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Journal of Clinical Microbiology, December 2000, p. 4367-4372, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Clonal Groups of Penicillin-Nonsusceptible Streptococcus
pneumoniae in Baltimore, Maryland: a Population-Based, Molecular
Epidemiologic Study
M. Catherine
McEllistrem,1,*
Margaret
Pass,2
John A.
Elliott,3
Cynthia G.
Whitney,3 and
Lee H.
Harrison1,2
Infectious Diseases Epidemiology Research Unit,
University of Pittsburgh Graduate School of Public Health and School of
Medicine, Pittsburgh, Pennsylvania1;
Department of International Health, Johns Hopkins University
School of Hygiene and Public Health, Baltimore,
Maryland2; and Division of Bacterial and
Mycotic Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia3
Received 7 July 2000/Returned for modification 31 August
2000/Accepted 25 September 2000
 |
ABSTRACT |
Few data are available on the molecular subtypes of all
penicillin-nonsusceptible Streptococcus pneumoniae (PNSP)
from a defined population base. Pulsed-field gel electrophoresis
(PFGE), serotyping, and antibiotic susceptibility testing were
performed for all available invasive PNSP isolates for which the
penicillin (MIC) was
0.1 µg/ml from Baltimore, Md., during
1995-1996 (n = 143). The dendrogram analysis of PFGE
patterns included 32 distinct clonal groups. Six major clonal groups
included two-thirds of the PNSP strains. Major clonal groups 2, 3, 4, and 6 strains were genetically related to four previously described
international clones and were all multidrug resistant. Major clonal
group 3 was genetically related to the Tennessee23F-4 clone
and contained all four strains for which the penicillin MIC was 8 µg/ml. Most of the clonal group 1 and 5 strains had intermediate
susceptibility to penicillin and were rarely multidrug resistant. The
latter clonal groups represent two previously undescribed penicillin-intermediate pneumococcal clones. Clonal group homogeneity was greater for serotype 9V, 19A, and 23F strains than for serotype 6A,
6B, 14, and 19F strains. The classification of PNSP strains into clonal
groups is essential for future population-based epidemiologic studies
of PNSP.
 |
INTRODUCTION |
Streptococcus pneumoniae
is responsible for an estimated 50,000 cases of bacteremia, 3,000 cases
of meningitis, 7 million cases of otitis media, and several hundred
thousand cases of pneumonia in the United States each year (4, 20,
32, 34). The overall yearly incidence of pneumococcal bacteremia
is estimated to be 15 to 35 cases per 100,000 population (2, 3,
14, 17, 18).
Penicillin-nonsusceptible S. pneumoniae (PNSP) was uncommon
in the United States until the 1990s, when the rates of antibiotic resistance rapidly increased. Although 90 serotypes of S. pneumoniae exist (15), a small number of serotypes
account for the majority of PNSP (17). The emergence of PNSP
in the United States appears to be partially related to the
dissemination of multidrug-resistant (MDR) international pneumococcal
clones (6-8, 10, 13, 19, 21-23, 26, 30).
Recent molecular studies of PNSP in the United States have shown that
the majority of strains can be subtyped into less than 10 clonal groups
by pulsed-field gel electrophoresis (PFGE) (9, 12). These
studies included a sample of isolates for which the penicillin MIC is
1 µg/ml from various areas of the United States.
The purpose of the present study was to characterize the phenotypic
characteristics of PNSP isolates associated with invasive disease in a
defined population base to facilitate our ongoing studies of the
epidemiology of PNSP.
 |
MATERIALS AND METHODS |
Active surveillance for invasive pneumococcal infection was
initiated in the Baltimore Metropolitan Area (BMA) on 1 January 1995 as
part of the Maryland Bacterial Invasive Disease Surveillance project
(BIDS) (14). BIDS is the Active Bacterial Core Surveillance component of the multistate Emerging Infections Program Network that is
coordinated by the Centers for Disease Control and Prevention (CDC).
