Streptococcus pneumoniae Strains Isolated in Brazil

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1994, p Vol. 32, No /94/$ Copyright C) 1994, American Society for Microbiology Distribution of Serotypes and Antimicrobial Resistance of Streptococcus pneumoniae Strains Isolated in Brazil from 1988 to 1992 JULIANA FURIAN SESSEGOLO,1 ANNA SARA S. LEVIN,2 CARLOS EMILIO LEVY,3 MARISE ASENSI,4 RICHARD R. FACKLAM,S AND LUCIA MARTINS TEIXEIRAI* Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941,1 Hospital das Clinicas da Universidade de Sdo Paulo, Sdo Paulo,2 Hospital das Clinicas, Faculdade de Medicina de Ribeirao Preto, Ribeirao Preto,3 and Instituto Fernandes Figueira, Fundaq4o Oswaldo Cruz, Rio de Janeiroj, Brazil, and Respiratory Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia Received 27 August 1993/Returned for modification 4 October 1993/Accepted 23 December 1993 Forty-two serotypes were identified among 288 Streptococcus pneumoniae strains isolated from patients living in Brazil. Serotyping was determined by the capsular typing test (Quellung reaction). Types 14 (10.4%), 6B (9.8%), 23F (8.0%), 5 (7.3%), 19F (6.9%), 6A (6.0%), and 1 and 4 (4.6%) were the most commonly identified strains. Two hundred twenty (76.4%) of the strains were of serotypes that are included in the 23-valent pneumococcal polysaccharide vaccine. If vaccine-related serotypes are also considered, the proportions of coverage in the vaccine are 82.3% (if type 6B alone is added) and 85.7% (if all the vaccine-related types are considered to be cross-protecting). Decreased susceptibility to penicillin, which was identified by using the l-,g oxacillin disk method as a screening test, was detected in 70 (26.7%) strains. The MICs of nine antimicrobial agents were determined by using the procedures recommended by the National Committee for Clinical Laboratory Standards. Seventy (35.9%) of the strains were resistant to tetracycline, 57 (29.2%) were resistant to sulfamethoxazole-trimethoprim, 3 (1.5%) were resistant to rifampin, 2 (0.80%) were resistant to penicillin, and 1 (0.5%) was resistant to chloramphenicol. The two penicillin-resistant strains were also resistant to or had decreased susceptibilities to cephalosporins. Forty-seven (17.9%) of the strains were intermediately resistant to penicillin, 17 (8.7%) were intermediately resistant to tetracycline, 13 (6.7%) were intermediately resistant to chloramphenicol, 12 (6.1%) were intermediately resistant to erythromycin, and 6 (3.1%) were intermediately resistant to rifampin. Although the introduction of penicillin as a therapeutic agent resulted in a significant decrease in the rates of mortality from pneumococcal infections, Streptococcus pneumoniae remains one of the agents most frequently associated with invasive infections. This microorganism is the most common cause of bacterial pneumonia and otitis media, the second most common cause of bacterial meningitis, and the third most common significant isolate from blood cultures (1, 2, 15, 21). For many years pneumococci were uniformly susceptible to penicillin. Even though most of the pneumococcal infections still respond to treatment with penicillin, isolation from patients and carriers of strains with decreased susceptibilities to this drug has been increasingly reported (4, 9, 10, 16-18, 21, 27, 29). Since the first description of pneumococci with decreased susceptibilities to penicillins in Australia in 1967 (9), strains with such a characteristic have been isolated from clinical specimens worldwide (8, 21, 25, 29). Resistance to alternative drugs such as chloramphenicol, erythromycin, tetracycline, clindamycin, rifampin, and sulfamethoxazole-trimethoprim has also been reported (1, 2, 13, 15, 16), and it is often associated with decreased susceptibility to penicillin (1). Multiple-antibiotic-resistant pneumococci were first reported in South Africa in 1977 (12), and in recent years, multidrug-resistant strains have been recovered from patients in several countries such as Spain, the United Kingdom, Italy, France, Belgium, Hungary, * Corresponding author. Mailing address: Respiratory Diseases Branch-MS-G07, Centers for Disease Control and Prevention, Atlanta, GA Phone: (404) Fax: (404) , (404) Pakistan, the former Czechoslovakia, the United States, and Canada (2, 5, 13, 19, 20, 24, 27). The continued frequency and severity of pneumococal infections and the increasing rates of pneumococcal