ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, Oct. 1992, p. 2176-2184

Vol. 36, No. 10

0066-4804/92/102176-09$02.00/0

Analysis of Multiply Antimicrobial-Resistant Isolates of Streptococcus pneumoniae from the United States LINDA K. McDOUGAL,l* RICHARD FACKLAM,2 MICHAEL REEVES,3 SUSAN HUNTER,3 JANA M. SWENSON,1 BERTHA C. HILL,1 AND FRED C. TENOVER1 Nosocomial Pathogens Laboratory Branch, Hospital Infections Program,1 Respiratory Diseases Branch,2 and Meningitis and Special Pathogens Branch,3 Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia 30333

Received 30 March 1992/Accepted 14 July 1992 Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotpes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillinsusceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotpe 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone ofS. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.

Streptococcus pneumoniae is a major cause of human morbidity and mortality in the United States. Pneumococcus is the most common cause of pneumonia, otitis media, and bacteremia in infants and children worldwide and is the third most common cause of meningitis in the same group (21). It is also the most frequent cause of bacterial meningitis and pneumonia in adults (47). Because pneumococci are relatively common pathogens, the emergence of high-level penicillin resistance, particularly when coupled with other resistance determinants, poses serious problems for antimicrobial therapy of such infections. Multiresistance is defined as resistance to at least three different classes of antimicrobial agents (19, 22). Multiresistant pneumococci have been reported for several different capsular types (serotypes), although the overwhelming majority of resistant strains are in serotypes 6, 14, 19, and 23 (21, 22, 32, 46). Pneumococci of these four serotypes account for the majority of disease in children (13). The first multiresistant pneumococci which demonstrated resistance to penicillin, chloramphenicol, tetracycline, erythromycin, clindamycin, and sulfamethoxazole-trimethoprim were isolated in South Africa in 1977 (2, 19). A high prevalence of drug-resistant strains was subsequently identified in Spain (5, 9, 26). In recent years, multiresistant pneumococci have been recovered from patients in the *

United Kingdom (10, 12, 31), Italy (22), France (11), Belgium (22, 44), Pakistan (8), Czechoslovakia (6, 30), and Hungary (29). In the United States, these strains are still unusual and are rarely isolated (20, 41). In 1991, Munoz et al. (34) reported the intercontinental spread of a multiresistant clone of S. pneumoniae serotype 23F. Those investigators examined several isolates of pneumococci from Ohio and Spain and demonstrated that the organisms had similar penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) patterns, and PBP 2b and PBP 2x gene restriction endonuclease profiles. We expanded those studies to determine whether all multiresistant serotype 23F strains in the United States disseminated from one resistant clone and to determine the extent of dissemination of the clone by analyzing the relatedness of 22 multiresistant serotype 23F pneumococci from 17 U.S. cities. To do this, we compared the isolates' PBP profiles, MLEE profiles, ribotypes, serotypes, and antimicrobial resistance patterns with those of other multiresistant and penicillin-susceptible pneumococci of several different serotypes from other parts of the United States and the world.

MATERIALS AND METHODS

Bacterial strains and growth conditions. S. pneumoniae isolates were selected from strains submitted to the Centers for Disease Control on the basis of identification, serotype, and antimicrobial resistance profile. The organisms were

Corresponding author. 2176

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2177

TABLE 1. Properties of resistant clinical isolates of S. pneumoniae Strain'

85UT23F 88AZ23F 88FL23F 89NY23F 900K23F 900H23F 90IN23F 90TX23F 91CA23F 91MO23F 91NC23F 91AZ23F 91AZ23FTcS 91MT23F 91FL23F 87SP23F 89SA23F 84PA23F 85PA23F 89CA23FEmr 89NB23FEmr

9ONB23FEmr 91DC23FEmr 91CA19FEmr 83MI23FEmr 89FL23FCms 91GA23FCmS 91FL23FCms 85NY23FPcS R6 81CO6B 85NY19A 90GA14 90MI14 90M06B 91VA14

Penicillin MIC

(Ig/ml)

