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Journal of Clinical Microbiology, September 2004, p. 4214-4222, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4214-4222.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Phenotypic and Molecular Characteristics of Streptococcus agalactiae Isolates Recovered from Milk of Dairy Cows in Brazil

Rafael S. Duarte,1 Otávio P. Miranda,1 Bruna C. Bellei,1 Maria Aparecida V. P. Brito,2 and Lúcia M. Teixeira1*

Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro,1 Empresa Brasileira de Agropecuária (Embrapa), Juiz de Fora, Minas Gerais, Brazil2

Received 23 December 2003/ Returned for modification 15 February 2004/ Accepted 31 May 2004


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ABSTRACT
 
Information on the characteristics of Streptococcus agalactiae obtained from bovine sources in Brazil is still very limited. The aim of this study was to assess the phenotypic and genotypic diversity among S. agalactiae isolates from milk of dairy cows presenting clinical or subclinical mastitis in the southeast region of Brazil. Phenotypic characterization was based on physiological and serological tests. Antimicrobial susceptibility tests were carried out by the disk method. Genetic diversity was evaluated by using random amplified polymorphic DNA-PCR (RAPD-PCR) (by using the primer 1254) and pulsed-field gel electrophoresis (PFGE) (by using SmaI as the restriction enzyme) and by PCRs for detection of genes associated with resistance to erythromycin and tetracycline as well as PCRs for detection of genes coding for cell surface-associated proteins. According to the results of physiologic tests, 45 (52.9%) isolates showed beta-hemolysis and 44 (51.7%) were susceptible to bacitracin. Fourteen different biotypes were detected. The two most frequent biotypes comprised strains that were non-beta-hemolytic; fermented galactose, lactose, and salicin; produced protease; and were negative for DNase production. Serotype III was predominant (66 isolates [77.6%]), followed by serotypes II, Ia, Ib, and VI. Resistance to tetracycline and erythromycin was found in 38 (44.7%) and 9 (10.5%) isolates, respectively, with tet(O) (31.7%) and erm(B) (100%) being the most frequently occurring resistance genes. Three genes coding for surface proteins, bca, lmb, and scpB, were detected in 55 (64.7%), 7 (8.2%), and 43 (50.5%) isolates, respectively. In most cases, isolates from animals in the same herd presented closely related genetic profiles (determined by either RAPD-PCR or PFGE), which were distinct from those of isolates from different herds.


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INTRODUCTION
 
Infectious mastitis is an important health problem affecting dairy cattle and constitutes a source of economic loss for the dairy industry due to the effects on milk quality and yield. Additionally, the impact of this disease extends to the financial cost of therapeutic strategies and laboratory and veterinary services involved in control programs (15, 16). The availability of information about the prevalence and the biological and epidemiological characteristics of the etiologic agents is essential for developing appropriate prevention programs and successful therapy (16, 20).

Streptococcus agalactiae, or group B Streptococcus (GBS), is a well-recognized worldwide etiological agent of mastitis in bovines, causing both clinical and mild subclinical mastitis of long duration (10, 20). Bacterial cells are shed in milk from infected quarters, and transmission to uninfected quarters and cows usually occurs during the milking period (15, 21).

The prevalence of GBS in cattle has been investigated in many areas, and the highest frequencies have been found in regions where appropriate control measures have not been implemented (20). In Brazil, studies have been developed by Empresa Brasileira de Agropecuária (Juiz de Fora city, Minas Gerais [MG] state) to investigate the frequency of bacterial agents of mastitis in regional dairy farms (3, 4). GBS was identified as one of the most frequent agents, being recovered from 60% of the dairy herds evaluated. Additional investigation, including the characterization of biological properties and antimicrobial susceptibility profiles of GBS isolates from bovine sources in that country, is still needed to evaluate the occurrence of antibiotic resistance and prevalent types. Such data can help in substituting the empirical therapeutic methods commonly applied in Brazil, which are often based on information gathered from other geographic areas, for more adequate control measures. Nevertheless, information on the molecular characteristics of GBS strains isolated from bovines in different regions is still limited, hindering a global understanding of the epidemiology of GBS infections. Studies exploring genomic traits of Brazilian isolates have not been reported.

The purpose of the present work was to investigate the phenotypic and genotypic characteristics of S. agalactiae isolates previously obtained from milk of dairy cows presenting clinical or subclinical mastitis in herds located in an area that comprises three major Brazilian states in the southeast region of the country.