BMA, with a population of 2.5 million, comprises Baltimore City and
Baltimore, Anne Arundel, Carroll, Harford, and Howard Counties. The
surveillance case definition is the isolation of S. pneumoniae from a normally sterile body fluid from a BMA resident of any age. All laboratories based in acute-care hospitals in BMA
participate, as do other microbiology laboratories that process blood
cultures. For each eligible patient, the hospital infection control
professional completes a one-page case report form, which includes
demographic (e.g., gender, age, and race) and brief clinical information, and the bacterial isolate is submitted for species confirmation and MIC testing. Biweekly telephone calls are made to
hospital infection control practitioners to ascertain cases not
reported spontaneously. Periodic laboratory audits are performed to
identify unreported cases.
Bacterial isolates and antibiotic susceptibility.
Available
BIDS pneumococcal isolates for which the penicillin MIC is
0.1
µg/ml isolated during 1995 and 1996 were included in the present
study. The MICs of penicillin, cefotaxime, erythromycin, tetracycline,
trimethoprim-sulfamethoxazole (TMP-SXZ), clindamycin, ofloxocin, and
vancomycin were determined by broth microdilution testing by a CDC
contract laboratory by methods recommended by the National Committee
for Clinical Laboratory Standards (27). Pneumococcal
serotypes were determined by the latex agglutination test and were
confirmed by the Quellung reaction with type-specific antiserum
prepared at CDC. Eleven PNSP international clones obtained from the
American Type Culture Collection were included for comparison: Spain23F-1 (strain Sp264, ATCC 700669) (8, 21),
Spain6B-2 (strain GM17, ATCC 700670) (6),
France9V-3 (strain TL7, ATCC 700671) (22),
Tennessee23F-4 (strain SP196, ATCC 51916) (7,
23), Spain14-5 (strain VH14, ATCC 700672)
(6), Hungary19A-6 (strain HUN663, ATCC 700673)
(10, 26), South Africa19A-7 (strain 17619, ATCC
700674) (30), South Africa6B-8 (strain 50803, ATCC 700675) (30), England14-9 (strain
PN93/872/B, ATCC 700676) (13), Slovakia14-10
(strain 29055, ATCC 700677) (19), and
Slovakia19A-11 (strain 6571, ATCC 700678) (10).
Isolates for which the penicillin MIC was between 0.1 and 1 µg/ml
were defined as penicillin intermediate (Peni), and those
for which the penicillin MIC was
2.0 µg/ml were defined as
penicillin resistant (Penr). The Peni and
Penr strains were collectively defined as PNSP. A strain
was defined as MDR if it was nonsusceptible to at least two of the
following antibiotics: penicillin and/or cefotaxime, erythromycin,
TMP-SXZ, tetracycline, ofloxacin, and chloramphenicol.
PFGE.
Two PFGE protocols that resulted in identical banding
patterns were used (24, 25). A simplified protocol deleted
the lysis step, eliminated proteinase K, and required shorter
incubation times (24). Equal amounts of bacterial suspension
and 2% low-melting-temperature agarose (Sea Plaque; FMC Bioproducts,
Rockland, Maine) were mixed and pipetted into 100-µl plug molds.
After solidification on ice for 10 min, the plugs were incubated in
lysis enzymes and buffer: 2 ml of buffer (1 M NaCl, 100 mM EDTA, 6 mM
Tris-HCl, 0.5% Brij 58, 0.5% deoxycholate, 0.5% N-lauroyl
sarcosine [pH 7.6]) supplemented with 1 mg of lysozyme per ml and 50 µg of RNase A per ml for 3 h at 37°C. Each plug was incubated
with 2 ml of ES buffer (0.5 M EDTA, 1% N-lauroyl sarcosine
[pH 8.5 to 9.3]) and 100 µg of proteinase K per ml for 6 to 18 h at 50°C. The plugs were washed three times with 10 ml of TE buffer
containing 10 mM Tris-HCl and 1 mM EDTA (pH 7.6) at 37°C for 15 to 30 min. After preincubation of a plug (2 by 10 mm) in buffer (NE #4; New
England BioLabs, Inc., Beverly, Mass.) for 20 min, the DNA was digested
with buffer (NE #4) mixed with 30 U of SmaI and 200 µg of
bovine serum albumin per ml at room temperature for 3 to 18 h.
