Distribution of Antimicrobial Resistance and Virulence-Related Genes ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Jan. 2005, p. 97–103 0066-4804/05/$08.00⫹0 doi:10.1128/AAC.49.1.97–103.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 49, No. 1

Distribution of Antimicrobial Resistance and Virulence-Related Genes among Brazilian Group B Streptococci Recovered from Bovine and Human Sources Rafael S. Duarte,1 Bruna C. Bellei,1 Ota´vio P. Miranda,1 Maria A. V. P. Brito,2 and Lu ćia M. Teixeira1* Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro,1 and Empresa Brasileira de Agropecua ´ria (Embrapa), Juiz de Fora, Minas Gerais,2 Brazil Received 25 February 2004/Returned for modification 8 May 2004/Accepted 19 September 2004

In the present report we describe the characteristics of 189 antimicrobial-resistant Streptococcus agalactiae isolates from bovine (38 isolates) and human (151 isolates) sources. All the strains were resistant to tetracycline (TET), and 16 (8.5%) were also resistant to erythromycin, corresponding to 23.7% of the TET-resistant bovine isolates and 4.6% of the TET-resistant human isolates. The tet(O), erm(B), and mreA resistance-related genes, as well as the bca and scpB virulence-related genes, were the most frequent among the bovine isolates, while the tet(M), erm(A), mreA, bca, lmb, and scpB genes were the most prevalent among the isolates from humans. Although a few major clusters were observed, pulsed-field gel electrophoresis results revealed a variety of profiles, reflecting the substantial genetic diversity among strains of this species isolated from either humans or bovines. Streptococcus agalactiae (group B Streptococcus [GBS]) is an important bovine pathogen, especially as a cause of both clinical and subclinical mastitis in dairy cows (23). In humans, GBS has been described as one of the most common agents of invasive infections in neonates, but it also causes invasive and noninvasive infections in adults (29). ␤-Lactam agents constitute the drugs of choice for the prophylaxis and treatment of GBS infections, since GBS isolates with confirmed resistance to these antimicrobial agents have not been observed to date (23, 29). Erythromycin and other macrolides are the recommended second-line agents and the first alternative in case of allergy to ␤-lactams. Several studies, however, have documented the emergence and spread of resistance of GBS to macrolides (2, 3, 9, 10), usually in association with resistance to tetracycline. In streptococci, the most frequent macrolide resistance mechanisms are ribosomal modification by a methylase encoded by an erm gene (37) and drug efflux by a membranebound protein encoded by a mef gene (24). The presence of the Erm methylase confers resistance to erythromycin and inducible or constitutive resistance to lincosamines and streptogramin B (the macrolide-lincosamine-streptogramin B [MLSB] phenotype), while the presence of the Mef pump confers resistance to 14- and 15-membered macrolides (M phenotype). An additional efflux mechanism, encoded by the mreA gene, has been described in GBS (8). The linB gene, described in Enterococcus faecium (4), was recently detected in a GBS isolate (10). Although resistance to tetracycline among GBS isolates is frequently found at high rates and, therefore, tetracycline is no

longer indicated for the treatment of GBS infections, tetracycline resistance genes are often found on the same motile unit as the erythromycin resistance genes (33), raising concern about the role of tetracycline-resistant strains in the spread of erythromycin-resistant strains. A variety of tetracycline resistance genes have been described to date, and most of them encode either a protein which pumps tetracycline out of the cell or a ribosomal protein which protects the ribosomes from the action of tetracycline (33). Different biotypes or ecovars of GBS have been detected when the phenotypic characteristics of isolates recovered from human and bovine sources were compared (13, 19, 22, 26, 38). While several previous studies have also compared the molecular characteristics of strains isolated from different hosts (6, 12, 15, 16, 20, 22, 25), only a few have explored the genetic mechanisms of antimicrobial resistance and the presence of genetic determinants that encode potential virulence factors. Recently, we have investigated the phenotypic and molecular characteristics of GBS strains obtained from bovine (17) and human (R. S. Duarte et al., unpublished data) clinical specimens in Brazil. Resistance to tetracycline was found among most of the isolates (151 isolates; 90.9%) from human sources and 38 (44.7%) of the bovine isolates, while erythromycin resistance was detected among 10.5% of the bovine isolates and 4.2% of the human isolates. In the present report we describe the characteristics of the macrolide- and/or tetracycline-resistant isolates recovered during these studies and compare the distributions of antimicrobial resistance genes and virulence-related genes among isolates from different sources.