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MATERIALS AND METHODS
 
Bacterial isolates. A total of 85 GBS isolates previously recovered from milk of bovines with clinical and subclinical mastitis were included in the present study. They were retrieved from a collection of isolates recovered from milk of lactating cows belonging to dairy herds distributed in an area that includes locations of three major states in the southeast region of Brazil (72 isolates from 18 herds located in the MG state, 7 isolates from a herd in the São Paulo state, and 6 from a herd in the Rio de Janeiro state). Most isolates were recovered during 1998 (14 isolates), 1999 (50 isolates), and 2000 (12 isolates). The remaining nine isolates were obtained during the period of 1995 to 1997. The isolates were identified on the basis of conventional methods, including colony morphology and hemolytic activity on sheep blood agar medium, Gram staining characteristics, catalase reaction, CAMP test, hippurate and esculin hydrolysis, susceptibility to bacitracin, leucine aminopeptidase and pyrrolidonyl arylamidase activities testing, and reactivity with Lancefield group B-specific antiserum (12). They were maintained as suspensions in 10% skim milk solution (Difco Laboratories, Detroit, Mich.) containing 10% glycerol at –20°C. For testing, the isolates were grown on Trypticase soy agar (BBL Microbiology Systems; Cockeysville, Md.) supplemented with 5% sheep blood at 37°C for 18 to 24 h.

Phenotypic characterization. The isolates were submitted to additional phenotypic characterization based on tests for production of acids from galactose, lactose, and salicin (12) and production of DNase and protease (25). Serological typing (serotypes Ia to VI) was performed by a coagglutination method with reagents prepared according to the recommendations of Christensen et al. (6). Typing antisera were kindly provided by R. R. Facklam, Streptococcus Laboratory, Centers for Disease Control and Prevention, Atlanta, Ga. The capillary precipitation method was used to serotype autoagglutinable isolates (12).

Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed by using the agar diffusion method according to NCCLS guidelines (26, 27). Disks containing ampicillin (10 µg), cefotaxime (30 µg), clindamycin (2 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (10 µg), levofloxacin (5 µg), rifampin (5 µg), tetracycline (30 µg), and trimethoprim-sulfamethoxazole (25 µg) were obtained from CECON (São Paulo, Brazil). Macrolide resistance phenotypes were determined by the double-disk test with erythromycin and clindamycin disks as previously described (30).

Detection of erythromycin and tetracycline resistance genes. Erythromycin-resistant and tetracycline-resistant GBS isolates were evaluated for the presence of the respective genetic determinants by single PCRs. Preparation of DNA extracts was based on a previously described method (10). With that method, 10 to 20 colonies of a fresh culture were suspended in 50 µl of distilled water and boiled for 5 min. For detection of erythromycin resistance genes, the following sets of primers (synthesized by Operon Technologies Inc., Alameda, Calif.) were used: 5'-GCA TGA CAT AAA CCT TCA-3' and 5'-AGG TTA TAA TGA AAC AGA-3', designed to amplify the erm(A) (previously named ermTR [31]) gene; 5'-GAA AAG GTA CTC AAC CAA ATA-3' and 5'-AGT AAC GGT ACT TAA ATT GTT TAC-3' for erm(B) (32); and 5'-AGT ATC ATT AAT CAC TAG TGC-3' and 5'-TTC TTC TGG TAC TAA AAG TGG-3' for mef(A) (32). The reaction mixtures, in final volumes of 50 µl, contained MgCl2 [2 mM for the erm(A) and erm(B) genes and 4 mM for the mef(A) gene], deoxynucleoside triphosphate (0.2 mM each), primers (0.5 µM each), Taq DNA polymerase (0.5 U), reaction buffer (10 mM), and 1 µl of DNA extract used as a template. Reaction chemicals were all from Boehringer Mannheim Corporation (Indianapolis, Ind.). Amplifications were performed by using a GeneAmp PCR System 2400 thermocycler (Perkin-Elmer Applied Biosystems, Branchburg, N.J.). Conditions used to detect the erm(A), erm(B), and mef(A) genes were as follows: initial denaturation step at 93°C for 3 min followed by 35 cycles of denaturation at 93°C for 1 min, primer annealing at 52°C for 1 min, and extension at 72°C for 1 min and a final elongation step at 72°C for 5 min (32). PCR products were resolved by electrophoresis on 1.2% agarose gels in 0.5x TBE buffer (1 mM Tris-0.01 M EDTA-1 M boric acid). Gels were stained with ethidium bromide and then visualized and photographed under UV light. The size of each PCR product was estimated by using standard molecular weight markers (100-bp ladder; Pharmacia Biotech, Uppsala, Sweden). Detection of genes tet(K), tet(L), tet(M), and tet(O) was performed according to the method of Trzcinski et al. (35). Briefly, each reaction was carried out in 50 µl of a mixture containing 20 mM Tris-HCl (pH 8.4) and 50 mM KCl, 3 mM MgCl2, 0.2 mM each nucleotide, 0.5 µM each primer, 2.5 U of Taq DNA polymerase, and 5 µl of DNA template. Primers used to amplify the tet determinants were 5'-TAT TTT GGC TTT GTA TTC TTT CAT-3' and 5'-GCT ATA CCT GTT CCC TCT GAT AA-3' for tet(K), 5'-ATA AAT TGT TTC GGG TCG GTA AT-3' and 5'-AAC CAG CCA ACT AAT GAC AAT GAT-3' for tet(L), 5'-AGT TTT AGC TCA TGT TGA TG-3' and 5'-TCC GCA TAT TTA GAC GAC GG-3'for tet(M), and 5'-AGC GTC AAA GGG GAA TCA CTA TCC-3' and 5'-CGG CGG GGT TGG CAA ATA-3' for tet(O). The PCR conditions consisted of 35 cycles of 1 min at 95°C, 1 min at 50°C, and 1 min 30 s at 72°C, followed by a final 5-min step at 72°C, except for tet(O), for which the annealing temperature was 55°C. PCR product detection was performed as indicated above.