Serotype

2.0 1.0 1.0 4.0 2.0 2.0 2.0 2.0 1.0 2.0 1.0 2.0 1.0 2.0 2.0 2.0 2.0 4.0 2.0 2.0 2.0 4.0 2.0 2.0 1.0 2.0 2.0 2.0 0.01 0.01 1.0 4.0 2.0 1.0 2.0 2.0

23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F 23F

19F 23F 23F 23F 23F 23F NTd 6B 19A 14 14 6B 14

Location

Isolation:

Utah Arizona Florida New York Oklahoma Ohio Indiana Texas California Missouri North Carolina Arizona Arizona Montana Florida

Spain South Africa Pennsylvania Pennsylvania California Nebraska Nebraska District of Columbia California Michigan Florida Georgia Florida New York New York Colorado New York Georgia Michigan Missouri Virginia

Year

Sitec

Cm

1985 1988 1988 1989 1990 1990 1990 1990 1991 1991 1991 1991 1991 1991 1991 1987 1989 1984 1985 1989 1989 1990 1991 1991 1983 1989 1991 1991 1985 ? 1981 1985 1990 1990 1990 1990

CSF BLD NP THT BLD EAR CSF CSF NP NP BLD SPU BLD SPU EYE ? ? ? SPU SIN SIN BLD BLD BLD ? SPU EAR BLD ? ? CSF NP SIN BLD BLD BLD

R R R R R R R R R R R R R R R R R R R R R R R R R S S S S S R R R R R R

Resistance to other drugs' Tc

SXT

Em

R R R R R R R R R R R R S R R R R R R R R R R R R S S S S S R R R R R S

R R R R R R R R R R R R R R R R R R R R R R R R R R R R

S S S S S S S S S S S S S S S S S S S R R R R R R S S S S S S S R R S R

S S S R R R R R

a Strains are coded to reflect the year of isolation, location, serotypes, and any unique characteristic; Tcs, tetracycline susceptible; Em', erythromycin resistant; cms, chloramphenicol susceptible; Pcs, penicillin susceptible. b Cm, chloramphenicol; Tc, tetracycline; SXT, sulfamethoxazole-trimethoprim; Em, erythromycin; R, resistant; S, susceptible. c CSF, cerebrospinal fluid; BLD, blood; NP, nasopharyngeal; THT, throat; SPU, sputum; SIN, sinus. d

NT,

nontypeable.

identified by susceptibility to ethylhydrocuprein and bile solubility; they were serotyped on the basis of capsular swelling (Quellung reaction) with type-specific antisera (Respiratory Diseases Branch, Centers for Disease Control) (4). Only one isolate was included from each cluster of similar isolates from the same locale or institution. All multiresistant strains were resistant to penicillin (MICs, 1 to 4 p,g/ml) and chloramphenicol (MICs, 28 ,ug/ml) and at least one other antimicrobial agent including tetracycline, sulfamethoxazole-trimethoprim, or erythromycin. Twenty-nine multiresistant strains from the United States were evaluated. The location (state or country); year; specimen site; serotype; penicillin MIC; and antimicrobial resistance patterns to chloramphenicol, tetracycline, sulfamethoxazole-trimethoprim, and erythromycin are listed in Table 1. Other penicillin-resistant isolates from the United States and multiresistant isolates from other countries, which were selected for PBP patterns, MLEE profiles, and ribotype comparisons, are also listed in Table 1. Penicillin-susceptible reference strains included the R6 derivative of the unencapsulated Rockefeller University strain S. pneumoniae R36A (derivative of a capsular type 2 strain, D39; American Type Culture

Collection, Rockville, Md.) and one other serotype 23F clinical isolate from the United States (from the Centers for Disease Control collection). Susceptibility testing. The antimicrobial susceptibility profiles of the organisms were determined by broth microdilution by using cation-adjusted Mueller-Hinton broth (Becton Dickinson Microbiology Systems, Cockeysville, Md.) supplemented with 3 to 5% lysed horse blood as described in the National Committee for Clinical Laboratory Standards publication M7-A2 (36). Labeling of PBPs. For labeling of PBPs, bacteria were grown statically in C medium (25) supplemented with 0.2% yeast extract (Difco Laboratories, Detroit, Mich.) at 35°C with an increased CO2 concentration. Growth was followed by determining the organism density with A-Just (Abbott Laboratories, Irving, Tex.) and by comparing the readings with a no. 0.5 McFarland standard. PBPs were labeled from samples of mid-logarithmic-phase cultures with equal optical densities (approximately 1.5 x 108 CFU/ml). For radioactive labeling of PBPs, [3H]benzylpenicillin, ethylpiperidinium salt (specific activity, 29.1 Ci/mM) (38), provided by Merck Sharp & Dohme Research Laboratories, Rahway, N.J., was

