MICROBIAL DRUG RESISTANCE Volume 10, Number 4, 2004 © Mary Ann Liebert, Inc.
Occurrence and Characteristics of Erythromycin-Resistant Streptococcus pneumoniae Strains Isolated in Three Major Brazilian States CLÁUDIA R.V. MENDONÇA-SOUZA,1 MARIA DA GLÓRIA S. CARVALHO,1,4 ROSANA R. BARROS,1 CÍCERO A. DIAS,2 JORGE LUIZ M. SAMPAIO,3 ANGELA C.D. CASTRO,1 RICHARD R. FACKLAM,4 and LÚCIA M. TEIXEIRA1
ABSTRACT We investigated the occurrence and phenotypic and genotypic characteristics of erythromycin-resistant Streptococcus pneumoniae strains isolated in three major states in Brazil, from 1990 to 1999. Of the 931 pneumococcal strains evaluated, 40 (4.3%) were erythromycin-resistant (Ery-R). Among the 40 Ery-R strains, 90.0%, 80.0%, 27.5%, 5.0%, and 2.5% were resistant to tetracycline, trimethoprim-sulfamethoxazole, penicillin, chloramphenicol, and rifampin, respectively. None of the strains were resistant to ofloxacin or to vancomycin. Most [37 (92.5%)] of the 40 Ery-R isolates presented the MLSB phenotype and 3 (7.5%) strains showed the M phenotype. PCR testing indicated that all MLSB phenotype isolates harbored the erm(B) gene only, whereas the mef(A/E) gene was present in all isolates presenting the M phenotype. The tet(M) gene was the most frequent (86.1%) among Ery-R isolates that were also resistant to tetracycline. Pulsed-field gel electrophoresis (PFGE) analysis after SmaI digestion revealed the occurrence of clonal relationships within groups of strains belonging to serotypes 14, 19A, and 23F. All Ery-R isolates belonging to serotype 14 were susceptible to penicillin and were included in a single clonal group (named Ery14-A) related to the England14-9 internationally spread clone.
INTRODUCTION
E
RYTHROMYCIN AND OTHER MACROLIDES represent important alternative drugs in the treatment of respiratory infections caused by Streptococcus pneumoniae. Isolation of macrolideresistant pneumococci (MRP) has been increasingly reported in many parts of the world,3,4,7 although the prevalence of MRP can vary according to the geographic area and period of time.5,6 Two major mechanisms are generally associated with macrolide resistance in pneumococci.19,20 One is associated with a target modification encoded by the erm(B) gene, leading to the MLSB phenotype, which can be either constitutive or inducible and is characterized by resistance to 14-, 15-, and 16-membered macrolides, lincosamides, and streptogramin B resistance. The other is associated with an efflux pump related to a membranebound protein, encoded by the mef(A/E) gene, which confers
the M phenotype characterized by resistance to 14- and 15membered macrolides only. More recently, MRP strains carrying the erm(A) gene, also associated with the MLSB phenotype, were reported.21 Three additional new mechanisms of resistance to macrolides in S. pneumoniae have also been described that are associated with mutations in the 23S rRNA genes, in the ribosomal protein L4, or in the ribosomal protein L22.16,22 Variation in the prevalence of the diverse genes and mechanisms of resistance to macrolides have been observed among pneumococci isolated in different regions.2,3,5,9,10,14,18 The aim of this study was to investigate the occurrence and the molecular mechanisms of macrolide resistance among a collection of pneumococcal strains isolated in three major Brazilian states, from 1990 to 1999. Genomic analysis by PFGE as well as determination of tetracycline resistance genotypes among macrolide-resistant isolates were also performed.
1Instituto
de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil. Faculdade de Ciências Médicas de Porto Alegre, Porto Alegre, Rio Grande do Sul 90050-170, Brazil. 3Fleury-Centro de Medicina Diagnóstica, São Paulo 04344-070, Brazil. 4Centers for Disease Control and Prevention, Atlanta, GA 30333. 2Fundação
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MENDONÇA-SOUZA ET AL.