Detection of cell surface protein genes. The presence of genes encoding surface proteins potentially associated with virulence of GBS strains was evaluated by PCR. Genomic DNA was prepared as described above, and 5-µl volumes of the supernatants were used for PCRs as previously described (10). The following primers (produced by Invitrogen Life Technologies, São Paulo, Brazil) were used for the detection of genes encoding the immunoglobulin A-binding ß-antigen (bac), the {alpha}-antigen (bca), the laminin-binding surface protein (lmb), and the C5a peptidase (scpB): 5'-TGT AAA GGA CGA TAG TGT GAA GAC-3' and 5'-CAT TTG TGA TTC CCT TTT GC-3' for bac (10), 5'-CAG GAG GGG AAA CAA CAG TAC-3' and 5'-GTA TCC TTT GAT CCA TCT GGA TAC G-3' for bca (11), 5'-GAC GCA ACA CAC GGC AT-3' and 5'-TGA TAG AGC ACT TCC AAA TTT G-3' for lmb (10), and 5'-ACA ATG GAA GGC TCT ACT GTT C-3' and 5'-ACC TGG TGT TTG ACC TGA ACT A-3' for scpB (10). PCR conditions consisted of an initial denaturation step at 94°C for 2 min followed by 30 cycles of denaturation (94°C, 30s), primer annealing (50°C, 1 min), and extension (72°C, 1 min). Electrophoresis and visualization of PCR products were performed as described above.

Analysis of DNA amplification products obtained by RAPD-PCR. Random amplified polymorphic DNA-PCR (RAPD-PCR) assays were carried out with primer 1254 (5'-CCGCAGCCAA-3'; synthesized by Gibco BRL, Gaithersburg, Md.) (1). Preparation of DNA extracts was based on the recommendations of Pacheco et al. (28). Briefly, 5 to 10 colonies of GBS were inoculated in 3 ml of Trypticase soy broth and incubated at 37°C for 18 to 24 h. A volume (200 µl) of a bacterial suspension with an optical density of 0.4 (at 600 nm) was centrifuged, and the sediment was suspended in 900 µl of Tris-EDTA buffer. Bacterial suspensions were boiled at 100°C for 10 min. After centrifugation, the supernatants were used for PCR testing. PCRs were performed in 30-µl final reaction volumes containing 5 µl of bacterial lysate, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 250 µM (each) deoxynucleoside triphosphate, 30 pmol of primer, and 1 U of Taq polymerase. The PCR conditions consisted of 4 cycles of 94°C for 5 min, 37°C for 5 min, and 72°C for 5 min followed by 30 cycles of 94°C for 1 min, 37°C for 1 min, and 72°C for 2 min and a final extension step at 72°C for 10 min. Reaction products were analyzed as described above. The RAPD amplification profiles were initially compared by visual inspection. Densitometric analysis, normalization of the densitometric traces, and interpolation of the profiles with the Dice coefficient and the unweighted pair group method with arithmetic averages were performed by using the Molecular Analyst Fingerprinting Plus software package, version 1.6, of the Image Analysis System (Bio-Rad Laboratories, Hercules, Calif.). Strains showing 80% similarity or higher were assumed to belong to the same clonal group. Clonal groups composed by three or more isolates were named by alphabetical letters.