2178

McDOUGAL ET AL.

used throughout the study. Broth cultures (1.5 ml) were centrifuged, and the intact cells were suspended in 25 ,ul of 10 mM potassium phosphate buffer (pH 7.0). Samples were incubated for 10 min at 37°C with [3H]benzylpenicillin (final concentration, 10 ,ug/ml). Cells were lysed with a 1.8% final concentration of Sarkosyl NL-97 (Sigma Chemical Co., St. Louis, Mo.). The samples were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (15, 48) (acrylamide and N,N'-methylenebisacrylamide concentrations, 7.5 and 0.16%, respectively, in the separating gel and 3.6 and 0.08%, respectively, in the stacking gel) with the Mini-PROTEAN II dual slab cell (Bio-Rad Laboratories, Richmond, Calif.) at a constant voltage of 200 V. The PBPs were detected by fluorography with Kodak X-Omat AR film (3) after exposure for 4 days at -70°C. Binding of [3H]benzylpenicillin to the PBPs was measured by scanning densitometry (LKB Ultroscan XL Laser Densitometer; LKBPharmacia Corp., Piscataway, N.J.) interfaced with an AT&T microcomputer programmed to integrate densitometric peaks. The PBP profile for each isolate was repeated several times with exponentially growing cultures to optimize comparison of PBP patterns and to confirm the stability of the pattern. MLEE. Strains were grown in 500 ml of CAT medium (37) for 8 h at 35°C in 5% CO2 until the cell density in the exponential growth phase equaled that of a no. 1 McFarland standard. The growth was pelleted and resuspended in breaking buffer (10 mM Tris HCl, 1 mM sodium EDTA [pH 7]) and stored at -70°C. Thawed cell suspensions were sonicated (model XL2020 with cup probe 431A containing an ice water slurry; Heat Systems Inc., Farmingdale, N.Y.) at 80% power with a 10-s pulse for 2 min. Cell debris was removed by centrifugation at 20,000 x g, and lysates were frozen at -70°C. Thawed lysates were absorbed into paper wicks and inserted into vertical slits cut in a 11.5% starch gel (Connaught Laboratories, Swiftwater, Pa.). After electrophoresis for 6 h, gel slices were stained for specific enzyme activities as described by Selander et al. (39). The 20 enzymes assayed were alcohol dehydrogenase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose 6-phosphate dehydrogenase, glutamate dehydrogenase NADP, diaphorase NADH, indophenol oxidase, aspartate carbamoyltransferase, nucleoside phosphorylase, glutamate oxaloacetate transaminase, hexokinase, fructose kinase, carbamylate kinase, adenylate kinase, phosphoglucomutase, esterase a-Pro, leucine aminopeptidase, peptidase Leu-GlyGly, peptidase Phe-Leu, and phosphoglucose isomerase. Each unique combination of alleles in the strains was designated an electrophoretic type (ET), and all ETs were compared with each other to determine genetic relatedness. The average-linkage method of clustering the ETs and principal coordinate analyses of the ETs were done as described by Selander et al. (39) by using an SAS macro developed by Jacobs (18). DNA isolation, restriction endonuclease digestion, fractionation by agarose gel electrophoresis, and hybridization with rRNA probe. Genomic DNA was prepared in situ in agarose blocks (40). The culture was grown in Todd-Hewitt broth (Becton Dickinson Microbiology Systems) with 10% horse serum at 35°C in increased CO2 until the mid- to late log phase of growth, when the turbidity of the culture equaled that of a no. 1 McFarland standard. Samples of 1.5 ml were harvested by centrifugation, washed with 1.5 ml of 1 M NaCl-10 mM Tris HCl (pH 7.6), resuspended in 700 ,ud of the same solution, and mixed with 1 ml of 1% (wt/vol) agarose before solidifying in molds. The cells were lysed in situ with

ANTIMICROB. AGENTS CHEMOTHER.

