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Journal of Clinical Microbiology, January 2000, p. 458-459, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bacteremic Pneumonia Caused by a Single Clone of
Streptococcus pneumoniae with Different Optochin
Susceptibilities
Hsiu-Yuan
Tsai,1
Po-Ren
Hsueh,2,3
Lee-Jene
Teng,4
Ping-Ing
Lee,5,*
Li-Min
Huang,5
Chin-Yun
Lee,5 and
Kwen-Tay
Luh2,3
Department of Pediatrics, Taipei Municipal
Ho-Ping Hospital,1 Departments of
Laboratory Medicine,2 Internal
Medicine,3 and
Pediatrics,5 National Taiwan University
Hospital, and School of Medical Technology, National Taiwan
University Medical College,4 Taipei, Taiwan
Received 6 July 1999/Returned for modification 24 August
1999/Accepted 22 September 1999
 |
ABSTRACT |
Two isolates of Streptococcus pneumoniae having
different optochin susceptibilities were recovered from a blood sample
of a 2-year-old boy with community-acquired pneumonia. The two isolates were documented to belong to a single clone on the basis of the isolates' identical serotype (23F), antibiograms by the E-test, random
amplified polymorphic DNA patterns generated by arbitrarily primed PCR,
pulsed-field gel electrophoresis, and restriction fragment length
polymorphism of the penicillin-binding protein genes pbp2b
and pbp2x.
| |
CASE REPORT |
A 2-year-3-month-old boy was
admitted to our ward with the chief problem of cough and rhinorrhea for
2 weeks and high fever with generalized tonic-clonic convulsion on the
day of admission. He had a history of febrile convulsion. Physical
findings were unremarkable except for an injected throat. His activity
was fair without any meningeal sign. A chest radiograph showed
infiltration over bilateral lung fields. Laboratory testing revealed a
leukocyte count of 37,650/mm3 with 88% neutrophils and a
markedly elevated C-reactive protein level (14.2 mg/dl, normal level
being <0.5 mg/dl). Two sets of blood cultures yielded
Streptococcus pneumoniae, with an inhibition zone around the
oxacillin disk (1-µg disk) (BBL Microbiology Systems, Cockeysville,
Md.) of 8 mm. Cefazolin (100 mg/kg of body weight/day) was given for 2 days, and the fever subsided before the blood culture result was
available. Since the patient's general condition was healthy,
cefazolin was given for a further 3 days, treatment was then shifted to
oral cephalexin (25 mg/kg/day) for 1 week, and the patient was
discharged in stable condition. The child did well in the following 6 months.
Microbiological investigation.
Two sets of blood cultures both
grew gram-positive cocci in BACTEC 6A aerobic bottles (Becton
Dickinson, Sparks, Md.). After subculture, the organisms grew well on
Trypticase soy agar supplemented with 5% sheep blood agar plates (BBL
Microbiology Systems) at 37°C in ambient air. The colonies were
alpha-hemolytic and nonmucoid. The optochin sensitivity test for the
organisms was performed on Trypticase soy agar supplemented with 5%
sheep blood agar according to a previous description (18).
An inhibition zone of 15 mm in diameter around the 6-mm optochin disk
was identified, and several colonies (isolate A) also grew within the
inhibition zone (7 to 9 mm from the center of the disk). When the
colonies grown inside (isolate A) and outside (isolate B) the
inhibition zone, respectively, were inoculated onto two plates of
Trypticase soy agar supplemented with 5% sheep blood and were
incubated for 24 h, no inhibition zone around the optochin disk
was found for isolate A and a complete inhibition zone of 15 mm around
the disk (no scattered colonies within the zone) was demonstrated for
isolate B. Isolates A and B were positive by the bile solubility test, and their biochemical profiles generated by the API 32 Strep System (bioMeriuex Vitek, Marcy l'Etoile, France) were in accordance with the
identification of S. pneumoniae.