strains resistant to antimicrobial agents underscore the need for the development of more effective therapeutic and preventive measures. Therefore, the recognition of prevalent serotypes of S. pneumoniae and their susceptibilities to antimicrobial agents in a given location is essential for guiding such prevention and control efforts. Data on such characteristics can be obtained and updated only by continuous local surveillance, since the data can vary according to several parameters such as geographical area, period of analyses, and kind of population. Serotyping and antimicrobial susceptibility information on the S. pneumoniae strains isolated from European and North American populations is widely available. However, very little is known about strains isolated from other populations. The purpose of the present study was to obtain information on the distributions of serotypes and antimicrobial susceptibilities of S. pneumoniae strains isolated from patients living in Brazil. MATERLALS AND METHODS Bacterial strains. A total of 345 S. pneumoniae strains were studied. Most of them were isolated from patients living in three large metropolitan areas in Brazil: Sao Paulo and Ribeirao Preto (Sao Paulo state) and Rio de Janeiro (Rio de Janeiro state) between 1988 and Most strains were consecutive isolates isolated at each hospital (Hospital das Clinicas da Universidade de Sao Paulo, Hospital das Clinicas

2 VOL. 32, 1994 da Faculdade da Medicina de Ribeirao Preto, and Instituto Fernando Figueiras, Rio de Janeiro), but there were periods of time when this did not occur. It was not possible to calculate the population served by each hospital. The strains were not preselected on the basis of their sources, phenotypic characteristics, or antimicrobial susceptibilities. The clinical sources included blood (n = 144), respiratory tract secretions (n = 74), cerebrospinal fluid (n = 50), ocular secretions (n = 13), and other secretions and body fluids (n = 64). Among the 74 respiratory tract isolates, 57 (75%) were recovered from patients with clinically suspected pneumonia, about 15% were from patients with upper respiratory infections such as sinusitis and rhinitis, and a few of them (about 10%) were isolated from individuals with suspected upper respiratory infections from which no specific agent was isolated. Identification. Pneumococci were identified by recognition of typical morphology on blood agar media, Gram staining characteristics, optochin susceptibility, and bile solubility (6). Two hundred eighty-eight strains were serotyped on the basis of capsular swelling (Quellung reaction) with type-specific pneumococcal antisera (Respiratory Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Ga.). The Danish system of nomenclature was used. Antimicrobial susceptibility testing. Two hundred sixty-two strains were screened for their susceptibilities to penicillin by the 1-,ug oxacillin disk method as recommended by the National Committee for Clinical Laboratory Standards (22). Each inoculum was prepared from growth on a Trypticase-soy-5% sheep blood agar plate (TSA-SB) after overnight incubation at 35 C. The microorganisms were suspended in sterile saline solution, and the turbidity was adjusted to match that of a 0.5 McFarland standard. The suspension was then streaked onto Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.) supplemented with 5% sheep blood (MHA-SB), and an oxacillin disk (BBL Microbiology Systems, Cockeysville, Md.) was applied. Zones of growth inhibition were measured after 20 to 24 h of incubation at 35 C. Isolates that had zones of inhibition of.20 mm around the oxacillin disks were considered penicillin susceptible, whereas those with zones of <20 mm were presumptively considered resistant (or as having decreased susceptibility to penicillin). MICs were determined by the agar dilution method recommended by the National Committee for Clinical Laboratory Standards (23) by using a Steers replicator. The MICs of penicillin for 262 strains were determined. The MICs of chloramphenicol, erythromycin, rifampin, sulfamethoxazoletrimethoprim, and tetracycline for 195 strains were determined. Eighty-eight of these strains were also tested for their susceptibilities to ampicillin, cephalothin, and vancomycin. Not all strains were available for testing with each antimicrobial agent because the tests were conducted at different times and we encountered storage problems. The drugs