MATERIALS AND METHODS Bacterial isolates and identification A total of 931 pneumococcal isolates, recovered from diverse clinical specimens obtained from patients living in Brazil during the period of 1990 to 1999, were evaluated for susceptibility to erythromycin. Most (98.4%) of the isolates were recovered from patients living in three major Brazilian states: Rio de Janeiro (327 isolates) and São Paulo (360 isolates), both located in the southeastern region of Brazil, and Rio Grande do Sul (229 isolates), located in the southern region of Brazil. The clinical sources included: blood (212 isolates), cerebrospinal fluid (196 isolates), respiratory tract secretions (total of 360 isolates, including 161 from upper respiratory tract, 82 from lower respiratory tract, and 117 from sputa specimens), eye (31 isolates) and ear (9 isolates) secretions, and other secretions and body fluids (37 isolates). Eight-six isolates were recovered from unknown sources. The identification of the isolates was based on the results of conventional testing, including colony morphology and -hemolytic activity on sheep blood agar media, Gram staining characteristics, catalase reaction, susceptibility to optochin, and bile solubility testing. They were serotyped on the basis of capsular swelling (Quellung reaction) with typespecific pneumococcal antisera (Centers for Disease Control and Prevention, Atlanta, GA). The Danish system of nomenclature was used. Nontypeable isolates were examined for reactivity with the AccuProbe S. pneumoniae culture identification test (Gen-Probe Inc., San Diego, Calif.), according to the manufacturer’s instructions.
Susceptibility testing All of the 931 isolates were screened for erythromycin resistance by the disk diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations.12 Susceptibilities to chloramphenicol, ofloxacin, penicillin (using 1-g oxacillin disks), rifampin, tetracycline, trimethoprim-sulfamethoxazole, and vancomycin were also determined by the disk (CECON, São Paulo, Brazil) diffusion test. MICs of clindamycin, erythromycin, and penicillin (Sigma Chemical Co., St. Louis, MO) were determined for all erythromycin-resistant (Ery-R) isolates by the broth dilution method according to the NCCLS guidelines.13 The phenotypes of Ery-R isolates were investigated by the double-disk test, as previously described.17,27 Blunting of the clindamycin inhibition zone near to the erythromycin disk was considered as an inducible type of MLSB resistance (iMLSB), whereas resistance to both drugs was interpreted as a constitutive type of MLSB resistance (cMLSB). Resistance to erythromycin associated with susceptibility to clindamycin was considered as indicative of the M phenotype.
Detection of erythromycin and tetracycline resistance determinants Preparation of genomic DNA was based on the recommendations of Beall et al.,1 with modifications. Briefly, each strain was grown on a sheep blood agar plate for 18–24 hr at 37°C. One loopfull of bacterial growth was resuspended in 300 l of 0.85% NaCl solution. After incubation at 60°C for 30 min, cells
were harvested by centrifugation, resuspended in 50 l of TE buffer containing 30 U of mutanolysin (Sigma) and incubated at 37°C for 10 min. Samples were then heated at 100°C for 3 min and centrifuged to pellet debris. DNA extracts were used in single PCR tests to detect the genetic determinants of erythromycin resistance. Ery-R isolates were screened for the presence of the erm(B) and mef(A/E) resistance determinants by PCR amplification, using gene-specific internal primers, as described elsewhere.19 Primers (Operon Technologies Inc., Alameda, CA) used for amplification were: erm(B) (5-GAA AAG GTA CTC AAC CAA ATA-3; and 5-AGT AAC GGT ACT TAA ATT GTT TAC3); mef(A/E) (5-AGT ATC ATT AAT CAC TAG TGC-3; and 5-TTC TTC TGG TAC TAA AAG TGG-3). The strains were also screened for the presence of the erm(A) gene by using the following pair of primers 5-GCA TGA CAT AAA CCT TCA-3 and 5-AGG TTA TAA TGA AAC AGA-3.6 Amplification was performed by using a DNA thermocycler (Gene Amp PCR System 2400; Perkin-Elmer Corporation, Branchburg, NJ), under the following conditions: one cycle of 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, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. An erythromycin-susceptible S. pneumoniae strain (Sp 526) was used as a negative control. S. pneumoniae strains Sp 643 [erm(B)], Sp 749 [mef(A/E)] and S. agalactiae strain 01394 [erm(A)] were used as control strains. Ery-R isolates that were simultaneously resistant to tetracycline were also tested for the presence of the following tetracycline resistance determinants: tet(K), tet(L), tet(M), and tet(O). Primers and PCR conditions for detection of the tetracycline resistance determinants were as described previously.28 A tetracycline-susceptible S. pneumoniae strain (Sp 1012) was used as a negative control. Escherichia coli strain 31 [tet(K)], S. agalactiae CL-5596 [tet(L)], S. pneumoniae Sp 557 [tet(M)], and S. pneumoniae Sp 749 [tet(O)] were used as positive-control strains.