Each section of plug (2 by 5 mm) was loaded into a 1% agarose gel.
PFGE was performed with a contour-clamped electrophoretic field gel
apparatus (DRIII) under the following conditions: pulse times, 1 to
30 s for 18 h and 5 to 9 s for 8 h; 198 V; flow rate, 1 liter/min; temperature, 14°C. After the gel was stained with ethidium
bromide, the image was digitized on a Gel Doc 2000 System (Bio-Rad,
Hercules, Calif.).
Statistical analysis.
Data were analyzed with Epi Info,
version 6.04, software (CDC). The
2 and Fisher exact
tests were used for the analysis of dichotomous variables. The genetic
relatedness of strains was determined by analyzing the PFGE patterns
with Molecular Analyst/Multi-Analyst computer programs (Bio-Rad).
Dendrograms were created by use of the unweighted pair group method
with arithmetic averages, the Dice coefficient, and a position
tolerance of 1.5%. The Molecular Analyst cophenetic correlation was
calculated for all dendrograms. The cophenetic correlation indicates
the degree of correlation between the computer-derived degree of
genetic relatedness and the visual display by a dendrogram. A minimum
70% correlation is necessary to ensure that the dendrogram faithfully
represents the actual degree of genetic relatedness between strains. A
PFGE-based clonal group was defined as a group of isolates with
genetically related PFGE patterns. The clonal groups were determined by
use of the criteria of Tenover et al. (33) and the
dendrogram-derived degree of genetic relatedness. In general, the PFGE
patterns of strains categorized within a clonal group had six or fewer
differences from each other (33) and
80% genetic
relatedness on the dendrogram. Strains were defined as genetically
related to an international clone if their PFGE patterns differed by
six or fewer bands from the PFGE patterns of the respective clones. Six
clonal groups had five or more strains per group; these were defined as
major clonal groups and were enumerated as groups 1 to 6. Twenty-six remaining clonal groups had less than five strains per group; these
were defined as minor clonal groups and were enumerated as groups 7 to 32.
To determine the degree of clonal group homogeneity for each serotype,
we summarized the distribution of the number of clonal groups for each
individual serotype using the formula
(X12 + X22 + ... Xn2)/N2, where
X denotes the number of isolates for each clonal group and
N represents the total number of isolates. This formula was derived by weighting the frequency distribution among the clonal groups
according to the number of isolates within each clonal group by use of
the formula X1(X1/N) + X2(X2/N) + ... Xn(Xn/N) and then adjusting for the sample
size by dividing by the total number of isolates (N) for
that serotype.
To determine whether differences in antimicrobial susceptibility
patterns were independent of serotype, we controlled for serotype in
the analysis. For the analysis of the antimicrobial susceptibilities of
the six major clonal groups, clonal group 1 was compared to clonal
group 2 and clonal group 3 was compared to clonal group 4. The majority
of the strains in clonal groups 1 and 2 were serotype 9V, and all of
the strains in major clonal groups 3 and 4 were serotype 23F. All of
the clonal group 5 and 6 strains were serotype 19A and serotype 6B,
respectively. No other major clonal group contained primarily serotype
19A or 6B strains. Therefore, the strains in clonal groups 5 and 6 were compared to nonclonal group 5 serotype 19A and nonclonal group 6 serotype 6B isolates, respectively.
 |
RESULTS |
Study isolates.
A total of 1,412 patients with invasive
pneumococcal infection were reported from 1 January 1995 to 31 December
1996. The pneumococcal isolates were available for 1,136 (80.5%) of
these patients, of which 86 (7.6%) and 81 (7.1%) were found to be
Peni and Penr, respectively. Of the 167 PNSP
isolates, 143 (86%) were available for subtyping.
