Download Serotype distribution of Streptococcus pneumoniae isolates

Indian J Med Res 124, July 2006, pp 99-104
Serotype distribution of Streptococcus pneumoniae isolates from
ophthalmic & systemic infections & of commensal origin
Upendra K. Kar, Gita Satpathy, N. Nayak, B.K. Das* & S.K. Panda**
Department of Ocular Microbiology, Dr R. P. Center for Ophthalmic Sciences, Departments of *Microbiology
& **Pathology, All India Institute of Medical Sciences, New Delhi, India
Received April 26, 2005
Background & objectives: Information regarding serotype distributions of Streptococcus pneumoniae
causing ophthalmic infections is scanty. This study was therefore undertaken to determine the
antimicrobial susceptibility status and serotypes of S. pneumoniae isolated from various ophthalmic
infections and to compare with those isolated from systemic infections and commensal
nasopharyngeal flora.
Methods: Thirty eight of S. pneumoniae isolates from ophthalmic infections, 9 from systemic
infections and 14 from the nasopharynx of apparently healthy school children were biochemically
characterized and tested for in vitro antimicrobial susceptibity to various antibiotics. Serotyping
of these 61 isolates was done by a rapid co-agglutination method.
Results: All the 61 isolates were sensitive to oxacillin (penicillin) and susceptibility against other
antimicrobials was variable. No multidrug resistance was observed. The 38 ophthalmic isolates
were distributed in 15 different serotypes. Most prevalent serotypes were 14, followed by 8 and
19F. The 9 systemic and 14 commensal. isolates of S. pneumoniae were distributed in 7 and 11
serotypes respectively. Three of the systemic and six of the commensal serotypes were observed in
ophthalmic infections whereas four of the commensal serotypes were observed in systemic infections.
Interpretation & conclusion: Resistance to penicillin was not observed. In ophthalmic infections, a
wide range of serotypes of S. pneumoniae were observed. More than half of the commensal serotypes
obtained in the study as well as majority of the systemic serotypes were observed in ophthalmic
infections.
Key words Commensals - in vitro antimicrobial susceptibility - ophthalmic infections - serotyping - Streptococcus pneumoniae systemic infections
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INDIAN J MED RES, JULY 2006
Streptococcus pneumoniae is an important
pathogen in ophthalmic infections like infective
keratitis, dachryocystitis and conjunctivitis 1-4. In
India, S. pneumoniae is the most common bacterial
cause of dachryocystitis, which if left untreated, leads
to nasolachrymal duct blockage with grave
sequelae 1,3 . S. pneumoniae has been shown to
constitute 11.74 per cent of all bacterial isolates
causing ocular infections in a study on school
children in Delhi5. S. pneumoniae is also the cause
of community acquired bacterial pneumonia, a
substantial proportion of bacterial meningitis, otitis
media and sinusitis worldwide6.
There are more than 90 serotypes of
S. pneumoniae described till date8. Of these, only a
limited number of serotypes are responsible for
majority of the infection, against which the currently
available 23 valent capsular polysaccharide vaccine
is designed6,7. Some of the serotypes involved in eye
infections may be different from those associated
with systemic infections 8. Moreover up to 77 per cent
of healthy individuals including children carry
S. pneumoniae as normal flora in the nasopharynx9,10.
Due to the anatomical proximity of the eye to
nasopharynx it is plausible that some of the
commensal serotypes may be involved in some of
the ocular infections.
We studied 61 well characterized isolates of
S. pneumoniae (38 from various ophthalmic infections,
9 from systemic infections and 14 commensal isolates
from normal nasopharynx) for in vitro antimicrobial
susceptibility pattern and serotype determination by
co-agglutination (COA) method.
Material & Methods
Isolation and characterization of Streptococcus
pneumoniae: Thirty-eight consecutive isolates of
S. pneumoniae ophthalmic isolates with no selection
bias included in this study were from patients
attending the outpatient department of Dr Rajendra
Prasad Ophthalmic Centre for Ophthalmic Sciences,
All India Institute of Medical Sciences (AIIMS),
New Delhi from January 2002 to June 2003. The
clinical diagnoses were;
dachryocystitis:
28 [18 were <12 yr of age (10 M and 8 F) and
remaining 10 were 16-81 yr of age (6 M and 4 F)],
infective keratitis: 7 (4 M and 3 F) 7-70 yr of age,
sporadic conjunctivitis: 2 and panophthalamitis: 1.
The systemic isolates of S. pneumoniae were from
9 patients with pneumococcal infections (4 with
pneumonia, 3 with meningitis, and 2 with
septicaemia) attending AIIMS hospital. Fourteen
commensal S. pneumoniae isolates were collected
from the nasopharynx of 30 apparently healthy
school children (5-10 yr of age) from Delhi.