Genotypic analysis The 40 Ery-R isolates were submitted to analysis of genomic DNA restriction profiles by PFGE. Randomly chosen erythromycin-susceptible isolates belonging to serotypes most frequently identified among Ery-R strains (seven serotype 14, seven serotype 23F, and four serotype 19A isolates), as well as the reference strains of several antimicrobial-resistant clones [ATCC 700669 (Spain23F-1), ATCC 700670 (Spain6B-2), ATCC 700671 (Spain9V-3), ATCC 51916 (Tennessee23F-4), ATCC 700902 (Spain14-5), ATCC 700673 (Hungary19A-6), ATCC 700674 (South Africa9A-7), ATCC 700675 (South Africa6B-8), ATCC 700676 (England14-9), ATCC 700677 (CSR14-10) and ATCC 700678 (CSR19A-11), ATCC (Finland6B-12), ATCC 700904 (South Africa19A-13), ATCC 700905 (Taiwan19F-14), and (Taiwan23F-15)], were also included for comparative purposes. DNA was prepared, treated with SmaI and analyzed by PFGE, as previously described,24 with the following modifications. Strains were grown on sheep blood agar plates for 15 hr at 37°C. PFGE was performed with a CHEF DR III (Bio-Rad Laboratories, Hercules, CA) and the following parameters were used: pulse time increasing from 1
ERYTHROMYCIN-RESISTANT PNEUMOCOCCI IN BRAZIL to 30 sec; running time, 20 hr; voltage, 6 V/cm; temperature, 11°C; and included angle, 120°. Analysis of genomic DNA fragmentation profiles was performed by visual inspection, and the criteria suggested by Tenover et al.26 was used for interpretation. Isolates presenting identical or related PFGE profiles were considered to compose a clonal group. Computer-assisted analysis was also performed by using the Molecular Analyst Fingerprinting Plus software, version 1.12 of the Image Analysis System (Bio-Rad). Comparison of the banding patterns was accomplished by the unweighted part group method with arithmetic mean (UPGMA) method, using the Dice coefficient of similarity.
RESULTS AND DISCUSSION The overall prevalence of macrolide resistance among the 931 pneumococcal strains evaluated was 4.3% (40 isolates). The distribution of the total number of isolates and the number of Ery-R isolates, according to the year of isolation was as follows: 1990, 77 isolates (no Ery-R isolates); 1991, 83 isolates (1 Ery-R isolate); 1992, 89 isolates (1 Ery-R); 1993, 7 isolates (1 Ery-R); 1994, 152 isolates (5 Ery-R); 1995, 74 isolates (3 Ery-R); 1996, 10 isolates (2 Ery-R); 1997, 120 isolates (4 EryR); 1998, 74 isolates (13 Ery-R); and 1999, 245 isolates (10 Ery-R). The prevalence rates of Ery-R strains, distributed over the 10-year period of time, were: 1990–1993 (3/256; 1.2%), 1994–1996 (10/236; 4.2%), and 1997–1999 (27/439; 6.2%), suggesting an apparent trend toward increasing incidence of this resistance trait. Ery-R strains were found among isolates from the three major Brazilian states represented in this study. The majority (21 isolates, 52.5%) of the 40 Ery-R strains were isolated in Rio de Janeiro state, followed by 32.5% (13 isolates) in the state of Rio Grande do Sul and 15% (6 isolates) in the state of São Paulo. Considering the occurrence in each individual state, Ery-R strains were more common among isolates from Rio de Janeiro (21/327; 6.5%), followed by isolates from Rio Grande do Sul (13/229; 5.7%) and isolates from São Paulo (6/360; 1.7%). The clinical sources of Ery-R isolates included respiratory tract secretions [18/40 isolates, representing 45% of the Ery-R isolates and 5% (18/360) of the total number of isolates recovered from these sources]; cerebrospinal fluid [9/40 isolates, representing 22.5% of the Ery-R isolates and 4.6% (9/196) of the isolates recovered from cerebrospinal fluid]; ear or eye secretions [7/40 isolates, representing 17.5% of the Ery-R isolates and 17.5% (7/40) of the isolates recovered from these sources]; blood [2/40 isolates, representing 5% of the Ery-R isolates and 0.9% (2/212) of the isolates recovered from blood], and other secretions or body fluids [2/40 isolates, representing 5% of the Ery-R isolates and 5.5% (2/37) of the isolates recovered from such sources]. Two Ery-R isolates were from unknown sources. Table 1 lists the characteristics of the 40 Ery-R isolates detected in the present study. Ery-R strains belonged to various serotypes, with serotype 14 (11/40; 27.5%) and serotype 23F (11/40; 27.5%) being the most frequent ones, followed by serotypes 19A (4/40; 10%), 19F (3/40; 7.5%), 6B and 10A (2/40; 5% each), and 5 and 37 (1/400; 2.5% each). Five strains (12.5%) were nontypeable (NT) isolates recovered from ocular secretions (3 isolates), tracheal (1 isolate) and oropharyngeal (1