EC buffer (6 mM Tris HCl [pH 7.6], 1 M NaCl, 100 mM sodium EDTA [pH 7.6], 1% [wt/vol] Sarkosyl, 1 mg of lysozyme per ml) overnight at 37°C. After digestion with proteinase K, the agarose blocks were treated twice for 12 h with 1 mM phenylmethylsulfonyl fluoride in TE (10 mM Tris HCl [pH 8.0], 1 mM sodium EDTA) at room temperature, washed three times with TE for 2 h at room temperature, and then stored at 4°C in TE. Slices 1 to 2 mm thick were transferred to 1.5-ml Eppendorf tubes containing 1 ml of the manufacturer's recommended lx enzyme reaction buffer (Gibco-Bethesda Research Laboratories, Gaithersburg, Md.) and were incubated overnight at room temperature. The agar slices were incubated overnight at 37°C in fresh reaction buffer with 100 Fg of bovine serum albumin per ml and 3 ,ul of HindIII enzyme. Samples were electrophoresed through 0.8% (wt/vol) agarose gels in TBE buffer (89 mM Tris, 89 mM boric acid [pH 8.3], 2.5 mM sodium EDTA) for 20 h at 35 V. The DNA was treated with 0.2 N NaOH in 0.5 M NaCl-4x TAE buffer and electroblotted onto a nylon membrane (MagnaGraph; Micron Separations, Inc., Westboro, Mass.) in 1 x TAE buffer (10 mM Tris [pH 7.8], 5 mM sodium acetate, 0.5 mM EDTA). The membrane was baked at 80°C for 2 h. Commercially available 16S-23S rRNA from Escherichja coli (Sigma Chemical Co., St. Louis, Mo.) was partially hydrolyzed in 10 mM Tris HCl (pH 9.5) for 5 min and quickly cooled on ice. The fragments generated were labeled at the 5' ends with [-y-32P]ATP (specific activity, 3,000 Ci/mmol) by using T4 polynucleotide kinase (Gibco-Bethesda Research Laboratories) (14, 42). After incubation at 37°C for 60 min, the labeled nucleic acids were separated from the unreacted nucleotides by chromatography on a BioGel P60 column. The specific activity of the probe was at least 8 x 106 cpm/50 ml of hybridization buffer. Hybridization of DNA fragments on nylon filters with rRNA was done as described by Maniatis et al. (28), except that prehybridization was in 6x standard saline citrate (SSC; lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-lOx Denhardt solution for 60 min, and hybridization was in 2x SSC-5x Denhardt solution0.02 M Tris hydrochloride (pH 7.4)-0.1% SDS-41% formamide. Hybridization was performed at 37°C overnight. Filters were washed three times for 15 min each time at 37°C in washing solution (2x SSC, 0.1% SDS) and were further washed twice for 15 min each time in a second solution (0.2x SSC, 0.1% SDS) (1). After drying at room temperature, the filters were autoradiographed for 24 h at -700C by using Kodak X-OMAT film and two intensifying screens. RESULTS Antimicrobial susceptibility profiles and serotypes. Twentynine isolates from the United States manifested resistance to penicillin (MICs, 1 to 4 pg/ml) and resistance to chloramphenicol, tetracycline (except isolates 91AZ23FTcS [susceptible] and 91VA14), and sulfamethoxazole-trimethoprim (except isolate 81CO6B) (Table 1). Five of the isolates were also resistant to erythromycin and clindamycin, while four others were resistant to erythromycin but not clindamycin (data not shown). Of the 29 isolates, 22 were serotype 23F, 3 were serotype 14, 2 were serotype 6B, 1 was serotype 19A, and 1 was serotype 19F. The first multiresistant pneumococcus was isolated in the United States in 1981. Five multiresistant strains (four serotype 23F and one serotype 19A) were isolated during 1983 and 1985. Most (n = 23) of the multiresistant isolates, however, were isolated since 1988, and 18