After detailed clinical examination and obtaining
informed consent, specimens with sterile cotton
tipped swabs were collected from lachrymal
canaliculus, cornea, and conjunctiva of patients with
dachryocystitis, infective keratitis and conjunctivitis
respectively. Pleural fluid, sputum and cerebrospinal
fluid were collected from patients suffering from
pneumonia and meningitis respectively. Five ml of
blood was collected aseptically from the patients of
septicaemia. Nasopharyngeal swabs were collected
from 30 apparently healthy school children.
However, ethical clearance was not required.
The specimens were cultured on tryptic soy agar
(TSA) plates supplemented with 5 per cent sheep
blood (for blood culture in 50 ml of tryptic soy broth
which was subcultured into plates after 48 h) and
incubated at 37 0C for 18-24 h in an atmosphere of
5 per cent CO 2 . a-haemolytic colonies with
morphology suggestive of S. pneumoniae in Gram
stain were subcultured to a fresh plate with an
KAR et al: SEROTYPE DISTRIBUTION OF S. PNEUMONIAE
optochin disk (5 mg/6 mm), (Difco Laboratories,
Detroit, USA) and were incubated at 370C for another
18-24 h. Isolates that exhibited an inhibition zone of
about 14 mm around the optochin disk and were
showing both bile-solubility and capability to ferment
inulin were identified as S. pneumoniae.
Antimicrobial susceptibility testing: The isolates
were tested for antimicrobial susceptibility with
antibiotic impregnated discs (Hi-Media, Mumbai,
India) against oxacillin (20 µg for penicillin),
tetracycline (30 µg), erythromycin (15 µg),
chloramphenicol (30 µg), co-trimoxazole (1.25/23.75
µg), ciprofloxacin (5 µg), gentamicin (100 µg),
cephalexin (30 µg) and vancomycin (30 µg), using
Kirby Bauer’s disc diffusion method11. The inhibition
zone was measured and interpreted according to the
recommendations of the National Committee for
Clinical Laboratory Standards12.
Serotyping of the S. pneumoniae isolates: The isolates
were sent to India CLEN infectious diseases
Reference Laboratory, Vellore, for serotyping. The
serotyping was carried out by a rapid co-agglutination
method using 12 pooled S. pneumoniae antisera from
Statens Serum Institut, Copenhagen, Denmark 13.
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Results & Discussion
High incidence of pneumococcal infections and
emergence of multidrug resistance are the major
reasons for continuous epidemiological
surveillance. In the present study, majority of
S. pneumoniae isolates from eye infections were
from patients with dachryocystitis 73.6 per cent,
followed by infective keratitis (18.4%) and sporadic
conjunctivitis (5.2%). All isolates were uniformly
sensitive to oxacillin (penicillin) (Table I), though
intermediate resistance to penicillin amongst
ophthalmic isolates was reported from south India 8.
Resistances to the other antibiotics tested were
variable i.e., co-trimoxazole (75.4%), ciprofloxacin
(29.5%), cephalexin (22.9%), tetracycline (26.22%),
chloramphenicol (6.5%), gentamicin (6.5%),
erythromycin (1.6%). No multidrug resistant
isolates were noted. However, 65 per cent of the
ophthalmic isolates, and almost all of the systemic
isolates were resistant to co-trimoxazole, which is
extensively used as a first line treatment for
pneumococcal infections. Majority of the
ophthalmic isolates were sensitive to ciprofloxacin
whereas the same was not observed for systemic
and commensal isolates.
Table I. Isolation source and antimicrobial susceptibility of 61 Streptococcus pneumoniae isolates
Antibiotics
Ophthalmic (38)
S
I
Penicillin (20 µg)
38
Co-trimoxazole (1.25/23.75 µg)
10
Ciprofloxacin (5 µg)
Systemic (9)
Commensal (14)
R
S
I
R
S
I
R
0
0
9
0
0
14
0
0
3
25
0
1
8
1
0
13
19
15
4
2
2
5
1
4
9
Cephalexin(30 µg)
29
4
5
5
0
4
9
0
5
Tetracycline (30 µg)
33
2
3
3
4
2
9
2
3
Chloramphenicol (30 µg)
36
0
2
9
0
0
12
0
2
Gentamicin (100 µg)
28
6
4
8
1
0
14
0
0
Erythromycin (15 µg)
37
0
1
9
0
0
14
0
0
Vancomycin (30 µg)
30
8
0
8
1
0
12
2
0
S, sensitive; I, intermediate; R, resistant
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INDIAN J MED RES, JULY 2006
Serotyping still remains the most import
epidemiological typing method 7,8, because currently
available vaccine is dependent on serotype
distribution of the strains causing major diseases6,14.