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isolate) secretions. All 5 NT isolates were positive for the AccuProbe S. pneumoniae culture identification test. Table 2 shows a comparison on the distribution of the different serotypes identified among the 40 Ery-R isolates and the occurrence of these serotypes among Ery-S isolates and the total number (931) of pneumococcal isolates studied. Serotypes 14, 6B, 19F, and 23F represented, respectively, the first, second, third, and fourth more frequent serotypes among the total number of isolates examined (data not shown). Among the 40 Ery-R strains, 36 (90%) were also resistant to tetracycline, 32 (80%) were resistant to trimethoprim-sulfamethoxazole, 11 (27.5%) were resistant to penicillin, 2 (5%) were resistant to chloramphenicol, and 1 (2.5%) to rifampin. None of the strains was resistant to ofloxacin or to vancomycin. According to the results of double-disk tests, 37 (92.5%) of the 40 Ery-R strains were found to present the constitutive MLSB phenotype and the remaining 3 (7.5%) displayed the M phenotype. The induced MLSB phenotype was not detected. All of the isolates with the constitutive MLSB phenotype harbored the erm(B) gene only, and the mef(A/E) gene was present in all of those expressing the M phenotype. None of the isolates carried the erm(A) gene. No strain was found to harbor more than one of the macrolide resistance determinants investigated. Therefore, our results show that methylation of the ribosomal target, one of the two most important mechanisms involved in the resistance of pneumococci to macrolides, predominated among the isolates tested. The predominance of the MLSB phenotype has also been reported from some European countries,2,9,14 as well as from Hong Kong,7 and South Africa.11 In contrast, data from the United States, Canada, and Germany indicate the prevalence of the M phenotype or a nearly equal distribution of both mechanisms.5,18,20 In the present study, erythromycin MICs for the 37 isolates with the MLSB phenotype ranged from 2 to 256 g/ml, although the majority of these strains had very high erythromycin MICs (256 g/ml). On the other hand, erythromycin MICs for all three isolates expressing the M phenotype were 4 g/ml. As expected on the basis of previous observations,10,11 the majority of the strains with the erm(B) gene presented highlevel erythromycin resistance whereas strains harboring the mef(A/E) gene presented low-level erythromycin resistance. All of the strains harboring the erm(B) gene were concomitantly resistant to erythromycin and to clindamycin, with clindamycin MICs ranging from 1 to 64 g/ml, whereas all the strains harboring the mef(A/E) gene were susceptible to clindamycin. No correlation between serotype and macrolide resistance phenotype and genotype and resistance to penicillin was observed among the isolates tested (Table 3). Ery-R isolates that also presented resistance to penicillin (Ery-R/Pen-R isolates) were distributed in different serotypes, including 5, 19A, 19F, 23F, 37, whereas two were NT. All four isolates belonging to serotype 19A were Ery-R/Pen-R, while none of the serotype 14 isolates and only two of the 11 isolates belonging to serotype 23F were Ery-R/Pen-R. The modification target mechanism of resistance was found among Ery-R strains that were either susceptible or nonsusceptible to penicillin. The macrolide efflux mechanism was found only among Ery-R isolates that were susceptible to penicillin. Tetracycline resistance (Tet-R) was high among Ery-R strains (36/40; 90%). Most (31 isolates; 86.1%) of the Ery-
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MENDONÇA-SOUZA ET AL. TABLE 1.