Analysis of the chromosomal DNA restriction profiles by PFGE. Chromosomal DNA was extracted in agarose plugs, treated with SmaI restriction endonuclease, and analyzed by pulsed-field gel electrophoresis (PFGE) as previously described (33), with a few modifications. Isolates were grown on sheep blood agar plates for 18 to 24 h at 37°C. The fragments were separated by PFGE in 1.2% agarose gels in a CHEF-DRIII system (Bio-Rad) with pulse times increasing from 2 to 30 s over 22.5 h at 11°C at a voltage gradient of 6 V/cm. The gels were stained with ethidium bromide and then photographed under UV light. Analysis of SmaI restriction profiles was performed by visual inspection, as suggested by Tenover et al. (34), followed by computer-assisted analysis performed as describe above for analysis of the RAPD-PCR profiles.

Discriminatory power of molecular typing techniques. The discriminatory power of RAPD and PFGE techniques for typing S. agalactiae strains was evaluated by using an index of discrimination as described by Hunter and Gaston (17) given by the equation D = 1 – [1/N (N – 1)] {Sigma} nj (nj – 1), where D is the numerical index of discrimination, N is the total number of isolates, and nj is the number of isolates belonging to the jth type. The discrimination indexes were obtained considering the comparison of the profiles by visual inspection and automated analysis.


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RESULTS
 
Phenotypic characteristics of the isolates. The 85 streptococcal isolates identified as S. agalactiae showed typical positive results in the CAMP tests as well as in the hippurate tests and negative results in esculin hydrolysis tests. They all reacted with Lancefield's serological group B antiserum. However, 45 (52.9%) isolates presented beta-hemolytic activity and 41 (48.2%) were resistant to bacitracin. All the isolates produced acids from galactose, and all isolates (except for one) produced acids from lactose. Production of acids from salicin was observed in 65 (76.5%) isolates. Except for one, all the isolates had protease activity and 31 (36.5%) showed DNase activity.

On the basis of the physiological characteristics tested, the GBS isolates were classified in biotypes as shown in Table 1. Fourteen different biotypes were detected. Biotype 1 was the most frequent, comprising 19 (22.3%) isolates that were susceptible to bacitracin; were non-beta-hemolytic; fermented galactose, lactose, and salicin; produced protease, and were negative for DNase production. The second most frequent biotype (biotype 2) included 14 (16.4%) isolates that differed from biotype 1 in relation to bacitracin susceptibility. Most of the isolates belonging to biotypes 1 and 2 were obtained from herds in two locations (Jaguará [JAG] and Juiz de For a [JUF]) of the MG state in different years.


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  		TABLE 1. Distribution of biotypes among S. agalactiae isolates recovered from bovines in Brazil

Serotype III was the predominant (66 isolates [77.6%]), followed by serotypes II (10 isolates [11.8%]), Ia (5 isolates [5.9%]), Ib (2 isolates [2.35%]), and VI (2 isolates [2.35%]). Protein C was detected in 24 (28.2%) isolates belonging to serotype II (3 isolates) or III (21 isolates). Serotype III was found to be the only or the predominant serotype in 15 (75%) of the 20 herds investigated. All the isolates from herds located in Campo Belo (CPB, MG state; 7 isolates), JAG (MG state; 21 isolates), and São João da Boa Vista (SJV, São Paulo state; 7 isolates) were identified as serotype III. Biotype distribution varied among the different serotypes, except for the five serotype Ia isolates that were all included in biotype 4.

Antimicrobial susceptibility. All the S. agalactiae isolates were resistant to gentamicin, and most of them (46 isolates [54.1%]) were susceptible to the other nine antimicrobials tested. Resistance to tetracycline and erythromycin was detected in 38 (44.7%) and 9 (10.5%) isolates, respectively. All the nine erythromycin-resistant isolates presented the constitutive phenotype (cMLSB), also conferring resistance to clindamycin, and were concomitantly resistant to tetracycline. Resistance to trimethoprim-sulfamethoxazole was found in one isolate (CL-5598) that was susceptible to tetracycline and erythromycin.