MULTIRESISTANT S. PNEUMONAE

VOL. 36, 1992

55 i ~~~ Nt AZ O 272 6B 2 T' Pe' f'PN

NYN1V4~~~~A?4Y 19A

23 :'

14

M

89 91 FL~ FL 23F 3 cm" Cm'

2179

~~~91

VA 14

FIG. 1. Fluorographs of electrophoretic profiles of polyacrylamide gels (3.6% stacking gel and 7.5% separating gel) of PBPs of penicillin-resistant isolates from the United States, Spain, and South Africa and susceptible strains R6 and 85NY23FPcS. See Table 1 for strain

nomenclature.

2180

ANTIMICROB. AGENTS CHEMOTHER.

McDOUGAL ET AL.

EET Strain Code 7 13 17 16 3 6 2

1

4 5 8 12 18 14 15 11 10 9

83MI23FEm r 81C06B 90M06B 90MI14 85PA23F

9ONB23FEm r 84PA23F 91 FL23F 91 CAl 9FEm r 91 MT23F 91 M023F 91AZ23F 91 NC23F 91 DC23FEm r 91AZ23FTcS 91CA23F 90TX23F 90IN23F 90OH23F 87SP23F 900K23F 89NY23F 89SA23F 88FL23F 85UT23F 88AZ23F 89CA23FEm r 89NB23FEm r 89FL23FCm s R6 91VA14 85NY19A 90GA14 85NY23FPcs 91 FL23FCm s 91GA23FCm -

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 0.32

Relatedness Index

FIG. 2. Dendrogram depicting genetic distance and overall relatedness of multiresistant, penicillin-resistant, and penicillin-susceptible S. pneumoniae isolates in the United States, Spain, and South Africa on the basis of MLEE. See Table 1 for strain nomenclature.

were serotype 23F. The isolates from Spain and South Africa were serotype 23F; both were resistant to penicillin (MIC, 2

,ug/ml), chloramphenicol, tetracycline, and sulfamethoxazole-trimethoprim and susceptible to erythromycin. PBP patterns. Figure 1 shows the PBP patterns of 30 strains. Separate gels are shown in each panel. At least six PBP profiles were identified among the multiresistant isolates. The PBP proffles of 21 multiresistant serotype 23F strains isolated in the United States and the multiresistant serotype 23F strains from Spain and South Africa were identical (Fig. 1A to E). The PBP patterns of the serotype 23F tetracycline-susceptible isolate (91AZ23FTcI; Fig. 1H, lane 2) and the erythromycin-resistant isolates (89NB23 FEmr; Fig. 1A, lane 1; 89CA23FEmr, Fig. 1C, lane 1; 9ONB 23FEmr, Fig. 1D, lane 5; and 91DC23FEmr, data not shown) were identical to those of the clonal group. The serotype 19F multiresistant isolate from California (91CA19FEmr) (Fig. 1D, lane 6) also had a PBP pattern identical to that of the clonal group. One multiresistant serotype 23F isolate (83MI 23FEmr) had a unique PBP profile (Fig. 1E, lane 2). Different PBP profiles were identified for serotype 23F isolates that manifested penicillin resistance but that were susceptible to chloramphenicol and tetracycline (Fig. 1E, lane 3; Fig. 1H, lane 3). The PBP patterns of multiresistant isolates of other

serotypes (serotypes 19A, 14, and 6B; Fig. 1F to H) were different. MLEE. Characterization of 36 pneumococcal isolates by MLEE with 20 enzymes is represented by a dendrogram depicting the genetic relatedness among strains (Fig. 2). Of the 22 multiresistant serotype 23F isolates from the United States, 16 had identical allelic profiles. The serotype 23F isolates from Spain and South Africa had MLEE proffles identical to the proffles of the clonal group of 16 isolates from the United States. Two strains (84PA23F and 85PA32F) with identical PBP patterns and ribotypes had slightly different MLEE allelic profiles. Three of the erythromycin-resistant serotype 23F isolates also varied slightly from the profile of the clonal group by one to three alleles. Two strains (resistant isolates 91DC23FEmr and 91CA19FEm') with identical PBP patterns but different ribotypes had MLEE profiles that were the same as those of the clonal group. The MLEE profiles of the other pneumococcal isolates tested exhibited even greater genetic diversity, with the resistant 83MI23 FEmr isolate representing the least relatedness (Fig. 2). rDNA gene restriction patterns. The rDNA patterns for 24 isolates determined by using the restriction enzyme HindIII are shown in Fig. 3 (9 isolates are repeated as reference points). Sections of different gels are shown in each panel.

VOL. 36, 1992

91 91

MULTIRESISTANT S. PNEUMONIE

91 91 Y9 99 91 91

9

AZ MT MO FL CA NB CA DC FL 23F 23F 23F 23F 2W 2W 19 23 23F Em' Emt Em' EmvCm' Te

I!6 90 MO 6B

9U 90 KO 89 83 85 89 87 91 91 90 90 89 88 VA GA FL MI NY SA SP NC AZ OH NB CA AZ 14 14 23F 23F 23F 23F 23F23F 23F 23F 23F 23F 23F