Strain Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp Sp
527 643 661 665 698 749 798 1141 1170 1408 1413 299 398 992 1107 1144 1146 1278 1375 557 755 957 862 863 915 1132 1119 1208 1012 1062 1168 1169 1405 1145 1424 1078 1113 1209 1241 1376
CHARACTERISTICS
Serotypea
Year
Local of isolationb
14 14 14 14 14 14 14 14 14 14 14 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 19A 19A 19A 19A 6B 6B 10A 10A NT NT NT NT NT 19F 19F 19F 5 37
1993 1994 1994 1994 1994 1995 1995 1998 1998 1999 1999 1991 1992 1997 1998 1998 1998 1999 1999 1994 1995 1997 1996 1996 1997 1998 1998 1999 1997 1998 1998 1998 1999 1998 1999 1998 1998 1999 1999 1999
RJ RJ RJ RJ RJ RJ RJ RJ RJ RJ RJ SP SP RS RS RJ RJ RS RJ RJ RJ RS RS RS RS RS RS SP RS RS RJ RJ RJ RJ SP RS RS SP SP RJ
OF
40 ERYTHROMYCIN-RESISTANT S. Source of isolationc
Resistance profiled
Ascitic fluid CSF CSF CSF CSF Bronchial secretion Bone marrow Bronchial secretion Unknown Unknown Eye secretion CSF Ear abscess Sputum Sputum Nasal secretion Nasal secretion Blood Nasal secretion CSF CSF CSF Sputum Ear secretion Ear secretion Blood CSF Oropharyngeal Sputum Sputum Eye secretion Eye secretion Eye secretion Tracheal secretion Oropharyngeal Sputum Nasal secretion Oropharyngeal Oropharyngeal Tracheal secretion
6* 6* 3* 2* 6* 4* 6* 6* 6* 6* 6* 6* 9* 7* 2* 6* 6* 2* 6* 2* 6* 6* 7* 7* 7* 7* 3* 8* 1* 1* 6* 6* 6* 5* 7* 7* 6* 8* 7* 5*
PNEUMONIAE ISOLATES FROM
BRAZIL
Erythromycin resistance genotype
Tetracycline resistance genotypee
PFGE clonal group or profile
erm(B) erm(B) erm(B) erm(B) erm(B) mef(A/E) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) mef(A/E) mef(A/E) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B) erm(B)
tet(M)/tet(K) tet(M) na tet(M) tet(M) tet(M)/tet(O) tet(M) tet(M) tet(M) neg tet(M)/tet(K) tet(M) tet(O) tet(M) tet(M) tet(M) tet(M) tet(M) tet(M) tet(M) tet(M) tet(M) tet(M)/tet(K) tet(M) tet(M) tet(M)/tet(K) na tet(M) na na tet(M) neg tet(M) tet(M) tet(M) neg tet(M) tet(M)/tet(O) neg tet(M)
Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery14-A Ery23F-B Ery23F-B Ery23F-B Ery23F-B Ery23F-B Ery23F-B Ery23F-B Ery23F-B Ery23F-D Ery23F-D Ery23F-E Ery19A-C Ery19A-C Ery19A-C Ery19A-C Ery6B-F Ery6B-F Ery10A-G Ery10A-G EryNT-H EryNT-H EryNT-H Ery-I Ery-J Ery-K Ery-L Ery-M Ery-N Ery-O
aNT,
Nontypeable. Rio de Janeiro; RS, Rio Grande do Sul; SP, São Paulo. cCSF, cerebrospinal fluid. dProfiles: 1, resistance to erythromycin only; 2, resistance to erythromycin, clindamycin, and tetracycline; 3, resistance to erythromycin, clindamycin, and trimethoprim-sulfamethoxazole; 4, resistance to erythromycin, tetracycline, and trimethoprimsulfamethoxazole; 5, resistance to erythromycin, clindamycin, penicillin, and tetracycline; 6, resistance to erythromycin, clindamycin, tetracycline, and trimethoprim-sulfamethoxazole; 7, resistance to erythromycin, clindamycin, penicillin, tetracycline, and trimethoprim-sulfamethoxazole; 8, resistance to erythromycin, clindamycin, chloramphenicol, tetracycline, and trimethoprimsulfamethoxazole; 9, resistance to erythromycin, clindamycin, penicillin, tetracycline, rifampicin and trimethoprim-sulfamethoxazole. *, Profile including resistance to penicillin. ena, Nonapplicable; neg, did not amplify with primers for the tet(M), tet(O), tet(K), and tet(L) genes. bRJ,
