Bacterial Genotype Affects the Manifestation and P
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微生物临床杂志 2005年第2期
Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, University of Helsinki
Department of Microbiology, National Public Health Institute, Helsinki, Finland
ABSTRACT
Two-hundred seventeen Staphylococcus aureus isolates from 116 dairy cows with intramammary infections were analyzed by pulsed-field gel electrophoresis to study the association between symptom severity, persistence of infection, and bacterial genotype. Among five main genotypes infecting 90% of the cows, one was associated with severe clinical symptoms but reduced persistence.
TEXT
Staphylococcus aureus is a common cause of bovine intramammary infection (IMI) worldwide. It is contagious and generally responds poorly to treatment. However, according to previous studies, virulence of this mastitis pathogen differs among strains (2, 3, 5, 11, 12). Those studies used a variety of methods of analysis based on phenotype and genotype, which makes comparisons difficult. Pulsed-field gel electrophoresis (PFGE), which is considered the best standard typing method, has been little used to investigate the possible associations between S. aureus genotypes and manifestation of bovine IMI (7, 12). Establishing a link between strain types and their pathogenity would help to improve treatment and prevention of staphylococcal mastitis. The objective of our study was to investigate the genetic relationships among S. aureus strains from bovine IMI, using PFGE, and to examine whether the manifestation and persistence of S. aureus IMI were genotype dependent.
The study material included the history, clinical data, and bacterial isolates of 116 dairy cows (134 mastitic mammary quarters) requiring veterinary care due to S. aureus IMI in 1993 to 1997 in 70 herds located in southern Finland, in the practice area of the ambulatory clinic of the University of Helsinki. Only the first IMI episode of the same cow was included. The attending veterinarians of the clinic visited the farm, recorded the clinical symptoms of the cow, and collected quarter milk samples by routine methods (4). A cell lysosome-originated indicator for udder inflammation, N-acetyl--D-glucosaminidase (NAGase) activity, was determined from the milk samples by a fluorogenic method (6). Gram-, catalase-, and coagulase-positive cocci were confirmed as S. aureus by PCR amplification of the thermonuclease (nuc) gene as described previously (1). Susceptibility to amoxicillin-clavulanate, cloxacillin, and penicillin G was tested by the disk diffusion method (9), and production of -lactamase was determined from all isolates by using nitrocefin (Becton Dickinson Microbiology Systems, Cockeysville, Md.). The isolates were preserved at –70°C in Protect Bacterial Preservers (Technical Service Consultants Limited, Heywood, United Kingdom) to await further studies.
The cows were treated for 5 days with penicillin G for IMIs caused by -lactamase-negative S. aureus strains (n = 69) or with amoxicillin-clavulanate (n = 28) or cloxacillin (n = 19) for IMIs caused by -lactamase-positive strains. The cows were revisited 2 and 4 weeks posttreatment for follow-up sampling and clinical examination. Isolated S. aureus strains were preserved for further studies. The symptoms of the cow were graded as subclinical (milk somatic cell count of >300,000/ml, no clinical symptoms), mild (visual abnormalities in milk and/or swelling or tenderness in the udder), or severe clinical, if elevated rectal temperature (>39.0°C) and other general symptoms (anorexia, decreased rumen motility, and general attitude) were also observed. An infection was considered to be persistent if the original S. aureus genotype was isolated from the follow-up milk samples.
Chromosomal DNA was prepared from the isolates and cleaved with SmaI (Boehringer, Mannheim, Germany) as described earlier (10). The DNA restriction fragments were separated according to the procedure described by Murchan et al. (8). PFGE fingerprints were stored, processed, and analyzed with Bionumerics software (version 10.1; Applied Maths, Kortrijk, Belgium) with Dice coefficients, using clustering by the unweighted pair group method with arithmetic mean. A position tolerance of 1.0 was used in the comparison of bands from different fingerprints. Genetic relatedness of the strains was defined visually as follows. Fingerprints with one to three band shifts were considered closely related and of the same pulsotype, and these were assigned a capital letter; the closely related clones of the respective pulsotype were accordingly numbered with a numeric suffix. Fingerprints with more than three band shifts were interpreted as being genetically unrelated and of a different pulsotype. The effect of pulsotype on severity of symptoms was tested by multinomial logistic regression (proportional odds model) by including time of infection (days from calving) in the model. The effects of pulsotype, time of infection, treatment (penicillin G versus other -lactam antibiotics), and severity of symptoms on persistence of infection were tested by logistic regression. The effects of pulsotype and time of infection on milk NAGase activity were tested by two-way analysis of variance, using log-transformed values for NAGase. The analyses were carried out with Stata 7.0 (Stata Statistical Software, College Station, Tex.). P values of <0.05 were considered to be significant.