Certain serotypes of S. pneumoniae are more
frequently associated with specific types of
infections 6,15 . Even though information regarding
serotype distribution of S. pneumoniae is available
for both commensal and systemic isolates, such
information is limited for the ophthalmic infections
from different geographical regions.
In the present study, of the 38 ophthalmic
isolates, 36 could be typed into 15 serotypes and
two were non-typeable. The 28 isolates from
dachryocystitis and the 7 isolates from keratitis
were distributed in 14 and 5 serotypes respectively
(Table II). Serotype 14 was found to be most
prevalent (n=6) followed by 8 (n=5) and 19 F (n=4)
which correlates with an earlier report suggesting
6A was more prevalent serotype in ophthalmic
infection 8. Five of the serotypes i.e., 14, 34, 22,
23A, and 23B were observed in both the studies 8.
An earlier study at our hospital reported that 19 of
20 ophthalmic isolates were typeable into
serotypes 3, 6, 8, 28, 29, 45, 21 and 18 1. Of these
only serotypes 18F and 28 were observed in the
present study. It is believed that serotypes of
S. pneumoniae causing a particular infection in a
geographical location may change over a period
of time and this is of interest for epidemiological
purposes.
All the serotypes observed in keralitits were also
in dachryocystitis. The serotypes observed in
panophthalamitis (serotype 14) and conjunctivitis
(serotypes 33) were also observed in dachryocystitis.
This suggests that the same serotypes may be
responsible for different types of ocular infections.
We did not observe non typable isolates from
conjuctivitis, though it is commonly reported from
Europe and other parts of the world4.
The 9 isolates from systemic infections and
14 commensal isolates from the nasopharynx were
distributed in 7 and 11 serotypes respectively
(Table III). Serotypes 1 and 4 were isolated from 2
patients each from systemic infections. These have been
reported as the predominant serotypes in systemic from
India14,15. Three of the systemic serotypes (8, 19 F and
23 A) were also observed in ophthalmic infections. Only
two serotypes i.e., 19 F, 23 A were observed amongst
the isolates from all the studied sites.
Table III. Serotype distribution of systemic and commensal
Streptococcus pneuminiae isolates
Source
Serogroup/type
No. of
isolates
Table II. Serotype distribution of ophthalmic Streptococcus
pneuminiae isolates
Pneumonia and
meningitis
1
1 (each)
Source
Serogroup/type
No. of isolates
Pneumonia
8
1
Dachryocystitis
14
5
Pneumonia
15 A
1
Dachryocystitis
8, 34
3 (each)
Dachryocystitis
18F, 19F, 22, 23A
2 (each)
Pneumonia
19 F
1
Dachryocystitis
10, 11, 16, 17,
1 (each)
Meningitis and
septicaemia
4
1 (each)
Meningitis
6
1
Septicaemia
23A
1
23B, 28, 33
Dachryocystitis
Non-typeable
2
Corneal ulcer
8, 19F
2 (each)
Corneal ulcer
10, 17, 33
1 (each)
Commensal
11,23A,34
2 (each)
Conjunctivitis
31,33
1 (each)
Commensal
6A,6B, 16, 18F, 19F,
1 (each)
Pan-ophthalmitis
14
1
Commensal
4, 18C, 39
1 (each)
KAR et al: SEROTYPE DISTRIBUTION OF S. PNEUMONIAE
From the 11 serotypes of commensal isolates, 6
(11, 16, 18F, 19F, 23A, 34) were observed in
ophthalmic infections (dachryocystitis and keratitis)
and 3 were seen in systemic infections. Because of
the anatomical proximity, the commensal isolates
may be frequently associated with ophthalmic
infections8. Some of the commensal serotypes 6, 11,
19, and 23 observed in this study, were also reported
in the nasopharynx of normal healthy children in
other Indian studies16,17.
In conclusion, no specific serotype
of
S. pneumoniae was found to have any special
predilection to cause eye infection. However,
majority of the nasopharyngeal isolates were
observed in ophthalmic infections suggesting a role
for anatomical proximity.
Acknowledgment
The authors thank Dr M.K. Lalitha, Department of Clinical
Microbiology, Christian Medical College and Hospital, Vellore,
for serotyping of the isolates, Prof. S Ghose ,the faculties and
resident doctors of Dr RP Centre for Ophthalmic Sciences for
clinical specimens. This study was funded by an Ad-hoc research
grant from Indian Council of Medical Research (ICMR), New
Delhi.
103
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Reprint requests: Dr Gita Satpathy, Professor, Ocular Microbiology, Dr R.P. Center for Ophthalmic Sciences
All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi 110029, India
e-mail: gita.satpathy@ gmail.com