* Corresponding author. Mailing address: Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, CCS, Bloco I, Cidade Universita´ria, Rio de Janeiro, RJ 21941-590, Brazil. Phone: 55 21 2260 4193. Fax: 55 21 2560 8344. E-mail: [email protected].

Bacterial isolates. A total of 189 antimicrobial-resistant GBS isolates, corresponding to 38 isolates from bovines and 151 from human sources, were included in the present study. The 38 bovine GBS isolates were all recovered from the milk of dairy cows with clinical and subclinical mastitis. The cows belonged to 16

MATERIALS AND METHODS

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TABLE 1. Target genes and oligonucleotide primers used to amplify antimicrobial resistance genes and virulence-related genes of S. agalactiae isolates Gene type and target gene

Erythromycin resistance genes erm(A) erm(B) mef(A) mreA linB Tetracycline resistance genes tet(K) tet(L) tet(M) tet(O) Virulence-related genes bac bca lmb scpB

Forward primer sequence (5⬘–3⬘)

Reverse primer sequence (5⬘–3⬘)

Amplicon length (bp)

Annealing temp (°C)

Reference

206 639

52 52

32 34

348

52

34

121

53

8

944

54

4

GCA TGA CAT AAA CCT TCA GAA AAG GTA CTC AAC CAA ATA AGT ATC ATT AAT CAC TAG TGC AGA CAC CTC GTC TAA CCT TCG CTC CCT ACC TAT TGT TTG TGG AA

AGG TTA TAA TGA AAC AGA AGT AAC GGT ACT TAA ATT GTT TAC TTC TTC TGG TAC TAA AAG TGG TTC AGT TAC TAC CAT GCC ACA GG ATA ACG TTA CTC TCC TAT TC

TAT TTT GGC TTT GTA TTC TTT CAT ATA AAT TGT TTC GGG TCG GTA AT AGT TTT AGC TCA TGT TGA TG AGC GTC AAA GGG GAA TCA CTA TCC

GCT ATA CCT GTT CCC TCT GAT AA AAC CAG CCA ACT AAT GAC AAT GAT TCC GAC TAT TTA GAC GAC GG

1,159

50

36

1,077

50

36

1,892

50

36

CGG CGG GGT TGG CAA ATA

1,723

55

36

TGT AAA GGA CGA TAG TGT GAA GAC CAG GAG GGG AAA CAA CAG TAC GAC GCA ACA CAC GGC AT

CAT TTG TGA TTC CCT TTT GC

530

50

15

GTA TCC TTT GAT CCA TCT GGA TAC G TGA TAG AGC ACT TCC AAA TTT G ACC TGG TGT TTG ACC TGA ACT A

183

50

16

300

50

15

255

50

16

ACA ATG GAA GGC TCT ACT GTT C

herds distributed in two major states located in the southeast region of Brazil: 32 isolates were from 15 herds located in Minas Gerais State, and 6 isolates were from one herd located in Rio de Janeiro State. Most bovine isolates were recovered during 1999 (15 isolates) and 2000 (12 isolates). The remaining 11 bovine isolates were obtained during the period from 1995 to 1997. The 151 human isolates were obtained from different sources, including 60 strains from genital tract secretions, 57 from urine, 15 from respiratory tract secretions, 7 from skin and superficial wounds, 6 from placenta or umbilical secretions, and 3 from blood; the remaining 3 strains were from other sources (2 from rectal secretions and 1 from a catheter). The isolates from human sources were all recovered during the period from 2000 to 2001 in Rio de Janeiro State. The isolates were preserved as heavy bacterial suspensions in 10% skim milk (Difco Laboratories, Detroit, Mich.) containing 10% glycerol at ⫺20°C. For most tests, GBS strains were grown on Trypticase soy agar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with 5% sheep blood (TSA-SB) at 37°C for 18 to 24 h. The isolates were identified on the basis of conventional morphological and physiological methods and reactivity with Lancefield group B-specific antiserum (18). Serological typing was performed by a coagglutination method with reagents prepared in our laboratory according to the recommendations of Christensen et al. (7). Autoagglutinable isolates were serotyped by the capillary precipitation method (18). Antimicrobial susceptibility testing. Susceptibilities to tetracycline and erythromycin were determined by the agar diffusion method according to the guidelines of the National Committee for Clinical Laboratory Standards (27). Macrolide resistance phenotypes were determined by the double-disk test on Mueller-Hinton agar supplemented with 5% sheep blood with erythromycin and clindamycin disks, as described previously (31). Detection of antimicrobial resistance genes. The strains were evaluated by single PCR tests for the presence of genetic determinants associated with resistance to erythromycin and resistance to tetracycline. Preparation of DNA extracts was based on a previously described method (15). Briefly, 10 to 20 colonies of a fresh culture on TSA-SB were suspended in 50 ␮l of distilled water and boiled for 5 min. The sets of primers used for detection of antimicrobial resistance genes are indicated in Table 1. For detection of erythromycin resistance