Resistance to tetracycline was found among all the isolates belonging to serotypes Ia, Ib, and VI and among the majority (9 out of 10) of serotype II isolates. In contrast, only 18 (27.3%) serotype III isolates were resistant to tetracycline. Erythromycin resistance was found in 5 (50%) of the 10 serotype II isolates and in only 4 (6%) of the 66 serotype III isolates.

Detection of antimicrobial resistance genes. PCR testing for detection of erythromycin resistance genes revealed that six out of the nine erythromycin-resistant isolates presented both the erm(A) and erm(B) genes, while the remaining three erythromycin-resistant isolates had the erm(B) gene only. Among the 38 tetracycline-resistant isolates, tet(O) was the most frequently found determinant (found in 27 isolates [71%]), followed by tet(M) (16 isolates [42.1%]) and tet(L) (3 isolates [7.8%]). Gene tet(K) was not detected. Seven (18.2%) isolates harboring tet(O) also presented tet(M), while two tet(O)-positive isolates also possessed the tet(L) gene simultaneously. Only one tetracycline-resistant isolate did not harbor any of the tet genes tested.

Detection of surface protein genes. Tests for the detection of surface protein genes indicated that 55 (64.7%) isolates harbored the bca gene, 43 (50.6%) contained the scpB gene, and 7 (8.2%) possessed the lmb gene. The bac gene was not detected. The distribution of surface protein gene profiles among S. agalactiae bovine isolates is shown in Table 2. The most common surface protein profiles were profile F, comprising 27 (31.7%) isolates that were positive for the bca and scpB genes and negative for the bac and lmb genes, and profile G, comprising 22 isolates that were positive for bca and negative for the other three genes tested. Additionally, a significant number of isolates (19 isolates [22.3%]) did not possess any of the surface protein genes investigated.


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  		TABLE 2. Occurrence of genetic determinants encoding cell surface-associated proteins among S. agalactiae isolated from bovines in Brazil

Analysis of RAPD-PCR and PFGE profiles. RAPD-PCR fingerprinting with the primer 1254 generated reproducible amplification profiles comprising 7 to 10 bands (Fig. 1A). The relationships among RAPD-PCR profiles of the 85 GBS isolates are represented in the dendrogram obtained with the Dice coefficient as shown in Fig. 2. The 85 S. agalactiae isolates were divided in 58 amplification profiles with percentages of similarity ranging from approximately 40 to 100%. The distribution of serotypes, biotypes, antimicrobial resistance genes, surface protein gene profiles, and localization and period of isolation among the isolates are also indicated in Fig. 2. One major clonal group (named as clonal group A) was identified, comprising 20 serotype III isolates with identical or highly related profiles recovered in two herds located in the MG state (19 isolates recovered in the JAG herd during 1998 and 1999 and 1 isolate recovered in the Coronel Pacheco [COP] herd). Several of the major clonal groups included isolates originating from the same herd or region and with similar characteristics (Fig. 2). In some cases, however, isolates belonging to different serotypes or biotypes had identical or highly related profiles and were included in the same clonal group.