were incorporated into the agar in serial twofold concentrations, as follows: ampicillin, to 4.0,ug/ml; cephalothin, to 8.0,ug/ml; chloramphenicol, 0.25 to 64.0,ug/ml; erythromycin, to 4.0,ug/ml; penicillin, to 4.0,g/ml; rifampin, to 4.0 i,g/ml, sulfamethoxazole-trimethoprim, 608 and 32 to 1.18 and 0.06 jig/ml, respectively; tetracycline, 0.06 to 128,ug/ml; vancomycin, 0.06 to 2.0,ug/ml. All of the antimicrobial agents used were obtained from Sigma Chemical Co. (St. Louis, Mo.). The inocula for the agar MIC tests were prepared as described above for the oxacillin disk test and were then additionally diluted 1:10. This dilution of bacteria was used to inoculate MHA-SB plates containing series of increasing concentrations of the drugs listed above. The MIC was determined as the S. PNEUMONL4E IN BRAZIL 907 lowest concentration of antimicrobial agent that inhibited growth, as observed with an unaided eye. We rationalized that penicillin-resistant and relatively penicillin resistant strains would be the most likely candidate strains that may demonstrate resistance to the cephalosporins, so we tested 18 strains with such characteristics for cephalosporin resistance. For that, cefaclor, cefadroxil, cefixime, cephalexin, ceftriaxone, and cefuroxime were tested by a broth microdilution assay by using the Sensititre Panel (Radiometer America Inc., Westlake, United Kingdom). Briefly, the cultures used to prepare the inocula were grown on TSA-SB for 18 h in a candle extinction jar. A suspension of cells equal to that of a McFarland 0.5 density standard was prepared in cation-adjusted Mueller-Hinton broth (BBL Microbiology Systems). One hundred microliters of the suspension of cells equal to that of a 0.5 McFarland density standard was used to inoculate 10 ml of Mueller-Hinton broth supplemented with lysed horse blood. The MIC panels were inoculated with 100 pi of this suspension, and the panels were incubated overnight in a carbon dioxide incubator. Growth was read by hand by holding the MIC panel in front of an incandescent lamp. The MIC was defined as the lowest concentration of drug that inhibited growth. RESULTS The type distributions of 288 pneumococcal strains collected from 1988 to 1992 are given in Table 1. Forty-two different types were identified. Types 14, 6B, 23F, 5, 19F, 6A, 1, and 4 were identified the most frequently. Two hundred twenty (76.4%) strains had polysaccharide-type antigens identical to those included in the 23-valent pneumococcal vaccine. Type 6A (6.0%) was the most common among those not included in the vaccine. Among the types identified, types 6A, 7B, 7C, 1 lb, 18F, 18A, and 23B are vaccine-related types. Differences in the distributions of the pneumococcal types most frequently identified in the three major Brazilian cities were observed in the study (Fig. 1). Types 6B, 19F, and 23F predominated in Rio de Janeiro city. In Ribeirao Preto, types 1, 5, 6B, and 7F were identified the most frequently. Type 14 predominated in Sao Paulo city; this was followed by types 23F, 6B, 9V, and 6A. Some types were frequently identified in all three cities (types 5, 6B, 14, and 23F), while other types were found in only one city (for example, 35A in Ribeirao Preto). However, the type distribution may be affected by our unequal sampling of the patients in each city. Use of the oxacillin disk screening test to detect decreased susceptibilities to penicillin allowed us to predict that 70 (26.71%) of the 262 pneumococcal strains tested would have some resistance to penicillin. When they were tested for MICs by the agar dilution procedure, 47 strains were relatively resistant to penicillin and 2 strains were fully resistant to penicillin. All of the remaining strains were susceptible to penicillin. Among the strains that were predicted to be susceptible to penicillin, none was resistant. Most of the strains for which there were discrepancies between the results of the oxacillin disk test and the MIC test had zones of inhibition with sizes ranging from 16 to 19 mm, and the MICs for the strains were 0.03 or 0.06,ug/ml. The MICs for 50% of isolates tested, the MICs for 90% of isolates tested, mode MICs, and MIC ranges of each of the nine antimicrobial agents tested are given in Table 2. Nearly 36% of the strains tested were fully resistant to tetracycline, and 29% were resistant to sulfamethoxazole-trimethoprim. Although most of the strains were susceptible to low levels of penicillin (MICs, <0.06 jig/ml), 47 (17.9%) were relatively