The 217 PFGE-typed isolates were divided into 22 different pulsotypes (A to V). Pulsotypes A to E were representative of those strains infecting the majority of the cows (104 of 116 [90%]) in multiple herds; they were found during consecutive years and comprised two to five closely related clones (A1 to A5, B1 and B2, C1 and C2, D1 and D2, and E1 to E4). Pulsotypes F to V were sporadic, being isolated from individual cows only. Spreading of pulsotypes A, D, and E from the originally infected quarter to a previously healthy quarter in seven cows was observed. The same clone was isolated if the cow had several infected quarters (Fig. 1).
Associations between pulsotypes and IMI severity were noted here, which supported the observations of Zadoks et al. (12). Infections caused by pulsotype B were related to severe symptoms. Compared with this genotype, infection of pulsotype A decreased the odds of severity of IMI by a factor of 0.12 (95% confidence interval [CI], 0.021 to 0.70), infection of pulsotype C by a factor of 0.012 (95% CI, 0.00052 to 0.28), and infection of pulsotype E by a factor of 0.037 (95% CI, 0.0043 to 0.32). However, pulsotype B was invariably eliminated from the udder (Table 1). These IMIs may have developed quickly with obvious clinical symptoms, whereas the others progressed more slowly, often being chronic when becoming clinical. It is likely that this affected the treatment response. The response among IMIs of pulsotype A was more favorable if penicillin was used (infections caused by -lactamase-negative strains), compared with other treatments (infections caused by -lactamase-positive strains) (P < 0.05), and if the symptoms were subclinical rather than mild clinical (P < 0.05).
The symptoms of the cows were more severe (P < 0.05) and milk NAGase values were higher (P < 0.05) within 1 month from calving than in later lactation. Unlike in the study by Middleton et al. (7), inflammation of the udder, as indicated by milk NAGase, was here pulsotype dependent and it supported the associations between pulsotypes and severity of symptoms. NAGase levels of IMIs promoted by pulsotype B were significantly higher (mean, 427 to 441 U/ml; standard error [SE], 45.7 to 48.0) compared with those promoted by pulsotype C (mean, 124 to 127 U/ml; SE, 27.0 to 28.2) (P < 0.05), and they tended to be higher than those associated with pulsotypes A (mean, 307 to 317 U/ml; SE, 32.5 to 33.3) (P 0.05) and E (mean, 282 to 307 U/ml; SE, 46.0 to 54.6). The NAGase values associated with pulsotype D (mean, 420 to 433 U/ml; SE, 60.5 to 62.8) were close to those for pulsotype B.
In conclusion, our findings provide evidence of differences in virulence potential between S. aureus genotypes causing bovine IMI, but further evaluation of the virulence requires more research.
ACKNOWLEDGMENTS
We wish to thank Elina Siren and other laboratory personnel in the National Public Health Institute for skillful technical assistance. We also thank all the veterinarians and laboratory personnel of the ambulatory clinic at the Faculty of Veterinary Medicine, University of Helsinki, for collecting the study material.
The Ministry of Agriculture and Forestry and Valio, Ltd., supported this study financially.
REFERENCES
Brakstad, O. G., K. Aasbakk, and J. A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654-1660.
Buzzola, F. R., L. Quelle, M. I. Gomez, M. Catalano, L. Steele-Moore, D. Berg, E. Gentilini, G. Denamiel, and D. O. Sordelli. 2001. Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina. Epidemiol. Infect. 126:445-452.
Fitzgerald, J. R., P. J. Hartigan, W. J. Meaney, and C. J. Smyth. 2000. Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection. J. Appl. Microbiol. 88:1028-1037.
Honkanen-Buzalski, T. 1995. Sampling technique, transportation and history, p. 111-114. In M. Sandholm, T. Honkanen-Buzalski, L. Kaartinen, and S. Pyrl (ed.), The bovine udder and mastitis. Gummerus Press, Jyvskyl, Finland.