genes, the reaction mixtures, in final volumes of 50 ␮l, contained MgCl2 [1.5 mM for the mreA gene; 2 mM for the erm(A), erm(B), and linB genes; and 4 mM for the mef(A) gene], deoxynucleoside triphosphates (0.2 mM each), primers (0.5 ␮M each), Taq DNA polymerase (0.5 U), reaction buffer (10 mM), and 1 ␮l of DNA extract, which was used as the template. The reaction mixtures for detection of tetracycline resistance genes contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 0.2 mM each nucleotide, 0.5 mM each primer, 2.5 U of Taq DNA polymerase, and 5 ␮l of DNA template (36). Conditions for amplification of the erm(A), erm(B), and mef(A) genes were as follows: initial denaturation 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, with a final elongation step at 72°C for 5 min (32, 34). Amplification conditions for detection of the mreA gene were initial denaturation at 95°C for 2 min, followed by 25 cycles at 94°C for 1 min, primer annealing at 53°C for 1 min, and extension at 72°C for 1 min, with a final elongation step at 72°C for 10 min (8). For detection of the linB gene the conditions were initial denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 45 s, primer annealing at 54°C for 45 s, and extension at 72°C for 1 min, with a final elongation step at 72°C for 5 min (4). Conditions for amplification of tetracycline resistance genes consisted of 35 cycles of 1 min at 95°C, 1 min at 50°C, and 1 min and 30 s at 72°C, followed by a final 5 min at 72°C for tests for all genes except tet(O), for which the annealing temperature was 55°C (36). Amplifications were performed in a GeneAmp PCR System 2400 thermocycler (Perkin-Elmer Applied Biosystems, Branchburg, N.J.). PCR products were resolved by electrophoresis on 1.2% agarose gels in 0.5⫻ TBE buffer (1 mM Tris, 0.01 M EDTA, 1 M boric acid). The 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 size markers (100-bp ladder; Pharmacia Biotech, Uppsala, Sweden). The following strains were included as positive controls for specific amplification of the different genes investigated: Streptococcus pyogenes CL-5062 [tet(K)], S. agalactiae CL-5957 [tet(L)], S. pyogenes CL-1218 [tet(M)], S. pyogenes CL-2185 [tet(O)], S. pyogenes CL-3760 [erm(A)], Streptococcus pneumoniae (Sp. 398) [erm(B)], E. faecium CL-1727 (linB), and S. pyogenes CL-3870 [mef(A)]. Anti-

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FIG. 1. Occurrence of resistance to erythromycin (Ery-R) and tetracycline (Tet-R) and distribution of virulence-related genes among S. agalactiae isolates by origin and serotype.