Cm' Emt Pc

2181

Ml DC CA PA PA NB NB CA SP NY 23F 23 23F 2W 23F 23F 23F 23F 23F 23F Pe Em' Emt Em' Em' E,m Em' FIG. 3. Autoradiographs of electroblots of HindIII-cleaved S. pneumoniae DNA hybridized with 32P-labeled E. coli 16S and 23S rRNA. Fragments were separated by electrophoresis through 0.8% agarose gels. See Table 1 for strain nomenclature.

23F isolates from Spain and South Africa had identical PBP profiles, ribotypes, and MLEE profiles. An additional four isolates had identical PBP profiles and either identical ribotypes or MLEE profiles. Three isolates had the same PBP pattern as that of the clonal group but had different ribotypes and MLEE profiles. Figure 5 is a map of the United States representing the locations of multiresistant S. pneumoniae isolates related by identical PBP patterns, ribotypes, and MLEE profiles (clonal) and the locations of closely related isolates with the

Emt Emt

All isolates with unique PBP patterns also had different ribotypes. Of 22 multiresistant serotype 23F isolates from the United States, 17 had identical ribotypes. This ribotype was identical to the ribotype of the multiresistant serotype 23F strains from Spain (Fig. 3B, lane 10) and South Africa (Fig. 3C, lane 7). This group included an isolate from Arizona (91AZ23FTcS; Fig. 3A, lane 1) which was susceptible to tetracycline. Although they had identical PBP profiles, three multiresistant serotype 23F isolates and the serotype 19F isolate from California, which were erythromycin resistant and clindamycin susceptible, had different ribotype patterns (Fig. 3A, lanes 6 to 8; Fig. 3B, lanes 3, 7, and 8). Strain 89CA23FEmr, which was erythromycin and clindamycin resistant, had a unique ribotype (Fig. 3A, lane 5). The multiresistant serotype 23F Michigan isolate (83MI23FEmr), which was isolated in 1983 and which had a unique PBP profile, had a unique ribotype (Fig. 3B, lane 2). Different ribotypes were also seen for a penicillin-resistant, chloramphenicol-susceptible serotype 23F isolate (Fig. 3A, lane 9), a penicillin-susceptible serotype 23F isolate (Fig. 3B, lane 11), the R6 control isolate (Fig. 3A, lane 10; Fig. 3B, lane 1), and all other multiresistant pneumococcal isolates of other serotypes (Fig. 3A, lane 11; Fig. 3C, lanes 1 and 2). Figure 4 is a Venn diagram representing the integration of the various sets of multiresistant isolates as related by the three typing methods: PBP pattern analysis, ribotyping, and MLEE analysis. Fifteen multiresistant serotype 23F isolates from the United States and the two multiresistant serotype

FIG. 4. Venn diagram depicting the integration of multiresistant strains of S. pneumoniae as related by the three typing methods: PBP patterns, MLEE profiles, and ribotypes. Other strains tested represent separate unrelated clones. See Table 1 for strain nomenclature.

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McDOUGAL ET AL.

loII7=2=

FIG. 5. A map of the United States showing the spread of the clonal multiresistant S. pneumoniae isolates (same PBP pattern, MLEE profile, and ribotype) and closely related isolates (indicated by asterisks; same PBP pattern but different MLEE profile and/or ribotype). See Table 1 for strain nomenclature. same PBP patterns but differing by their ribotypes or MLEE profiles.

DISCUSSION

Although previously reported as rare (41), multiresistant isolates of S. pneumoniae have been recovered from patients from many areas of the United States. Munoz et al. (34) established an intercontinental link between pneumococci isolated in Spain and Ohio; and our PBP, MLEE, and ribotyping data indicate the widespread dissemination of this multiresistant strain of serotype 23F in the United States. Worldwide, there are several regions where multiresistant pneumococci are particularly prevalent, including South Africa, Hungary, and Spain. In South Africa, where 14.1% of isolates from blood or cerebrospinal fluid were resistant to one or more antimicrobial agents, multiresistant strains were confined predominantly to serotype 6, 14, or 19 (23). Most of the multiresistant Hungarian strains belong to serotype 19A and share a common PBP pattern that contains the unusual feature of an atypically sized PBP 3, suggesting that these isolates share a common genetic