R/Tet-R isolates harbored the tet(M) gene. The majority (25 isolates; 80.6%) had the tet(M) gene only, whereas four had both tet(M) and tet(K), and two strains were positive for both tet(M) and tet(O). One isolate presented the tet(O) gene only. This strain (Sp 398) was also the only rifampin-resistant (MIC
> 4 g/ml) isolate identified in the present study. Four of the Ery-R/Tet-R isolates were negative for all four tetracycline resistance genes tested. The predominance of the tet(M) gene among Tet-R pneumococci has been documented previously.10,23 On the other hand, to our knowledge, the associa-
317
ERYTHROMYCIN-RESISTANT PNEUMOCOCCI IN BRAZIL TABLE 2. Categorya of pneumococcal isolates Ery-R (40 isolates) Ery-S (891 isolates) Total (931 isolates) aEry-R,
DISTRIBUTION OF THE SEROTYPES IDENTIFIED AMONG ERYTHROMYCINRESISTANT PNEUMOCOCCAL ISOLATES FROM BRAZIL Number (%) of isolates according to serotype
5
6B
10A
14
19A
19F
23F
37
Nontypeable
1 (2.5) 29 (3.3)
2 (5.0) 87 (9.8)
2 (5.0) 18 (2.0)
11 (27.5) 92 (10.3)
4 (10.0) 17 (1.9)
3 (7.5) 64 (7.2)
11 (27.5) 54 (6.1)
1 (2.5) 5 (0.6)
5 (12.5) 10 (1.1)
30 (3.2)
89 (9.6)
20 (0.2)
103 (11.1)
21 (2.3)
67 (7.2)
65 (7.0)
6 (0.6)
15 (1.6)
Erythromycin-resistant isolates; Ery-S, erythromycin-susceptible isolates.
tion of both the tet(M) and the tet(K) genes in a single pneumococcal isolate has not been previously described. The only Ery-R isolate exhibiting the M phenotype and Tet-R was found to have both the tet(M) and the tet(O) genes. Molecular analysis by PFGE showed 26 profiles among the 40 Ery-R isolates distributed among seven clonal groups (composed of at least two isolates presenting indistinguishable or related profiles) and eight individual profiles (expressed by single isolates). In general, the isolates included in a given clonal group clustered according to the serotype (Table 1 and Fig. 1). Most of the isolates were grouped into three major clonal groups
TABLE 3. DISTRIBUTION OF SEROTYPES AMONG BRAZILIAN ERYTHROMYCIN-RESISTANT PNEUMOCOCCAL ISOLATES ACCORDING TO MACROLIDE RESISTANCE PHENOTYPE AND PENICILLIN SUSCEPTIBILITY Susceptibilitya to penicillin (number of isolates within each category) Serotype and macrolide resistance phenotype Serotype 14 MLSB M Serotype 23F MLSB M Serotype 19A MLSB M Serotype 6B MLSB M Nontypeable MLSB M Other serotypesb MLSB M Total number of isolates aS,
S
I
R
10 1
0 0
0 0
9 0
1 0
1 0
0 0
0 0
4 0
2 0
0 0
0 0
3 0
2 0
0 0
2 2 29
3 0 6
0 0 5
Susceptible; I, intermediate; R, resistant. serotypes 5, 10A, 19F, and 37.