microbial-susceptible S. agalactiae strains (strains CL-5736 and CL-5953) were used as negative controls. Detection of virulence-related genes. PCR was also used to evaluate the strains for the presence of genes encoding surface proteins of GBS potentially associated with virulence. Genomic DNA was prepared as described above, and 5-␮l volumes of the supernatants were used for PCRs, as described previously (15). The sets of primers (synthesized by Invitrogen Life Technologies Brazil, Saõ Paulo, Brazil) used for the detection of genes encoding immunoglobulin A-binding ␤-antigen (bac), ␣-antigen (bca), laminin-binding surface protein (lmb), and C5a peptidase (scpB) are listed in Table 1. PCR conditions consisted of an initial denaturation step at 94°C for 2 min, followed by 30 cycles of denaturation (94°C, 30 s), primer annealing (50°C, 1 min), and final extension (72°C, 1 min). Electrophoresis and visualization of PCR products were performed as described above. Analysis of chromosomal DNA restriction profiles by PFGE. The genetic diversity of the resistant GBS isolates was also evaluated by analysis of the chromosomal DNA restriction profiles after digestion with SmaI and separation of the fragments by pulsed-field gel electrophoresis (PFGE). For that, chromosomal DNA was extracted in agarose plugs and treated with SmaI restriction endonuclease as previously recommended by Teixeira et al. (35). The fragments were separated by PFGE in 1.2% agarose gels in a CHEF-DRIII system (BioRad Laboratories, Hercules, Calif.) 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. The SmaI restriction profiles were initially compared by visual inspection. Computer-assisted analysis was also performed by using the Molecular Analyst Fingerprinting Plus software (version 1.6) of the Image Analysis System (Bio-Rad). Comparison of the banding patterns was accomplished by the unweighted pair group method with arithmetic averages by using the Dice similarity coefficient. Statistical analysis. Epi-Info (Database and Statistics Program for Public Health) software (version 6.04; Centers for Disease Control and Prevention, Atlanta, Ga.) was used to analyze the data on the distributions of the different genes by source by the chi-square test for categorical variables. P values of 0.05 or less were considered statistically significant.

RESULTS AND DISCUSSION All 189 strains included in this study were resistant to tetracycline, and 8.5% were also resistant to erythromycin, corresponding to 23.7% of the bovine isolates and 4.6% of the human isolates. The distribution of strains resistant to erythromycin and/or tetracycline by origin and serotype of GBS is

shown in Fig. 1. In our previous study (17) in which the collection of 38 bovine isolates included in the present work originated, serotype III was the most frequent (66 strains; 77.6%), followed by serotypes II (10 strains; 11.8%), Ia (5 strains; 5.9%), Ib (2 strains; 2.35%), and VI (2 strains; 2.35%). Resistance to tetracycline was found in all the isolates belonging to serotypes Ia, Ib, and VI and among the majority (9 of 10) of serotype II isolates. In contrast, only 18 (27.3%) serotype III strains were resistant to tetracycline. Resistance to erythromycin was observed in 10.5% of the bovine strains that we previously studied; these represented 23.7% of the 38 tetracyclineresistant isolates selected for inclusion in the present study. The nine erythromycin-resistant isolates were obtained from six different herds located in Minas Gerais State. Resistance to erythromycin was predominately associated with serotype II isolates (5 serotype II isolates; 50%) and was also found in 4 (22.2%) serotype III isolates. Tetracycline resistance was found in most (90.9%) of the strains identified in our recent study on GBS isolated from human sources (Duarte et al., unpublished data). The 151 tetracycline-resistant human isolates included in the present study comprised 62 isolates of serotype Ia, 17 isolates of serotype Ib, 40 isolates of serotype II, 21 isolates of serotype III, and 11 isolates of serotype V. Macrolide resistance was found in seven (4.6%) tetracycline-resistant strains of human origin, comprising four isolates belonging to serotype V and three isolates belonging to serotype Ia. These isolates represented 36.4% of the tetracycline-resistant serotype V strains and 4.8% of the tetracycline-resistant serotype Ia strains. All erythromycin-resistant strains of both bovine and human origin were also resistant to tetracycline. The frequencies of the different genes associated with erythromycin and tetracycline resistance varied according to the origin of the GBS isolates (Table 2). The tet(O) gene was the most frequent determinant among the 38 tetracycline-resistant isolates of bovine origin and was found in 27 strains (71%),

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TABLE 2. Distribution of erythromycin and tetracycline resistance genes among S. agalactiae strains recovered from bovine and human sources in Brazil No. (%) of resistant strains harboring resistance genesb Source