Matsunaga, T., S. Kamata, N. Kakiichi, and K. Uchida. 1993. Characteristics of Staphylococcus aureus isolated from peracute, acute and chronic bovine mastitis. J. Vet. Med. Sci. 55:297-300.
Mattila, T., and M. Sandholm. 1986. Milk plasmin, N-acetyl-beta-D-glucosaminidase, and antitrypsin as determinants of bacterial replication rates in whey. J. Dairy Sci. 69:670-675.
Middleton, J. R., L. K. Fox, J. M. Gay, J. W. Tyler, and T. E. Besser. 2002. Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl--D-glucosaminidase activity in cattle from eight dairies. J. Dairy Sci. 85:1133-1140.
Murchan, S., M. E. Kauffmann, A. Deplano, R. de Ryck, M. Struelens, C. E. Zinn, V. Fussing, S. Salmenlinna, J. Vuopio-Varkila, N. El Solh, C. Cuny, W. Witte, P. T. Tassios, N. Legakis, W. van Leeuwen, A. van Belkum, A. Vindel, I. Laconcha, J. Garaizar, S. Haeggman, B. Olsson-Liljequist, U. Ramsjo, G. Coombes, and B. Cookson. 2003. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J. Clin. Microbiol. 41:1574-1585.
National Committee for Clinical Laboratory Standards. 1993. Performance standards for antimicrobial disk susceptibility tests, 5th ed. Approved standards M2 to M5. NCCLS, Villanova, Pa.
Salmenlinna, S., O. Lyytikainen, P. Kotilainen, R. Scotford, E. Siren, and J. Vuopio-Varkila. 2000. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Finland. J. Clin. Microbiol. Eur. Infect. Dis. 19:101-107.
Smith, T. H., L. K. Fox, and J. R. Middleton. 1998. Outbreak of mastitis caused by one strain of Staphylococcus aureus in a closed dairy herd. J. Am. Vet. Med. Assoc. 212:553-556.
Zadoks, R., W. van Leeuwen, H. Barkema, O. Sampimon, H. Verbrugh, Y. H. Schukken, and A. van Belkum. 2000. Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates. J. Clin. Microbiol. 38:1931-1939.(M. Haveri, S. Taponen, J.)
Department of Microbiology, National Public Health Institute, Helsinki, Finland
ABSTRACT
Two-hundred seventeen Staphylococcus aureus isolates from 116 dairy cows with intramammary infections were analyzed by pulsed-field gel electrophoresis to study the association between symptom severity, persistence of infection, and bacterial genotype. Among five main genotypes infecting 90% of the cows, one was associated with severe clinical symptoms but reduced persistence.
TEXT
Staphylococcus aureus is a common cause of bovine intramammary infection (IMI) worldwide. It is contagious and generally responds poorly to treatment. However, according to previous studies, virulence of this mastitis pathogen differs among strains (2, 3, 5, 11, 12). Those studies used a variety of methods of analysis based on phenotype and genotype, which makes comparisons difficult. Pulsed-field gel electrophoresis (PFGE), which is considered the best standard typing method, has been little used to investigate the possible associations between S. aureus genotypes and manifestation of bovine IMI (7, 12). Establishing a link between strain types and their pathogenity would help to improve treatment and prevention of staphylococcal mastitis. The objective of our study was to investigate the genetic relationships among S. aureus strains from bovine IMI, using PFGE, and to examine whether the manifestation and persistence of S. aureus IMI were genotype dependent.
The study material included the history, clinical data, and bacterial isolates of 116 dairy cows (134 mastitic mammary quarters) requiring veterinary care due to S. aureus IMI in 1993 to 1997 in 70 herds located in southern Finland, in the practice area of the ambulatory clinic of the University of Helsinki. Only the first IMI episode of the same cow was included. The attending veterinarians of the clinic visited the farm, recorded the clinical symptoms of the cow, and collected quarter milk samples by routine methods (4). A cell lysosome-originated indicator for udder inflammation, N-acetyl--D-glucosaminidase (NAGase) activity, was determined from the milk samples by a fluorogenic method (6). Gram-, catalase-, and coagulase-positive cocci were confirmed as S. aureus by PCR amplification of the thermonuclease (nuc) gene as described previously (1). Susceptibility to amoxicillin-clavulanate, cloxacillin, and penicillin G was tested by the disk diffusion method (9), and production of -lactamase was determined from all isolates by using nitrocefin (Becton Dickinson Microbiology Systems, Cockeysville, Md.). The isolates were preserved at –70°C in Protect Bacterial Preservers (Technical Service Consultants Limited, Heywood, United Kingdom) to await further studies.