3 908 SESSEGOLO ET AL. TABLE 1. Relationship between serologic types of strains from patients in Brazil and serologic types included in the pneumococcal polysaccharide vaccine Serotype No. isolates (%) of Vaccine type (4.6) (3.1) (4.6) (7.3) 6B (9.8) 7F... 9 (3.1) (1.0) 9N... 9 (3.1) 9V (3.5) 11A... 8 (2.7) (10.4) 17F... 6 (2.1) 18C... 6 (2.1) 19A... 9 (3.1) 19F (6.9) 22F... 3 (1.0) 23F (8.0) Total (76.4) Vaccine-related type 6A (6.0) 7C... 3 (1.0) 18A... 2 (0.7) 18F... 2 (0.7) Othersa... 3 (1.0) Total (9.4) Non-vaccine-related type 15C... 4 (1.4) (2.1) 24A... 2 (0.7) 24F... 3 (1.0) 28A... 3 (1.0) 28F... 3 (1.0) 35A... 4 (1.4) (0.7) (0.7) Othersb... 8 (2.7) Nontypeable... 4 (1.4) Total (14.2) a One each of serotypes 7B, lib, and 23B. b One each of serotypes IOF, 15A, 21, 29, 31, 34, 39, and 40. resistant to penicillin (MICs, 0.12 to 1.0 p.g/ml) and 2 strains were fully resistant to penicillin (MICs, 2.0,ug/ml). Intermediate resistance to chloramphenicol, erythromycin, and rifampin was detected among several strains. The two strains that were fully resistant to penicillin were also resistant to multiple antimicrobial agents (Table 3). Strain Sp-65 (type 19A) was isolated from the blood of a patient with pneumonia in Ribeirao Preto in 1988, and it was resistant to rifampin, tetracycline, and cefaclor. It was also intermediately resistant to several other antimicrobial agents. Strain Sp-129 (type 19F) was isolated from a bronchial aspirate in Rio de Janeiro in This strain was resistant or had decreased susceptibility to several of the antimicrobial agents tested, including cephalosporins with different spectra of activity. Resistance to other antimicrobial agents was also observed among 91 (57.23%) penicillin-susceptible and 20 (55.55%) relatively penicillin-resistant strains (Table 4). Sulfamethoxazole-trimethoprim- and tetracycline-resistant, penicillin-susceptible strains and sulfamethoxazole-trimethoprim-resistant, relatively penicillin-resistant strains were the most frequently observed patterns among strains resistant to multiple antimicrobial agents. Strains relatively resistant to penicillin were more frequently associated with serotypes 6B (11 strains), 23F (7 strains), 14 (6 strains), and 19F (5 strains); this was followed by serotypes 9N and 19A (3 strains each), 35A (2 strains), and serotypes 1, 3, 4, 6A, 11A, and 18C (1 strain each). Although they were identified quite frequently, all of the type 5 strains were susceptible to penicillin. DISCUSSION J. CLIN. MICROBIOL. Determination of the serotype distributions among pneumococcal strains, as well as their associations with different infections, including those of the respiratory tract (12, 29), is very important for estimating the efficacy of the 23-valent antipneumococcal vaccine. Considering that the type distribution can vary according to the geographical areas and period of time (29), it may be necessary to monitor the type distribution periodically or continuously. A total of 76.4% of the Brazilian strains analyzed in the present study were of serotypes that are included in the pneumococcal vaccine. However, it is believed that there may be cross-protection against strains with vaccine-related serotypes because of their close antigenic relationship to serotypes included in the vaccine. Therefore, if cross-protection is realized, the proportions of coverage in the vaccine are 82.3% if serotype 6B alone is added and 85.7% if all of the vaccinerelated serotypes are cross-protecting. This is lower than that previously found in Europe and the United States (89%) (14, 26). The formulation of the 23-valent pneumococcal vaccine was based on the serotype distributions of strains isolated in a limited number of locations worldwide (26). In a previous report from our laboratory, 82.2% of the strains isolated from patients with meningitis in the city of Rio de Janeiro belonged to serotypes included in the vaccine (28). Overall, the most frequently identified S. pneumoniae serotypes were 14, 6B, 23F, 5, 19F, 6A, 1, 4, 9V, 3, 7F, 9N, 19A, and 1lA. This type distribution does not seem to differ appreciably from that found in other parts of the world (1, 14, 16). The one exception may be the increased number of type 5 strains in our study; type 5 strains ranked as the 4th most common, whereas worldwide data rank them 15th (26). Nevertheless, the type profiles of the S. pneumoniae