Erythromycin resistance phenotypea

Bovine milk Human

Erythromycin resistance genes Total

erm(A)

erm(B)

mreA

9 3 4

6 (66.6) 2 (66.6) 4 (100)

9 (100) 1 (33.3) 2 (50.0)

9 (100) 3 (100) 4 (100)

cMLSB cMLSB iMLSB

Tetracycline resistance genes erm(A) and Total erm(B)

6 (66.6) 0 2 (50.0)

tet(L)

tet(M)

tet(O)

tet(M) and tet(L) and tet(L) and tet(O) tet(M) tet(O)

38 3 (7.8) 16 (42.1) 27 (71.0) 7 (18.2) 151 1 (0.6) 139 (92.0) 21 (13.9) 10 (6.6)

0 1 (0.6)

2 (5.2) 0

a Phenotype M was not detected among isolates obtained from either bovine or human sources. cMLSB, constitutive MLSB resistance phenotype; iMLSB, inducible MLSB resistance phenotype. b The mef(A), linB, and tet(K) genes were not detected among the antimicrobial-resistant S. agalactiae isolates.

followed by tet(M) (16 strains; 42.1%) and tet(L) (3 strains; 7.8%). Seven (18.2%) strains harboring tet(O) also harbored 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 for which tests were conducted. The tet(M) gene was the predominant tet gene among the 151 tetracycline-resistant isolates from humans (139 isolates; 92%), followed by tet(O) (21 isolates; 13.9%) and tet(L) (1 strain; 0.6%). Only one tetracycline-resistant strain of human origin did not harbor any of the tetracycline resistance genes for which tests were conducted, and 10 isolates harbored both the tet(M) and the tet(O) genes. Statistically significant differences (P ⬍ 0.001) in the distributions of tet(M) and tet(O) were found between the strains isolated from bovine milk and those isolated from human sources. The tet(K) gene was not detected among GBS isolates of bovine or human origin. All nine erythromycin-resistant isolates of bovine origin presented the constitutive MLSB phenotype, which indicates concomitant constitutive resistance to clindamycin. They all had

the erm(B) and the mreA genes, and six of them also had the erm(A) gene. All erythromycin-resistant GBS isolates of bovine origin carried the tet(O) gene; two of them also carried the tet(M) gene simultaneously and one carried the tet(L) gene simultaneously. Most of the seven human GBS strains (four isolates) had the inducible MLSB phenotype and harbored the erm(A) gene, and two of them also harbored the erm(B) genetic determinant. Three human strains had the constitutive MLSB phenotype and were found to contain the erm(A) gene (two strains) or the erm(B) gene (one strain). The mreA gene was found in all erythromycin-resistant strains as well as in all erythromycin-susceptible strains. This finding confirms previous evidence of the ubiquitous presence of this gene in resistant and susceptible isolates (10, 28), supporting the hypothesis that the mreA gene may not be directly related to erythromycin resistance. The absence of the mef(A) and linB genes among Brazilian GBS isolates confirms previous evidence indicating that these genes are not frequently associated with MLSB resistance in S. agalactiae isolates (2, 10, 28). Resistance to tet-

TABLE 3. Phenotypic and genotypic characteristics of macrolide-resistant S. agalactiae isolates from bovine and human sources in Brazil Amplification reaction resultb Isolate origin and strain

Source

Bovine isolates CL-5592 CL-5595 CL-5599 CL-5600 CL-5651 CL-5652 CL-5654 CL-5675 CL-5676

Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine Bovine

Human isolates CL-5570 CL-5784 CL-5812 CL-5823 CL-5886 CL-5965 CL-6071

Wound Urine Urine FGT sec.c Urine FGT sec. Urine

a b c

milk milk milk milk milk milk milk milk milk

Yr of isolation

Serotype

Macrolide resistance phenotypea

Macrolide resistance gene

Tetracycline resistance genes

Virulence-related genesb

erm(A)

erm(B)

mreA

tet(L)

tet(M)

tet(O)

bca

lmb

scpB

1995 1996 1997 1997 1999 1999 1999 2000 2000

II III II III II II II III III

cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB cMLSB

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺

⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹

2000 2000 2000 2000 2000 2001 2001

Ia V V V V Ia Ia

iMLSB iMLSB cMLSB cMLSB iMLSB iMLSB cMLSB

⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

cMLSB, constitutive MLSB resistance phenotype; iMLSB, inducible MLSB resistance phenotype. ⫹, positive amplification reaction; ⫺, negative amplification reaction. FGT sec., female genital tract secretion.