bIncludes
arbitrarily named as clonal group Ery14-A, Ery23F-B and Ery19A-C, which were composed by strains belonging to serotypes 14 (11 isolates), 23F (8 isolates), and 19A (4 isolates), respectively. PFGE profiles of SmaI-digested genomic DNA representative of these three major clonal groups identified among Ery-R S. pneumoniae isolates from Brazil are shown in Fig. 2. All of the Ery-R strains belonging to serotype 14 were included in the same clonal group: most of them (9/11; 82%) presented indistinguishable PFGE profiles and two had closely related PFGE profiles. The clonal group Ery23F-B, composed by 8 (72.7%) of the 11 of the serotype 23F isolates, was more variable (6 PFGE profiles). All four 19A isolates presented identical PFGE profiles. A large proportion of all Ery-R strains (11/40; 27.5%) in this study belonged to serotype 14 and clustered in a single clonal group (Ery14-A). Except for one isolate possessing the mef(A/E) gene, the erm(B) gene was the erythromycin resistance determinant found in all of them. Isolates included in the clonal group Ery14-A presented susceptibility to penicillin and resistance to tetracycline and trimethoprim-sulfamethoxazole. They were all obtained from patients living in Rio de Janeiro state. Similarly, most (6/8; 75%) of the serotype 23F isolates belonging to the clonal group Ery23F-B were penicillin susceptible. All of them also presented tetracycline resistance, and the majority also presented trimethoprim-sulfamethoxazole resistance. Clonal group Ery23F-B was observed among isolates from Rio Grande do Sul, Rio de Janeiro, and São Paulo states. In contrast, the four serotype 19A strains (clonal group Ery19A-C) were resistant to penicillin. All of them were also resistant to tetracycline and to trimethoprim-sulfamethoxazole and were obtained from patients living in Rio Grande do Sul state. Seven erythromycin-susceptible (Ery-S) strains belonging to serotype 14 were analyzed by PFGE. Four of them were also penicillin susceptible (Pen-S) and the other three were Pen-R. The four Ery-S/Pen-S isolates presented PFGE profiles indistinguishable from those of isolates belonging to the clonal group Ery14-A, whereas the three Ery-S/Pen-R isolates presented different profiles, not related to the clonal group Ery14-A profiles or to any of the profiles identified among the other isolates. The PFGE profile obtained for the reference strain of the England149 clone was closely related to the PFGE profiles of isolates included in the clonal group Ery14-A (Fig. 1). PFGE analysis was also performed in seven Ery-S serotype 23F isolates (four PenS and three Pen-R) and four Ery-S serotype 19A isolates (three
318
MENDONÇA-SOUZA ET AL. serotype and susceptibility to penicillin are major markers of this clonal group. The PFGE analysis also revealed that strains included in clonal group Ery14-A were related to the England14-9 clone.11 The presence of this clone has already been documented in several European countries (United Kingdom, Portugal, Italy, Greece, Belgium, Germany), United States, Australia, and Argentina (http://www.pneumo.com). Interestingly, all of the Brazilian Ery-R isolates belonging to clonal group Ery14-A identified in this study presented tetracycline and trimethoprim-sulfamethoxazole resistance and all but one harbored the erm(B) erythromycin resistance genetic determinant, whereas the England14-9 clone was originally characterized by susceptibility to these drugs and by harboring the mef(A/E) erythromycin resistance gene (http://www.pneumo.com). The clonal group Ery23F-B was also found among Ery-S isolates; however, only the Pen-S serotype 23F strains analyzed presented PFGE profiles related to those of strains belonging to the clonal group Ery23F-B. These results suggest that penicillin susceptibility and serotype are also major characteristics for this group. Notably, the PFGE profiles of isolates included in the clonal group Ery23F-B were different from those of previously reported serotype 23F clones.11 On the other hand, the clonal group Ery19A-C was not detected among serotype 19A Ery-S strains investigated. These results suggest that resistance to erythromycin and to penicillin, as well as serotype, are important characteristics of this particular clonal group. Except for clone Ery14-A, none of the other clonal groups identified in the present study were clonally related to the reference strains of the antimicrobial-resistant clones included for comparison. In conclusion, the results of the present study indicate the low incidence of resistance to macrolides among pneumococci
FIG. 1. Dendrogram resulting from a computer-assisted analysis of PFGE profiles of 40 erythromycin-resistant S. pneumoniae isolates from Brazil and the reference strain of the England14-9 clone. The three major clonal groups identified are indicated. The scale represents the average percentage of similarity.