origin (35). Spain has been a major focus of penicillin resistance for more than a decade: 65.5% of pneumococci in Spain have shown resistance to one or more drugs (9). In that study (9), analysis of the most frequent resistance patterns throughout the years revealed a progressive decrease of strains in serotypes 3, 5, and 7 resistant to tetracycline and chloramphenicol and a progressive increase in strains (predominantly in serotype 23) resistant to penicillin, tetracycline, and chloramphenicol. These strains have been imported into the United Kingdom (7, 31), where they are the most prevalent type of penicillin-resistant pneumococci (10, 12), and to other parts of Europe (22). In 1991, Spika et al. (41) reported that most of the penicillin-resistant pneumococci isolated in the United States appeared sporadically. In that study of 5,469 pneumococcal isolates submitted to the Centers for Disease Control from 1979 through 1987, MICs were >0.1 pLg/ml for only 5% of the isolates, and of these, MICs were >1 ,ug/ml for only 5 isolates. None of the isolates were resistant to chloramphenicol. A separate study during 1987 to 1989

ANTIMICROB. AGENTS CHEMOTHER.

found 4.1% of pneumococci to be penicillin resistant and 1.2% to be chloramphenicol resistant (20). These data indicate that antimicrobial resistance among pneumococcal isolates in the U.S. general population appears to be at low levels compared with the levels in other countries. Many of the isolates in this study, however, were recovered in 1991 and, as documented in a recent abstract by Thornsberry et al. (43), may represent a more recent trend of increasing resistance. Jabes et al. (17) demonstrated that PBP patterns can be used to characterize strain relatedness among penicillinresistant pneumococci. They identified 11 different groups or families of penicillin-resistant pneumococci from various parts of the world on the basis of PBP profiles, serotypes, and antibiotic resistance patterns. They identified a group of pneumococci from Spain that had four PBPs with molecular sizes corresponding to those of PBPs lb, 2a, and 3 plus a PBP with a greater molecular size than that of PBP 2a. The MIC of penicillin was 1.5 jjg/ml, and all but one strain belonged to serotype 23. All of the strains were resistant to chloramphenicol and tetracycline and susceptible to erythromycin. The Spanish serotype 23F strain, in our hands, had a PBP pattern similar to that described by Jabes et al. (17). This PBP pattern was typical of 21 multiresistant serotype 23F isolates from the United States, with the notable exception of the first multiresistant serotype 23F isolate from Michigan in 1983, which had a unique PBP pattern. PBP patterns differentiated other penicillin-resistant serotype 23F strains, which were susceptible to chloramphenicol, and multiresistant isolates of other serotypes from the pattern of the clonal group. The PBP patterns of the multiresistant serotype 23F isolates which were resistant to erythromycin were indistinguishable from the pattern of the clonal group. Our data and those of others (17, 34) have shown that resistant strains of pneumococci for which the penicillin MIC is the same not only have different PBP patterns but different penicillin affinity profiles as well. These data indicate that PBP patterns are stable and can be an important tool for characterizing changes in proteins involved in penicillin resistance. However, as a typing method, PBP pattern analysis does not discriminate genetic variations owing to the acquisition of other resistance markers (e.g., erythromycin resistance). MLEE analysis detects subtle mutations in a variety of genes for metabolic enzymes which may not alter protein function but which cause an electrophoretically detectable change in the protein's charge. By this method, the strain is characterized by analyses of loci throughout the entire chromosome and is not limited to changes in genes encoding penicillin resistance proteins. The analysis of 20 enzyme loci identified a cluster of isolates with the same allelic profiles at all loci. All of these isolates were characterized by an identical PBP pattern and included the isolates from Spain and South Africa. These results are in agreement with results from the assay of 14 enzymes by Munoz et al. (34) for the serotype 23F isolates from Ohio and Spain. Five other multiresistant serotype 23F isolates which were related by PBP pattern (three isolates were resistant to erythromycin, two isolates were susceptible to erythromycin) differed by only a few alleles and were considered highly related. As a method for determining strain relatedness in pneumococci, MLEE analysis appears to be more discriminating than does PBP pattern analysis. Chromosomal restriction endonuclease analysis is highly discriminatory in fingerprinting pneumococcal strains (45). However, because of the large number of bands, comparison