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TABLE 4. Occurrence of different profiles of virulence-related genetic determinants among antimicrobial-resistant S. agalactiae strains recovered from bovine and human sources in Brazil No. (%) of isolates Profile

A B C D E F G H a

Virulence gene profilea

bac⫺ bac⫺ bac⫺ bac⫺ bac⫺ bac⫺ bac⫺ bac⫹

bca⫹ bca⫺ bca⫺ bca⫺ bca⫹ bca⫹ bca⫹ bca⫹

lmb⫹ lmb⫹ lmb⫺ lmb⫺ lmb⫹ lmb⫺ lmb⫺ lmb⫹

scpB⫹ scpB⫹ scpB⫹ scpB⫺ scpB⫺ scpB⫹ scpB⫺ scpB⫹

Bovine isolates (total n ⫽ 38)

Human isolates (total n ⫽ 151)

5 (13.1) 1 (2.6) 2 (5.3) 5 (13.1) 0 (0) 17 (44.7) 8 (21.0) 0 (0)

101 (66.9) 34 (22.5) 0 (0) 1 (0.7) 1 (0.7) 0 (0) 3 (2.0) 11 (7.3)

⫺, negative for the gene; ⫹, positive for the gene.

racycline among the erythromycin-resistant isolates recovered from human sources was due to the presence of the tet(M) gene. Several virulence profiles were found among erythromycin-resistant isolates. Table 3 shows the characteristics of GBS strains simultaneously resistant to erythromycin and tetracycline. Most of the studies on the mechanisms and genetic determinants of erythromycin and tetracycline resistance in GBS usually focus on the macrolide resistance of isolates recovered from human sources, due to the clinical importance of these antimicrobial agents (1, 2, 3, 5, 9, 10, 11, 14, 21, 28, 30). However, although tetracycline is no longer used for the treatment of streptococcal infections, studies are necessary to elucidate whether high rates of tetracycline resistance constitute a potential risk factor for the acquisition of other resistance genes, since elements that transfer tetracycline resistance genes can also carry genes encoding resistance to macrolides, lincosamides, and chloramphenicol (9, 33). As previously reported for strains isolated in other countries (1, 2, 9, 10, 28), constitutive and inducible MLSB phenotypes predominate among human isolates, although differences in the frequencies of these phenotypes were noted. The M phenotype was not detected in the present investigation, but its

101

occurrence has been reported among human GBS isolates at low rates (2, 9, 10, 21, 28). Overall, the distribution of the different antimicrobial resistance genetic determinants in human GBS isolates was also similar to those described in previous reports (1, 2, 9, 10, 28). As expected, the erm(A) and the erm(B) genes were the most prevalent among erythromycinresistant GBS strains, as were the tet(M) and the tet(O) genes among the tetracycline-resistant isolates from human sources. On the other hand, studies on erythromycin resistance in GBS isolates from other hosts are lacking, and only a few have investigated the distribution of tet genetic determinants among tetracycline-resistant GBS strains obtained from animal sources. Our results showed that erm(B) and tet(O) were the most frequent genes associated with erythromycin and tetracycline resistance, respectively, in GBS isolates from bovines. The higher frequency of the tet(O) and the tet(M) genes among GBS strains recovered from dairy cows was described previously (5, 30). The virulence genes bac, bca, lmb, and scpB were detected in 0 (0%), 30 (78.9%), 6 (15.8%), and 25 (65.8%) of the tetracycline-resistant GBS strains from bovines, respectively, and in 13 (7.3%), 116 (76.8%), 147 (97.3%), and 146 (96.7%) of the tetracycline-resistant isolates from human sources, respectively. The distribution of virulence genes among antimicrobial-resistant GBS strains of different serotypes by origin is shown in Fig. 1. Table 4 shows the frequencies of the different virulence profiles among antimicrobial-resistant GBS strains by origin. The majority of the tetracycline-resistant GBS strains from human sources (146 isolates; 96.7%) had both the lmb and scpB genes simultaneously, and a large proportion (101 isolates; 66.9%) had the bca, lmb, and scpB genes simultaneously. The presence of these three genes characterized the prevalent virulence profile among human isolates. Among the bovine isolates resistant to tetracycline, the most frequent profile consisted of simultaneous carriage of both the bca and the scpB genes and was found in 17 isolates (44.7%). All six lmbpositive bovine strains were also scpB positive. The distinct distribution of the virulence-related genes investigated among the GBS isolates recovered from human and bovine sources indicates that different virulence traits may be involved in the