Of the 143 PNSP isolates, 140 (98%) were from blood and <1% each
were from pleural fluid, joint fluid, and cerebrospinal fluid. Blacks
between the ages of 5 and 64 years with PNSP infections had a lower
proportion of Penr isolates (38.5%) than whites of the
same age (64.3%) (P = 0.04). Twenty-eight (40.6%) of
the isolates from patients from Baltimore City were resistant to
penicillin whereas 44 (59.5%) of the isolates from patients from the
remaining BMA counties were resistant to penicillin (P = 0.02) (Table 1).
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TABLE 1.
Patient characteristics and serotype distribution of
isolates from patients with invasive pneumococcal infection due to
Peni and Penr strains
|
|
Clonal groups.
The 143 PNSP strains were classified into 32 clonal groups (Table 2). The six major
clonal groups (Fig. 1) accounted
for 95 (66.4%) of the PNSP isolates. The remaining 48 (33.6%) strains were categorized into 26 minor clonal groups.

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FIG. 1.
Dendrogram analysis of 104 PNSP strains in the six major
clonal groups; the cophenetic correlation is 89.4%. Asterisks indicate
the PFGE patterns for the international clones. All strains within a
major clonal group have the same serotype except as indicated. NT,
nontypeable.
|
|
Four of the major clonal groups were genetically related to an
international pneumococcal clone and accounted for 58 (40.6%) of all
PNSP isolates; clonal group 2 was related to the France9V-3
clone, clonal group 3 was related to the Tennessee23F-4
clone, clonal group 4 was related to the Spain23F-1 clone,
and clonal group 6 was related to the Spain6B-2 clone. Most
of the clonal group 2 and 5 strains were Peni serotype 9V
and 19A strains, respectively. These isolates accounted for 35 (49.3%)
of the Peni strains.
In general, strains categorized into a major clonal group were of the
same serotype. Clonal groups 1 and 2 consisted of 87.2% serotype 9V
strains, clonal groups 3 and 4 consisted of 100% serotype 23F strains,
and clonal groups 5 and 6 consisted of 100% 19A and 6B strains (Table
2). The cophenetic correlation for the dendrogram was 89.4% (Fig. 1).
The majority of strains of serotypes 6A, 14, and 19F were present in
multiple minor clonal groups. One major clonal group and multiple minor
clonal groups contained serotype 6B and 19A strains. Accordingly, the
measure of clonal group homogeneity was higher for serotypes 9V, 19A,
and 23F (range, 0.40 to 0.68) than for serotypes 6A, 6B, 14, and 19F
(range, 0.18 to 0.26) (Table 2).
Antibiotic susceptibility.
The penicillin MICs for the study
isolates were distributed as follows: for 62 (43.4%) isolates the
penicillin MIC was 0.1 to 0.5 µg/ml, for 9 (6.3%) isolates the
penicillin MIC was 1.0 µg/ml, for 43 (30%) isolates the penicillin
MIC was 2.0 µg/ml, for 25 (17.5%) isolates the penicillin MIC was
4.0 µg/ml; and for 4 (2.8%) isolates the penicillin MIC was 8.0 µg/ml. Of all 143 PNSP strains, 101 (79%) were nonsusceptible to
TMP-SXZ, 35 (24.5%) were nonsusceptible to erythromycin, 68 (47.6%)
were nonsusceptible to cefotaxime, 30 (21%) were nonsusceptible to
tetracycline, 29 (20.3%) were nonsusceptible to chloramphenicol, 12 (8.4%) were nonsusceptible to clindamycin, and 9 (6.3%) were
nonsusceptible to ofloxocin. None of the isolates were nonsusceptible
to vancomycin.
Serotype was strongly associated with the level of penicillin
resistance. For example, 23 (71.9%) of the serotype 23F isolates but
only 2 (7.4%) of the serotype 19A isolates were Penr
(P < 0.001) (Table 1). Serotype 19A was less likely to
be associated with MDR strains and serotype 23F was more likely to be
associated with MDR strains compared to the associations for the
remainder of the serotypes.