The cows were treated for 5 days with penicillin G for IMIs caused by -lactamase-negative S. aureus strains (n = 69) or with amoxicillin-clavulanate (n = 28) or cloxacillin (n = 19) for IMIs caused by -lactamase-positive strains. The cows were revisited 2 and 4 weeks posttreatment for follow-up sampling and clinical examination. Isolated S. aureus strains were preserved for further studies. The symptoms of the cow were graded as subclinical (milk somatic cell count of >300,000/ml, no clinical symptoms), mild (visual abnormalities in milk and/or swelling or tenderness in the udder), or severe clinical, if elevated rectal temperature (>39.0°C) and other general symptoms (anorexia, decreased rumen motility, and general attitude) were also observed. An infection was considered to be persistent if the original S. aureus genotype was isolated from the follow-up milk samples.
Chromosomal DNA was prepared from the isolates and cleaved with SmaI (Boehringer, Mannheim, Germany) as described earlier (10). The DNA restriction fragments were separated according to the procedure described by Murchan et al. (8). PFGE fingerprints were stored, processed, and analyzed with Bionumerics software (version 10.1; Applied Maths, Kortrijk, Belgium) with Dice coefficients, using clustering by the unweighted pair group method with arithmetic mean. A position tolerance of 1.0 was used in the comparison of bands from different fingerprints. Genetic relatedness of the strains was defined visually as follows. Fingerprints with one to three band shifts were considered closely related and of the same pulsotype, and these were assigned a capital letter; the closely related clones of the respective pulsotype were accordingly numbered with a numeric suffix. Fingerprints with more than three band shifts were interpreted as being genetically unrelated and of a different pulsotype. The effect of pulsotype on severity of symptoms was tested by multinomial logistic regression (proportional odds model) by including time of infection (days from calving) in the model. The effects of pulsotype, time of infection, treatment (penicillin G versus other -lactam antibiotics), and severity of symptoms on persistence of infection were tested by logistic regression. The effects of pulsotype and time of infection on milk NAGase activity were tested by two-way analysis of variance, using log-transformed values for NAGase. The analyses were carried out with Stata 7.0 (Stata Statistical Software, College Station, Tex.). P values of <0.05 were considered to be significant.
The 217 PFGE-typed isolates were divided into 22 different pulsotypes (A to V). Pulsotypes A to E were representative of those strains infecting the majority of the cows (104 of 116 [90%]) in multiple herds; they were found during consecutive years and comprised two to five closely related clones (A1 to A5, B1 and B2, C1 and C2, D1 and D2, and E1 to E4). Pulsotypes F to V were sporadic, being isolated from individual cows only. Spreading of pulsotypes A, D, and E from the originally infected quarter to a previously healthy quarter in seven cows was observed. The same clone was isolated if the cow had several infected quarters (Fig. 1).
Associations between pulsotypes and IMI severity were noted here, which supported the observations of Zadoks et al. (12). Infections caused by pulsotype B were related to severe symptoms. Compared with this genotype, infection of pulsotype A decreased the odds of severity of IMI by a factor of 0.12 (95% confidence interval [CI], 0.021 to 0.70), infection of pulsotype C by a factor of 0.012 (95% CI, 0.00052 to 0.28), and infection of pulsotype E by a factor of 0.037 (95% CI, 0.0043 to 0.32). However, pulsotype B was invariably eliminated from the udder (Table 1). These IMIs may have developed quickly with obvious clinical symptoms, whereas the others progressed more slowly, often being chronic when becoming clinical. It is likely that this affected the treatment response. The response among IMIs of pulsotype A was more favorable if penicillin was used (infections caused by -lactamase-negative strains), compared with other treatments (infections caused by -lactamase-positive strains) (P < 0.05), and if the symptoms were subclinical rather than mild clinical (P < 0.05).