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  		FIG. 1. Representative genomic profiles identified among S. agalactiae isolates recovered from bovines in Brazil. (A) RAPD-PCR profiles. Lanes 1 and 12, molecular size markers (in base pairs; DNA ladder ranging from 100 to 1,000 bp); lane 2, isolate CL-5594 (serotype II; RAPD clonal group G; Santos Dumont [SAD] herd, 1996); lane 3, isolate CL-5601 (serotype III; RAPD clonal group not nominated; Matozinho [MAT] herd, 1998); lane 4, isolate CL-5637 (serotype III; RAPD clonal group I; CPB herd, 1999); lane 5, isolate CL-5618 (serotype III; RAPD clonal group A; JAG herd, 1999); lane 6, isolate CL-5608 (serotype III; RAPD clonal group A; JAG herd, 1998); lane 7, isolate CL-5612 (serotype III, RAPD clonal group A; JAG herd, 1998); lane 8, isolate CL-5657 (serotype III; RAPD clonal group B; JUF herd, 1999); lane 9, isolate CL-5598 (serotype III; RAPD clonal group E; CPB herd, 1997); lane 10, isolate CL-5664 (serotype III; RAPD clonal group not nominated; CPB herd, 1999); lane 11, isolate CL-5676 (serotype III; RAPD clonal group F; Visconde do Rio Branco [VRB] herd, 2000). (B) PFGE profiles of SmaI-digested DNA. Lane 1, molecular size markers (in kilobases; lambda DNA concatemers ranging from 48.5 to 1,018.5 kb); lane 2, isolate CL-5648 (serotype Ia; PFGE clonal group C; Carmo de Paranaíba [CAP] herd, 1999); lane 3, isolate CL-5669 (serotype Ia; PFGE clonal group not nominated; Propriedade Correia e Silva [PCS] herd, 2000); lane 4, isolate CL-5663 (serotype Ib; PFGE clonal group B; JUF herd, 1999); lane 5, isolate CL-5665 (serotype Ib; PFGE clonal group not nominated; Propriedade Maria Ratto [PMR] herd, 2000); lane 6, isolate CL-5651 (serotype II; PFGE clonal group not nominated; Dona Euzébia [DEU] herd, 1999); lane 7, isolate CL-5660 (serotype II; PFGE clonal group B; JUF herd, 1999); lane 8, isolate CL-5644 (serotype III; PFGE clonal group not nominated; COP herd, 1999); lane 9, isolate CL-5608 (serotype III; PFGE clonal group A; JAG herd, 1998); lane 10, isolate CL-5604 (serotype VI; PFGE clonal group D; Barra Mansa [BAM] herd, 1998). Only clonal groups represented by three or more isolates were named by capital alphabetical letters.



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  		FIG. 2. Dendrogram resulting from a computer-assisted analysis of the RAPD-PCR profiles of S. agalactiae isolates recovered from bovines in Brazil. The Dice coefficient and a tolerance of 1.5% were used for calculating the similarities and clustering among the profiles. Location refers to the herd and the state where the isolates originated. RJ, Rio de Janeiro; SP, São Paulo. Asterisks indicate isolates that were not restricted by SmaI in PFGE experiments. –/– indicates the absence of resistance genes.

PFGE analysis revealed 41 SmaI digestion profiles among 64 isolates. Representative PFGE profiles are shown in Fig. 1B. SmaI digestion was not achieved for a significant proportion of the isolates (21 isolates [24.7%]) that did not display PFGE profiles by the method used. Similarly to the RAPD results, a few major clonal groups comprising isolates with higher levels of similarity were observed. Most of isolates from the same herd had PFGE profiles more closely related to each other than to those of isolates obtained from animals in distinct herds, with a few exceptions (Fig. 3). In general, isolates with a large number of similar characteristics were included in the same clonal group. However, in some cases, isolates from animals in the same herd were highly related despite differences in phenotypic characteristics or carriage of antimicrobial and surface protein genes. Although the structure of the dendrogram derived from analysis of PFGE profiles was slightly different from that obtained by analysis of RAPD-PCR profiles, in most cases, the isolates composing the major clonal groups were the same.



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  		FIG. 3. Dendrogram resulting from a computer-assisted analysis of the PFGE profiles of S. agalactiae isolates recovered from bovines in Brazil. The Dice coefficient and a tolerance of 1.5% were used for calculating the similarities and clustering among the profiles. Location refers to herd and the state where the isolates originated. RJ, Rio de Janeiro state. –/– indicates the absence of resistance genes.

The discrimination indexes obtained for the two molecular techniques used were 0.961 (for RAPD-PCR) and 0.960 (for PFGE).


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DISCUSSION
 
S. agalactiae represents one of the most important causes of mastitis in bovines throughout the world (15, 20). In Brazil, data on prevalence rates of mastitis due to GBS, as well as the biological characteristics of S. agalactiae isolates, are still scarce. In the present study, we investigated the phenotypic and genotypic properties for a collection of GBS isolates from bovine milk in herds of the southeast region of Brazil, a highly populated area where many of the most important dairy farms and dairy industries in the country are located.