strains identified in the present study indicate that the 23-valent pneumococcal vaccine would provide potential protection against pneumococcal infections in the locations that were included in the present study. However, considering the large size of Brazil, additional studies on the pneumococcal type distribution in other regions of Brazil are necessary to determine potential vaccine coverage Ṫhe acquisition of resistance to penicillin and other antimicrobial agents, such as tetracycline, chloramphenicol, erythromycin, sulfamethoxazole-trimethoprim, rifampin (15), and, more recently, cephalosporins (3), by S. pneumoniae has caused there to be a reduction in the number of effective antimicrobial treatment regimens for pneumococcal infections. The increasing levels of resistance of S. pneumoniae to various antibiotics points out the importance of epidemiologic monitoring of pneumococcal infections. Relative or full resistance to penicillin has been associated with 22 different serotypes worldwide (15, 29). In the present study, penicillin resistance was associated with 13 different serotypes. Serotypes 6B, 35A, 23F, 19A, 19F, and 9N were the types most frequently identified with antimicrobial resistance;

4 VOL. 32, 1994 S. PNEUMONMIE IN BRAZIL A 6B 7F 9N 9V 14 l9a19f 23F 35A Serotypes FIG. 1. Distributions of the most frequently occurring serotypes of S. pneumoniae in the following three Brazilian cities: Rio de Janeiro, 67 strains; Ribeirao Preto, 72 strains; and Sao Paulo, 136 strains, which are indicated by the bars from left to right, respectively. this was followed by types 14, 18C, 4, 1lA, 3, 1, and 6A. Strains with resistance to multiple antibiotics have most frequently been reported to be type 6, 19, or 23 (1). The two fully penicillin-resistant strains identified in the present study, which were also resistant to other antimicrobial agents, belonged to serotype 19 (19A and 19F, respectively). The numbers of pneumococcal strains with decreased susceptibilities to penicillin have been increasing worldwide (2). Our results (28) support this observation; we compared the results for penicillin resistance among strains collected from patients residing in Rio de Janeiro between January 1981 and August 1982 with the current results for strains collected from the same population. Penicillin resistance, either relative or full, was not detected in 1981 and 1982, whereas the strains isolated from 1988 to 1992 show relative resistance (13 [19.40%] of the strains) and full resistance (1 strain [1.50%]). These results point out the importance of continuous monitoring of S. pneumoniae strains for antimicrobial resistance in Brazil. We found only intermediate resistance to erythromycin in about 6% of the strains in our current study. This agrees with the results of other studies (15). Although resistance to chloramphenicol is not a serious problem worldwide, it has been reported in Spain, Hungary, and Pakistan (7, 18, 20). In our study, only one pneumococcal strain was fully resistant and three strains were intermediately resistant to chloramphenicol. Only one of the strains intermediately resistant to chloramphenicol was relatively resistant to penicillin. The most frequently identified antimicrobial resistance in the present study was to tetracycline (44.6%) and sulfamethoxazole-trimethoprim (29.2%). This level of tetracycline resistance has also been observed in Europe, Asia, and the Middle East (15) and may reflect the widespread use of this drug. The rate of resistance to tetracycline observed in the present study compared with that found in the previous study (28) of the same population did not change. The resistance to sulfamethoxazole-trimethoprim, however, showed a dramatic increase over that observed in our previous study of the same population (28). In 1981 and 1982, only one strain (1%) with resistance to sulfamethoxazole-trimethoprim was identified, TABLE 2. Antimicrobial susceptibilities among S. pneumoniae strains isolated in Brazil between 1988 and 1992 Antimicrobial agent Total no. of MIC (p.g/ml)" % of strains' strains tested Range Mode 50% 90% S I (RR) R Ampicillin Cephalothin Chloramphenicol Erythromycin Penicillin 262 < Rifampin 195 <0.015-> Sulfamethoxazole-trimethoprim 195 <608/ / / / / Tetracycline 195 < Vancomycin a 50% and 90%, MICs for 50 and 90% of strains tested, respectively. b S, susceptible; I (RR), intermediate or relatively resistant; R, resistant.