VOL. 36, 1992

of different fingerprints is tedious and does not allow for quantitative or objective appraisal. Conversely, ribotyping reduces the number of bands but still provides discriminating data. In this study, ribotyping identified the clonal group with a unique ribotype and also distinguished the isolates that were resistant to erythromycin and clindamycin from those that were moderately resistant to erythromycin and susceptible to clindamycin. On the other hand, two isolates identified as clonal by ribotyping had different MLEE profiles. All three typing methods (PBP patterns, MLEE profiles, and ribotypes) were able to identify a highly related group of multiresistant pneumococci isolated from throughout the United States. MLEE analysis and ribotyping, however, provided greater sensitivity than did PBP patterns in assessing the clonal nature of these strains. One isolate with a different serotype (serotype 19F) was identified as being highly related to the clonal group. The PBP pattern and MLEE profile were the same as those of the clonal group. The ribotype of this erythromycin-resistant isolate was identical to the ribotype of the multiresistant serotype 23F isolates that were erythromycin resistant and clindamycin susceptible. This observation was also made by Jabes et al. (17), who suggested that the transformation of capsular genes could account for the difference in serotypes. The emergence and successful spread of pneumococci with a particular resistance pattern can be correlated with exposure to certain antimicrobial agents in a particular population (16, 23, 24, 33). The absence of erythromycin resistance in strains from Spain, probably because of the infrequent use of erythromycin in Spain (9), suggests that penicillin and erythromycin resistance may evolve separately in response to different antimicrobial pressures. The use of erythromycin for therapy of community-acquired lower respiratory infections is increasing not only because of its indication in penicillin-allergic patients but also because of an increased awareness of Legionella pneumophila and mycoplasma infections (27). The close relatedness of the multiresistant serotype 23F strains, which are also resistant to erythromycin, suggests subsequent acquisition of erythromycin resistance in response to different antibiotic pressures in different communities. In conclusion, even though the overall prevalence of resistant pneumococci remains low in the United States, our data suggest that most of the penicillin-resistant, multiresistant strains isolated in the United States have disseminated from a multiresistant serotype 23F clone of S. pneumoniae that was isolated in Spain as early as 1978. ACKNOWLEDGMENT We thank Regine Hakenbeck, Max Planck Institut fir Molekulare Genetik, Berlin, Federal Republic of Germany, for helpful discussions on MLEE. REFERENCES 1. Altwegg, M., R. Altwegg-Bissig, A. Demarta, R. Peduzzi, M. W. Reeves, and B. Swaminathan. 1988. Comparison of four typing methods forAeromonas species. J. Diarrhoeal Dis. Res. 6:6-14. 2. Appelbaum, P. C. 1987. World-wide development of antibiotic resistance in pneumococci. Eur. J. Clin. Microbiol. 6:367-377. 3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 4. Broome, C. V., and R. R. Facklam. 1981. Epidemiology of clinically significant isolates of Streptococcus pneumoniae in the United States. Rev. Infect. Dis. 3:277-280. 5. Casal, J. 1982. Antimicrobial susceptibility of Streptococcus

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Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with i...
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