Approximately half of the bovine GBS isolates included in the study did not present phenotypic characteristics that are common among GBS isolates from other sources, particularly from humans, such as beta-hemolytic activity and resistance to bacitracin. These aspects support the hypotheses of a bovine-adapted ecovar of S. agalactiae compared to strains from other hosts (9). Similar results were previously found when isolates from bovine milk were investigated in Kenya, New Zealand, and North America (14, 23, 24, 36). In accordance with earlier observations (14, 24), production of acids from galactose, lactose, and salicin was a trait found frequently among bovine strains. Protease activity, as detected by casein hydrolysis, was also found to be a usual characteristic of GBS from bovine milk. This physiological property, as well as galactose fermentation, may be essential for the acquisition of nutrients through milk degradation, leading to survival of bacteria inside the udder.

Serotype III was the most frequently occurring serotype among GBS isolates recovered from milk of the dairy cow population investigated. The high frequency of serotype III among isolates from bovine sources was also previously observed in Canadian herds (8, 22). In contrast, serotypes Ia and II were observed to be the prevalent types in Kenya and New Zealand, respectively (14, 24). These findings indicate that serotype distribution can vary according to geographical region and that it may be an important characteristic to be considered for the development of vaccine strategies, although GBS spread and infection can be eradicated by appropriate herd management and antimicrobial treatment, which has been the most widely used method for prevention.

Over the last decade, a number of reports on the antimicrobial susceptibility of GBS from veterinary sources have been published (5, 16, 29). Data on antimicrobial susceptibility are valuable for monitoring the emergence of resistance traits and for guiding the selection of a more judicious and effective therapy. In the present work, resistance to tetracycline (44.7%) and erythromycin (10.5%) was observed in considerable proportion among GBS isolates from bovine milk. These frequencies were higher than those recently detected in French herds (16). Genes tet(O) and tet(M) were identified as the most frequent among tetracycline-resistant isolates, similar to the results obtained in several states in the United States (5) and in Germany (29). Erythromycin resistance in the bacterial population investigated was found to be related to the presence of the erm(B) gene alone (in three isolates) or in association with the erm(A) gene (in six isolates). The presence of both the erm(A) and erm(B) genes was identified in an isolate (CL-5592) collected as far back as 1995. The significant prevalence of GBS isolates resistant to tetracycline and erythromycin indicates the limitation of the therapeutic use of these antimicrobials for the treatment of infected cows and suggests that measures of control and prevention, including the control of the use of antimicrobials, may not have been effectively applied. On the other hand, the homogeneous susceptibility to ampicillin corroborates the concept that ß-lactams still constitute good prophylactic and therapeutic options since resistance to these antimicrobial agents has not emerged in S. agalactiae strains.

The distribution of genes encoding cell surface proteins potentially associated with virulence was in general accordance with previous findings (10, 11). A significant number (22.3%) of the isolates did not harbor any of the surface protein genes evaluated, which may indicate that the proteins codified by these genetic elements may not be essential for the development of mastitis caused by GBS. Other factors related to the bacteria and/or host may be involved in the pathogenesis of infections caused by this microorganism in the bovine host.

Except for the finding that resistance to tetracycline or erythromycin was predominantly observed among isolates belonging to serotypes other than the most frequently occurring one (serotype III), no other apparent association was identified among phenotypic traits, such as biotype and serotype, and the molecular characteristics investigated.

In addition to the characterization of the genetic determinants associated with important biological markers, such as those involving resistance to antimicrobials and surface protein properties, we also investigated the genetic diversity of the isolates by using two different molecular typing systems that evaluate overall DNA polymorphism. One system (PFGE using SmaI) is now considered a "gold standard" technique for typing several bacterial pathogens including the streptococci associated with bovine mastitis (2, 23). The other system (RAPD-PCR using primer 1254) has been used for typing different microorganisms (1, 28) but has not previously been applied to type GBS isolates. Different RAPD-PCR approaches with a variety of primers have been used for typing GBS recovered from bovine or human sources (7, 22).

Both RAPD-PCR and PFGE typing systems showed a good discriminatory power (as indicated by discriminatory indexes of >0.95), although SmaI DNA restriction profiles could not be obtained for all isolates. The SmaI unrestricted isolates included all seven isolates from SJV (in the São Paulo state) and all seven isolates from CPB (in the MG state). This was unexpected because we have obtained enzyme restriction for other isolates with identical biotypes and serotypes and also because we have not experienced difficulties with SmaI restriction during an ongoing study involving a large number of GBS isolates from human sources. On the other hand, the occurrence of failures with PFGE typing of GBS isolates has already been documented in previous reports, frequently associated with isolates obtained from the same herd and harboring similar phenotypic and genotypic characteristics (13, 23). Methylation of the cutting sequences seems to be related to the isolates showing unrestricted PFGE profiles. The fact that difficulties with SmaI restriction in the present study were observed only among GBS isolates recovered from a few herds, predominantly from two of them, additionally suggests that such an occurrence may be related to some peculiar characteristic of the isolates.