Major clonal groups 1 and 2 differentiated Peni from
Penr strains among the serotype 9V strains (Table
3). The major clonal groups differed
substantially by antibiotic susceptibility pattern. The susceptibility
patterns of clonal group 2 to 4 and 6 strains mirrored the
susceptibility patterns of the respective international clones with a
few exceptions. Specifically, the erythromycin susceptibility patterns
of the Spain23F-1 and Spain6B-2 clones and the
penicillin susceptibility pattern of the Tennessee23F-4
clone differed from those of most of the strains in these clonal groups. The clonal groups which were genetically related to an international clone were more likely than the remainder of the clonal
groups to contain MDR strains (Table 3).
Major clonal group 3, which was genetically related to the
Tennessee23F-4 clone, included two subsets of unique MDR
strains. The only four isolates for which the penicillin MIC was 8 µg/ml and the only two isolates that were both Peni and
cefotaxime resistant were present in this clonal group. All six of
these strains were also resistant to cefotaxime and TMP-SXZ. Six (46%)
of the clonal group 3 strains were Peni, whereas none of
the major clonal group 4 strains were Peni (Table 3).
 |
DISCUSSION |
This is the first study that has described the molecular
epidemiology of all invasive PNSP isolates from an entire metropolitan area. This approach provides a complete representation of the genetic
diversity of invasive PNSP isolates for a defined population base.
Other studies which have described the molecular epidemiology of PNSP
in the United States have focused on a select sample of PNSP isolates
for which the penicillin MIC is
1 µg/ml (9, 12).
In this study, we found that two-thirds of invasive PNSP isolates in
Baltimore could be characterized into six major clonal groups and that
nearly half of the PNSP isolates were genetically related to one of
four recognized international clones. Furthermore, antibiotic
susceptibility patterns differed substantially between major clonal
groups, even among isolates of the same serotype. PNSP strains which
were genetically related to an international clone were MDR.
To the best of our knowledge, two of the major clonal groups that we
identified (clonal groups 1 and 5) have not been previously described
as international clones or identified in other studies of the molecular
epidemiology of PNSP. This is most likely because previous studies have
focused on Penr isolates, whereas these clonal groups
largely consisted of Peni strains (9, 12).
Nearly all of the clonal group 1 and 5 strains were serotype 9V or 19A,
and for 95% of these strains the penicillin MICs were <1 µg/ml. As
would be expected, the majority of the strains were not MDR.
We also found that the degree of genetic diversity varied by serotype.
Each of the six major clonal groups largely comprised strains of one
serotype. Serotypes 9V, 19A, and 23F were associated with more clonal
group homogeneity than serotypes 6A, 6B, 14, and 19F. Whether these
differences in genetic diversity by serotype are due to different rates
of recombination, different lineages of S. pneumoniae, or
some other mechanism is not known.
As has been described for other geographic areas, the majority of PNSP
strains in BMA were of serotypes 6A, 6B, 9V, 14, 19A, and 23F
(17). The present data are in accordance with those from two
recent studies which found that 70 to 93% of the strains for which the
penicillin MIC was
1.0 µg/ml could be subtyped into 1 of 10 PFGE
types (9, 12).
PFGE is a standardized method for the subtyping of bacteria and has
been used effectively to track the worldwide spread of major
international clones. The dendrogram has become an essential component
of the analysis of the genetic relatedness of a large number of
isolates. Unfortunately, no standardized method for the grouping of
isolates into clonal groups has been developed. To complicate matters,
computer-based analyses are operator dependent (11) and the
genetic relatedness of isolates can be substantially altered by
modifying the band sensitivity parameters. We defined genetic
relatedness by a combination of dendrogram and visual inspection. These
two methods resulted in a classification which was supported by the
phenotypic characteristics of the clones and a dendrogram with a high
degree of cophenetic correlation. Strains within a major clonal group
had a
80% correlation on the dendrogram. In addition, our clonal
groups generally had at least seven-band differences between groups and
less than seven-band differences within groups. For clonal groups which
contained an international clone, all strains were genetically related
to the clone by the criteria of Tenover et al. (33) and
often had the susceptibility pattern of the respective clone. The
notable exceptions were the strains of major clonal groups 1 and 2. Some of the clonal group 1 strains had differences of five to six bands
compared to the pattern for the France9V-3 clone of major
clonal group 2. However, the dendrogram categorized these strains into
two clonal groups which were phenotypically distinct. Our high degree
of cophenetic correlation confirmed that the dendrogram faithfully
represented the computer-derived degree of genetic relatedness between strains.