The symptoms of the cows were more severe (P < 0.05) and milk NAGase values were higher (P < 0.05) within 1 month from calving than in later lactation. Unlike in the study by Middleton et al. (7), inflammation of the udder, as indicated by milk NAGase, was here pulsotype dependent and it supported the associations between pulsotypes and severity of symptoms. NAGase levels of IMIs promoted by pulsotype B were significantly higher (mean, 427 to 441 U/ml; standard error [SE], 45.7 to 48.0) compared with those promoted by pulsotype C (mean, 124 to 127 U/ml; SE, 27.0 to 28.2) (P < 0.05), and they tended to be higher than those associated with pulsotypes A (mean, 307 to 317 U/ml; SE, 32.5 to 33.3) (P 0.05) and E (mean, 282 to 307 U/ml; SE, 46.0 to 54.6). The NAGase values associated with pulsotype D (mean, 420 to 433 U/ml; SE, 60.5 to 62.8) were close to those for pulsotype B.
In conclusion, our findings provide evidence of differences in virulence potential between S. aureus genotypes causing bovine IMI, but further evaluation of the virulence requires more research.
ACKNOWLEDGMENTS
We wish to thank Elina Siren and other laboratory personnel in the National Public Health Institute for skillful technical assistance. We also thank all the veterinarians and laboratory personnel of the ambulatory clinic at the Faculty of Veterinary Medicine, University of Helsinki, for collecting the study material.
The Ministry of Agriculture and Forestry and Valio, Ltd., supported this study financially.
REFERENCES
Brakstad, O. G., K. Aasbakk, and J. A. Maeland. 1992. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. J. Clin. Microbiol. 30:1654-1660.
Buzzola, F. R., L. Quelle, M. I. Gomez, M. Catalano, L. Steele-Moore, D. Berg, E. Gentilini, G. Denamiel, and D. O. Sordelli. 2001. Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina. Epidemiol. Infect. 126:445-452.
Fitzgerald, J. R., P. J. Hartigan, W. J. Meaney, and C. J. Smyth. 2000. Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection. J. Appl. Microbiol. 88:1028-1037.
Honkanen-Buzalski, T. 1995. Sampling technique, transportation and history, p. 111-114. In M. Sandholm, T. Honkanen-Buzalski, L. Kaartinen, and S. Pyrl (ed.), The bovine udder and mastitis. Gummerus Press, Jyvskyl, Finland.
Matsunaga, T., S. Kamata, N. Kakiichi, and K. Uchida. 1993. Characteristics of Staphylococcus aureus isolated from peracute, acute and chronic bovine mastitis. J. Vet. Med. Sci. 55:297-300.
Mattila, T., and M. Sandholm. 1986. Milk plasmin, N-acetyl-beta-D-glucosaminidase, and antitrypsin as determinants of bacterial replication rates in whey. J. Dairy Sci. 69:670-675.
Middleton, J. R., L. K. Fox, J. M. Gay, J. W. Tyler, and T. E. Besser. 2002. Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl--D-glucosaminidase activity in cattle from eight dairies. J. Dairy Sci. 85:1133-1140.
Murchan, S., M. E. Kauffmann, A. Deplano, R. de Ryck, M. Struelens, C. E. Zinn, V. Fussing, S. Salmenlinna, J. Vuopio-Varkila, N. El Solh, C. Cuny, W. Witte, P. T. Tassios, N. Legakis, W. van Leeuwen, A. van Belkum, A. Vindel, I. Laconcha, J. Garaizar, S. Haeggman, B. Olsson-Liljequist, U. Ramsjo, G. Coombes, and B. Cookson. 2003. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J. Clin. Microbiol. 41:1574-1585.
National Committee for Clinical Laboratory Standards. 1993. Performance standards for antimicrobial disk susceptibility tests, 5th ed. Approved standards M2 to M5. NCCLS, Villanova, Pa.
Salmenlinna, S., O. Lyytikainen, P. Kotilainen, R. Scotford, E. Siren, and J. Vuopio-Varkila. 2000. Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Finland. J. Clin. Microbiol. Eur. Infect. Dis. 19:101-107.
Smith, T. H., L. K. Fox, and J. R. Middleton. 1998. Outbreak of mastitis caused by one strain of Staphylococcus aureus in a closed dairy herd. J. Am. Vet. Med. Assoc. 212:553-556.
Zadoks, R., W. van Leeuwen, H. Barkema, O. Sampimon, H. Verbrugh, Y. H. Schukken, and A. van Belkum. 2000. Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates. J. Clin. Microbiol. 38:1931-1939.(M. Haveri, S. Taponen, J.)