Our observations reinforce the importance of using more than a single molecular technique as the basis for assessment of the genetic relationship among isolates, given that isolates that are concordantly grouped into similar types by different systems are increasingly more likely to be highly related. This was of particular significance in relation to interpretation of RAPD-PCR profiling, since criteria for the interpretation of results obtained by the use of this technique have not been proposed. The results obtained with the method used in the present investigation were in good agreement with those of the gold standard technique (PFGE) when a cutoff of 80% similarity among profiles was applied. Therefore, the results indicate that the PCR-based technique used is a reliable alternative method to type GBS of bovine origin; it allowed typing of all the isolates and showed high reproducibility, since isolates tested at least two times showed identical profiles and were assigned to the same type upon repeat testing (data not shown).

Analysis of both the RAPD-PCR and PFGE profiles and of the resulting dendrograms showed that isolates obtained from the same herd presented identical or highly related DNA profiles and composed the same groups of similarity, with a few exceptions. This observation was consistent even when isolates were obtained in different years. Otherwise, for most cases, different profiles were observed when isolates recovered from the various dairy farms were compared, suggesting no epidemiological relationship. The relatively low degree of variation between DNA profiles of GBS isolates within each farm for years indicates the presence of one or only a few strains infecting each herd. Similar results were found among GBS isolates from Australia (2) and Germany (23). These aspects are also in accordance with the concept of direct transmission of an obligate parasite of the bovine udder from animal to animal in a closed herd at milking. The use of these DNA-based techniques is likely to allow detection of significant epidemiological features, including virulent clones.

The detection of groups of higher genetic similarity composed by isolates harboring different type antigens may be related to recombination events that do not significantly affect the evolutionary relationship among the isolates. The occurrence of isolates belonging to different serotypes but showing high levels of genetic relatedness has already been described (18, 19, 22). These findings indicate that although valuable, classification based on serological traits, largely used for epidemiological investigations to date, may not reflect the genetic relatedness of bovine GBS isolates. Therefore, epidemiological studies of bovine GBS will greatly benefit from the discriminatory power of molecular techniques, which is higher than that of phenotypic typing methods.

In summary, the collection of bovine GBS included in the present study was heterogeneous in regard to phenotypic characteristics and the presence of antimicrobial resistance and surface protein genetic markers. Although a few major clonal groups composed by highly related isolates were observed, molecular typing by either RAPD-PCR or PFGE revealed a variety of profiles, reflecting the substantial genetic diversity among GBS isolates recovered from bovines. It should be pointed out, however, that multiple GBS isolates obtained from the same herd on different occasions were included in this study, and in most of these cases, they were shown to constitute the same strain due the higher degree of similarity than the isolates that originated from a distinct herd. Their characteristics are presented individually to show that differences in a variety of phenotypic and genotypic traits can be detected even among highly related isolates belonging to a given GBS strain. We conclude that mastitis caused by GBS in the different Brazilian herds investigated is due to the dissemination of multiple strains within the bacterial population rather than to the epidemic spread of a single strain. On the other hand, within each particular herd, there was a tendency for strains to spread and the persistence of isolates sharing several characteristics and belonging to the same clonal group. To our knowledge, this is the first report to describe phenotypic and genotypic characteristics, as well as the antimicrobial resistance and the respective genetic determinants, of S. agalactiae isolates from milk of dairy cows in Brazil. Detailed genetic analysis of GBS strains isolated in other areas of the country and around the world may contribute to a better understanding of the biological diversity and epidemiological aspects involved in the transmission of GBS diseases from a regional and global perspective.


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ACKNOWLEDGMENTS
 
This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Ministério da Ciência e Tecnologia (MCT/PRONEX), Brazil.

We thank Carlos Ausberto B. de Souza and Filomena Soares Pereira da Rocha for technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil. Phone: 55 21 2260 4193. Fax: 55 21 2560 8344. E-mail: lmt2{at}micro.ufrj.br. Back


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Journal of Clinical Microbiology, September 2004, p. 4214-4222, Vol. 42, No. 9
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.9.4214-4222.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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