The two isolates of S. pneumoniae (isolates A and B) both
belonged to serotype 23F as determined by the capsular swelling test
(quellung reaction) by specific antisera as previously described (8). For both isolates, penicillin and cefotaxime MICs were 2 and 1 µg/ml, respectively, determined by means of the E-test (PDM
Epsilometer; AB Biodisk, Solna, Sweden). MICs of other agents for these
two isolates were also identical: erythromycin, 
256 µg/ml;
rifampin, 1 µg/ml; clindamycin, 256 µg/ml; and vancomycin, 0.5 µg/ml. Molecular typing of the two isolates by random amplified polymorphic DNA (RAPD) analysis of chromosomal DNA generated by arbitrarily primed PCR and pulsed-field gel electrophoresis and restriction fragment length polymorphism (RFLP) profiles of
penicillin-binding protein genes (pbp2b and
pbp2x) after digestion with restriction enzymes
HinfI and AluI (Gibco BRL, Gaithersburg, Md.)
were performed in accordance with previous descriptions (7, 12,
19). For RAPD analysis, three primers were used: ERICI
(5'-GTGAATCCCCAGGAGCTTACAT-3'), M13
(5'-GAGGGTGGCGGTTCT-3'), and OPA-7 (5'-GAAACGGGTG-3').
For molecular typing studies, one S. pneumoniae
isolate recovered from another patient was included as a control
strain. The two isolates had identical RAPD patterns (Fig.
1), pulsotypes, and RFLP profiles of
pbp2b and pbp2x (Fig.
2).
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FIG. 1.
RAPD patterns generated by arbitrarily primed PCR by
three primers, ERICI, M13, and OPA-7. Lane M, molecular markers; lanes
C, control strain; lanes A and B, isolates A and B, respectively.
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FIG. 2.
RFLP patterns of the pbp2b and
pbp2x genes generated with the restriction enzymes
HinfI and AluI. See the legend to Fig. 1 for lane
definitions.
|
|
Discussion.
S. pneumoniae is a major cause of
community-acquired pneumonia, otitis media, paranasal sinusitis,
bacteremia, and meningitis (16). The emergence of
drug-resistant strains of S. pneumoniae has complicated
treatment of these common infections (3, 4, 6, 8, 10, 11).
Pneumococci are identified in the Clinical Microbiology Laboratory of
the National Taiwan University Hospital by the following three
reactions: alpha-hemolysis on sheep blood agar, catalase negativity,
and solubility in bile salts or susceptibility to ethylhydrocupreine
(optochin) (18). In recent years, a number of isolates have
been found to be optochin resistant, which has led cautious
microbiologists to rely more on the use of bile solubility for
definitive identification (9, 15, 17). In fact, it has been
reported that up to 5% of S. pneumoniae strains may be optochin indeterminate or resistant (1, 15). Furthermore, the bile solubility test is not always specific for S. pneumoniae (5). The use of these standard tests can
result in ambiguous phenotypes for certain organisms, leading to
identification difficulties for routine microbiology laboratories
(2, 14).
To our knowledge, bacteremia due to a single clone of S. pneumoniae which simultaneously possessed two isolates with
different optochin susceptibilities has never been documented
previously. By use of the antibiotyping and molecular typing methods,
this report described an episode of bacteremia caused by a single clone of serotype 23F and penicillin-resistant S. pneumoniae that
obviously possessed two subpopulations with different optochin susceptibilities.
In summary, we describe a case with invasive infection caused by two
isolates of multidrug-resistant S. pneumoniae exhibiting different optochin susceptibilities which were later documented to
belong to a single clone. Clinical microbiologists need not only to be
aware of a continued increase in antimicrobial drug resistance in
clinical isolates of S. pneumoniae but also to understand the potential difficulties of identification of this organism by
conventional methods, particularly the optochin susceptibility test.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pediatrics, National Taiwan University Hospital, No. 7, Chung-Shan
South Rd., 10016 Taipei, Taiwan. Phone: 886-2-23970800-5138. Fax:
886-2-23934749. E-mail:
pinging{at}ha.mc.ntu.edu.tw.
| |
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Journal of Clinical Microbiology, January 2000, p. 458-459, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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