Pen-R and one Pen-S). The four Ery-S/Pen-S, serotype 23F isolates presented PFGE profiles related to those of the clonal group Ery23F-B, whereas the three Ery-S/Pen-R isolates had PFGE profiles unrelated to profiles of the isolates included in clonal group Ery23F-B. All of the Ery-S serotype 19A isolates presented PFGE profiles different from each other and not related to profiles of the clonal group Ery19A-C. Ery-R strains belonging to serotype 14 and included in clonal group Ery14-A were detected since 1993 in Rio de Janeiro state. However, previous studies in our laboratory revealed that serotype 14 isolates belonging to the clonal group Ery14-A are circulating at least since 1989, in different Brazilian locations, and that the majority of these strains were characterized by erythromycin and penicillin susceptibility.25 These results suggest that resistance to erythromycin was acquired later and that the
FIG. 2. PFGE profiles of SmaI-digested genomic DNA representative of the three major clonal groups identified among erythromycin-resistant S. pneumoniae isolates from Brazil. Lanes 1 and 14, Molecular size markers (in kilobases, lambda DNA concatemers ranging from 48.5 to 1,018.5 kb); lanes 2–4, serotype 14 Ery-R isolates, Sp 643, Sp 665, and Sp 698; lane 5, serotype 14 Ery-S isolate, Sp 139; lanes 6–9, serotype 23F Ery-R isolates, Sp 299, Sp 992, Sp 1107, and 1144; lane 10, serotype 23F Ery-S isolate, Sp 373; lanes 11–12, serotype 19A Ery-R isolates, Sp 862 and Sp 915; lane 13, serotype 19A EryS isolates, Sp 264.
ERYTHROMYCIN-RESISTANT PNEUMOCOCCI IN BRAZIL isolated in Brazil over the last decade. Even though the fact that the isolates were not recovered consecutively and the small number of Ery-R strains preclude a more consistent evaluation of temporal trends regarding the evolution of macrolide resistance among the bacterial population studied, the present report documents the occurrence of this resistance trait among isolates circulating in the areas studied and provides information on the diversity of Ery-R pneumococcal isolates from these areas. This constitutes a body of useful information for future studies, and also for comparative purposes with characteristics of isolates from other regions. The results demonstrated the predominance of the constitutive MLSB resistance phenotype among the S. pneumoniae isolates examined, whereas the M phenotype was not a frequent characteristic. Molecular analysis based on PFGE revealed the occurrence of three predominant clonal groups associated with serotypes 14, 19A, and 23F, suggesting that resistance to macrolides in the pneumococcal population investigated might be related to the spread of these three successful Ery-R clones. Nevertheless, horizontal spread of macrolide resistance genes should not be excluded. All Ery-R isolates belonging to serotype 14 were susceptible to penicillin and were included in a single clonal group (named Ery14-A) related to the England14-9 internationally spread clone. Continuous monitoring of the emergence and spreading of macrolide resistance among pneumococcal isolates from different Brazilian locations is necessary for a better understanding of the evolution of this resistance trait from a regional and global perspective.
ACKNOWLEDGMENTS This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Financiadora de Estudos e Projetos (FINEP), Fundação Universitária José Bonifácio (FUJB), and Ministério da Ciência e Tecnologia (MCT/PRONEX), Brazil. We thank Carlos Ausberto B. de Souza and Filomena Soares Pereira da Rocha of the Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, for technical assistance. We also thank Alma Ruth Franklin, Delois Jackson, and Terry Thompson of the Centers for Disease Control and Prevention for assistance with serotyping.
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