FIG. 2. Dendrogram resulting from a computer-assisted analysis of the PFGE profiles of SmaI-digested DNA of erythromycin-resistant Streptococcus agalactiae strains isolated from bovine and human sources in Brazil.

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pathogenesis of infections caused by this microorganism in these hosts and that diverse evolutionary lineages of GBS are associated with different hosts. None of the erythromycin-resistant isolates had the mef(A), linB, or bac gene. Although a few major clusters were observed, PFGE revealed a variety of profiles (data not shown), reflecting the substantial genetic diversity among antimicrobial-resistant GBS strains isolated from either humans or bovines. When the PFGE profiles of erythromycin-resistant isolates were compared separately, two major clusters were observed, corresponding to most of the isolates of bovine and human origin, respectively (Fig. 2). Clustering was particularly observed among human isolates of serotype V resistant to erythromycin. We conclude that resistance to erythromycin and tetracycline among S. agalactiae strains may occur predominantly due to the multiclonal spread of resistance genes instead of being related to the epidemic dissemination of a few clones. This study is the first to correlate the distribution of the genetic mechanisms of macrolide and tetracycline resistance as well as the occurrence of genetic determinants encoding for surface proteins potentially associated with virulence among the different serotypes of S. agalactiae from bovines and humans in Brazil. Among the human isolates, tetracycline resistance was associated with most of the serotypes identified and was predominantly due to the presence of the tet(M) gene. This resistance marker was also found in a large proportion of bovine isolates, predominantly due to the presence of the tet(O) gene, and in association with several serotypes. Differences in the frequencies of genes coding for potential virulence factors were also noted among bovine and human isolates, reinforcing the concept that distinct GBS populations circulate among bovine and human hosts. While the knowledge of the most prevalent serotypes and other molecular characteristics of GBS isolates from a given geographical area is essential to trace the epidemiological course of infections, surveillance of the genetic backgrounds of antimicrobial-resistant GBS strains circulating in different hosts and regions is relevant to guiding the design of more appropriate procedures for infection control and prevention. ACKNOWLEDGMENTS This study was supported in part by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo ´gico, Financiadora de Estudos e Projetos, Fundação Universita´ria Jose´ Bonifaćio, Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, and Ministe´rio da Cieˆncia e Tecnologia (MCT/PRONEX) of Brazil. Typing antisera were kindly provided by R. R. Facklam, Streptococcus Laboratory, Centers for Disease Control and Prevention. We thank Carlos Ausberto B. de Souza and Filomena Soares Pereira da Rocha for technical assistance. REFERENCES 1. Aracil, B., M. Min ˜ ambres, J. Oteo, M. de la Rosa, J. L. Go´mez-Garce´s, and J. I. Alo ´s. 2002. Susceptibility of strains of Streptococcus agalactiae to macrolides and lincosamides, phenotype patterns and resistance genes. Clin. Microbiol. Infect. 8:745–748. 2. Betriu, C., E. Culebras, I. Rodrı´guez-Avial, M. Go ´mez, B. A. Sa ńchez, and J. J. Picazo. 2004. In vitro activities of tigecycline against erythromycinresistant Streptococcus pyogenes and Streptococcus agalactiae: mechanisms of macrolide and tetracycline resistance. Antimicrob. Agents Chemother. 48: 323–325. 3. Bingen, E., C. Doit, P. Bidet, N. Brahimi, and D. Deforche. 2004. Telithromycin susceptibility and genomic diversity of macrolide-resistant serotype III group B streptococci isolated in perinatal infections. Antimicrob. Agents Chemother. 48:677–680.

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