype. The S. aureus 8325 background is widely used in genetic studies [20]. In addition, most readily available information on the S. aureus genome to the general scienti¢c community was obtained from the 8325 background [21]. Therefore, our study provides information to researchers using the S. aureus 8325 strain or its derivatives in studies in which the research subject, e.g. an attenuated mutant of S. aureus, may be released to the environment. We are currently screening strains from di¡erent sources worldwide in order to extend the geographical validity of our observations. In conclusion, SmaI macrorestriction PFGE typing and automated ribotyping demonstrated to be useful methods
for discrimination of our prototypic vaccine strains from S. aureus bovine isolates in controlled studies performed on isolated and small size herds. The signi¢cant concordance between the most prevalent types by PFGE and ribotyping supports these ¢ndings. Acknowledgements This work was supported in part by Grants from UBACYT Argentina (integrado IM-05 ; trienal TM-55), CONICET Argentina (PIP 0944/98), ANPCyT (PICT 047460) and the `A.J. Roemmers Foundation'. The authors thank Scott Frischell and Eileen Cole (Qualicon, Wilmington, DE, USA) for providing the RiboPrint ribotyping kits and for her help with data analysis, respectively.
References [1] Yancey Jr., R.J. (1999) Vaccines and diagnostic methods for bovine mastitis: fact and ¢ction. Adv. Vet. Med. 41, 257^273. [2] Calzolari, A., Giraudo, J.E., Rampone, H., Odierno, L., Giraudo, A.T., Frigerio, C., Bettera, S., Raspanti, C., Herna¨ndez, J., Wehbe, M., Mattea, M., Ferrari, M., Larriestra, A. and Nagel, R. (1997) Field trials of a vaccine against bovine mastitis 2. Evaluation in two commercial dairy herds. J. Dairy Sci. 80, 854^858. [3] O'Brien, C., Guidry, A., Fattom, A., Shepherd, S., Douglass, L. and Westho¡, D. (2000) Production of antibodies to Staphylococcus aureus serotypes 5, 8 and 336 using poly(DL-lactide-CO-glycolide) microspheres. J. Dairy Sci. 83, 1758^1766. [4] McKeenney, D., Pouliot, K., Wang, Y., Murthy, V., Ulrich, M., Doring, G., Lee, J.C., Goldmann, D. and Pier, G. (2000) Vaccine potential of poly-1,6 L-DN-succinylglucosamine, an immunoprotective surface polysaccharide of Staphylococcus aureus and Staphylococcus epidermidis. J. Biotechnol. 29, 37^44. [5] Go¨mez, M.I., Garc|¨a, V.E., Gherardi, M.M., Cerquetti, M.C. and Sordelli, D.O. (1998) Intramammary immunization with live-attenuated Staphylococcus aureus protects mice from experimental mastitis. FEMS Immunol. Med. Microbiol. 20, 21^27. [6] Sordelli, D.O., Cerquetti, M.C., Buzzola, F.R., Garc|¨a, V.E., Go¨mez, M.I. and Morris Hooke, A. (1998) Attenuated mutants of Staphylococcus aureus with two temperature-sensitive lesions: isolation and characterization. Microbios 94, 95^102. [7] Tenover, F.C., Arbeit, R.D., Archer, G., Biddle, J., Byrne, R., Goering, R., Hancock, G., Herbert, A., Hill, B., Hollis, R., Jarvis, W., Kreiswirth, B., Eisner, W., Maslow, J., McDougal, L., Miller, M., Mulligan, M. and Pfaller, M. (1994) Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus. J. Clin. Microbiol. 32, 407^415. [8] Fiztgerald, J.R., Hartigan, P.J., Meaney, W.J. and Smyth, C.J. (2000) Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection. J. Appl. Microbiol. 88, 1028^1037. [9] Mullarky, I.K., Su, C., Frieze, N., Park, Y.H. and Sordillo, L.M. (2001) Staphylococcus aureus agr genotypes with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect. Immun. 69, 45^51. [10] Kloos, W.E. and Bannerman, T.L. (1999) Staphylococcus and Micrococcus. In: Manual of Clinical Microbiology (Murray, P.R., Baron, E.J., Pfaller, M.A., Tenover, F.C. and Yolken, R.H., Eds.), pp. 264^ 282. ASM Press, Washington, DC. [11] Maslow, J.N., Mulligan, M.E. and Arbeit, R.D. (1993) Molecular
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F.R. Buzzola et al. / FEMS Microbiology Letters 202 (2001) 91^95
[12]
[13]
[14]
[15]
[16]
epidemiology: application of contemporary techniques to the typing of microorganisms. Clin. Infect. Dis. 17, 153^164. Nei, M. and Li, W.H. (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76, 5269^5273. Hunter, P.R. and Gaston, M.A. (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J. Clin. Microbiol. 26, 2465^2466. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H. and Swaminathan, B. (1995) Interpreting chromosomal DNA restriction patterns produced by pulse-¢eld gel electrophoresis : criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233^2239. Galdbart, J.O., Morvan, A. and El Sohl, N. (2000) Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in France from 1995 to 1997. J. Clin. Microbiol. 38, 185^190. Bruce, J. (1996) Automated system rapidly identi¢es and characterizes microorganisms in food. Food Technol. (Chicago) 50, 77^81.
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[17] Fitzgerald, J.R., Meaney, W.J., Hartigan, P.J., Smyth, C.J. and Kapur, V. (1997) Fine-structure molecular epidemiological analysis of Staphylococcus aureus recovered from cows. Epidemiol. Infect. 119, 261^269. [18] Quelle, L.S., Buzzola, F.R. and Sordelli, D.O. (2001) Phylogenetic relationship of bovine mastitis Staphylococcus aureus from America and Europe. 101th General Meeting of the American Society for Microbiology, Abstract C-407, p. 246, Orlando, FL. [19] Hooke, A.M., Bellanti, J.A. and Oeschger, M.P. (1985) Live-attenuated vaccines: new approaches for safety and e¤cacy. Lancet 8444, 1472^1474. [20] Novick, P.R. (1989) The Staphylococcus as a molecular genetic system. In: Molecular Biology of the Staphylococci (Novick, R.P., Ed.), pp. 1^37. VCH Publishers. [21] Iandolo, J.J. (2000) Genetic and physical map of the chromosome of the Staphylococcus aureus 8325. In: Gram-Positive Pathogens (Fischetti, V.A., Novick, R.P., Ferretti, J.J., Portnoy, D.A. and Rood, J.I., Eds.), pp. 317^325. ASM Press, Washington, DC.
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