5 910 SESSEGOLO ET AL. TABLE 3. Antimicrobial resistance to penicillin of two S. pneumoniae strains isolated in Brazil MIC (pug/mi) for strain: Antimicrobial agent Sp-65 Sp-129 (type 19A) (type 19F) Penicillin 2 2 Ampicillin 2 2 Chloramphenicol 4 16 Erythromycin Rifampin >4 >4 Tetracycline 64 8 Vancomycin Sulfamethoxazole-trimethoprim 38/2 > 76/4 Cephalexin 8 >16 Cephalothin 4 16 Cefaclor >32 >32 Cefadroxil 4 > 16 Cefixime > 1 > 1 Ceftriaxone Cefuroxime 4 16 whereas nearly 30% of the strains in the current study were resistant to sulfamethoxazole-trimethoprim. The rate of resistance to rifampin was low, and all strains were susceptible to vancomycin, in agreement with recent reviews (1, 11, 15). These drugs may be important alternatives for use in the treatment of infections caused by S. pneumoniae strains that are resistant to penicillin and other antimicrobial agents. Although the cephalosporins represent important alternatives for the treatment of pneumococcal infections (1, 11), especially for meningitis, the first therapeutic failures with the use of these drugs have been reported (3). In the present study, we tested the penicillin-resistant and some of the relatively penicillin-resistant strains for cephalosporin resistance. The two multidrug-resistant strains showed different levels of TABLE 4. Antimicrobial resistance profiles of penicillin-susceptible and relatively penicillin-resistant S. pneumoniae strains Antimicrobial No. of isolates resistance Penicillin Relatively penicillin profile' susceptible" resistant' C 3 C/E 1 C/Ra/SXT/Te C/SXT/Te 3 C/Te 3 E 1 2 E/SXT/Te 1 E/Te 4 Ra 1 Ra/SXT/Te 2 Ra/Te I SXT SXT/Te 26 1 Te 35 5 Total " C, chloramphenicol; E, erythromycin; Ra, rifampin; SXT, sulfamethoxazoletrimethoprim; Te, tetracycline. 'The total number of strains in this category that were tested for their susceptibilities to other antimicrobial agents, 159. ' The total number of strains in this category that were tested for their susceptibilities to other antimicrobial agents, 36. susceptibility to narrow-spectrum, expanded-spectrum, and broad-spectrum cephalosporins. The results of the present study show that the distributions of pneumococcal types found in Brazilian patients are not much different from those in other populations that have been studied. Most of the serotypes of the strains recovered from Brazilian patients are found in the 23-valent pneumococcal vaccine, indicating the potential usefulness of the vaccine in the Brazilian population. The present study also documents the increasing problem of antimicrobial resistance, especially to penicillin, sulfamethoxazole-trimethoprim, and tetracycline, among S. pneumoniae strains isolated from patients in Brazil. 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