The use of only one molecular subtyping technique and one restriction
enzyme is a limitation of this study that could have led to an
underestimation of the genetic diversity of our isolates. PFGE provides
discriminatory power equal to that of BOX fingerprinting or restriction
fragment end labeling and discriminatory power higher than those of PCR
and ribotyping (16). However, PNSP strains with a different
penicillin-binding protein gene (dhf) can have identical
PFGE patterns (12).
Despite the substantial degree of MDR among isolates in Baltimore, all
PNSP isolates were susceptible to vancomycin and there was nearly
complete susceptibility to ofloxacin (1, 28). However,
fluoroquinolone-nonsusceptible pneumococci are increasing in frequency
in Canada, most likely due to the increasing use of fluoroquinolones
(5). In addition, the emergence of vancomycin-nonsusceptible Staphylococcus aureus raises the possibility that
vancomycin-resistant S. pneumoniae may develop in the future
(29, 31). The changing epidemiology of drug resistance in
the United States underscores the need to use these antibiotics
prudently and to continue to monitor resistance rates to ensure the
development of appropriate antibiotic therapy guidelines. Since
geographic variations in the penicillin susceptibilities of S. pneumonia isolates have been detected, these data may not be able
to be generalized to other areas of the United States.
In summary, we defined the clonal groups and phenotypic characteristics
of PNSP isolates responsible for invasive infection in BMA. The
classification that we used to define PFGE-based clonal groups resulted
in groups with distinctive phenotypic features that were characteristic
of those of the previously reported international clones and were often
serotype specific. The identification of two previously undescribed
clonal groups was most likely due to the inclusion of S. pneumoniae isolates for which penicillin MICs were
0.1 and <1
µg/ml. Future studies will determine whether new clonal groups have
emerged or whether the frequencies of the present clonal groups have
changed since 1996. The definition of these clonal groups will be
crucial for our ongoing studies to understand the demographic,
geographic, and seasonal differences in PNSP (B. A. Albanese,
Z. H. Reed, J. C. Roche, M. A. Pass, C. G. Whitney,
and L. H. Harrison, Abstr. 39th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. 1047, p. 152, 1999).
 |
ACKNOWLEDGMENTS |
We thank the participating hospital infection control
practitioners and microbiology laboratory personnel in BMA for
identifying patients with pneumococcal infections and providing the
bacterial isolates; Yvonne Dean-Hibbert for assistance in conducting
surveillance; Kim Holmes for assistance with data collection; and
Althea Glenn, Laboratories Administration, Maryland Department of
Health and Mental Hygiene, for processing the isolates. We gratefully
acknowledge Victor Yu for expertise and David McDevitt for
susceptibility testing of selected isolates. We thank Terry Thompson
and Lashondra Shealey for assistance with the serotype testing of
selected isolates, Linda McDougal for providing the susceptibility
patterns of the international pneumococcal clones, and Susan Hunter for
guidance with the dendrogram analysis. We also thank the Emerging
Infections Project in Maryland, especially the staff of the
Epidemiology and Disease Control Program of the Maryland Department of
Health and Mental Hygiene, for support.
This work was supported by the National Foundation for Infectious
Diseases (to M.C.M.) and the National Vaccine Program, Emerging Infections Program Network, National Center for Infectious Diseases, CDC (to L.H.H).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Infectious
Diseases Epidemiology Research Unit, Public Health Infectious Diseases
Laboratory, University of Pittsburgh, 3471 Fifth Ave., 501 Kaufmann
Building, Pittsburgh, PA 15213. Phone: (412) 648-6401. Fax: (412)
648-6399. E-mail:
mcellistremc{at}msx.dept-med.pitt.edu.
 |
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Journal of Clinical Microbiology, December 2000, p. 4367-4372, Vol. 38, No. 12
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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