DIAGN MICROBIOLINFECTDIS 1990;13:63-66
63
NOTES
Optochin-Resistant Variants of
Streptococcus pneumoniae Rosario Mufioz, Asunci6n Fenoll, Dolores Vicioso, and Julio Casal
Ten Streptococcus pneumoniae clinical isolates, possessing physiologically typical pneumococcal characteristics, showed optochin-susceptible and optochin-resistant colonies around the
optochin disk, when tested for optochin susceptibility. Equivocal optochin disk test results should be confirmed by bile solubility, agglutination tests, or both.
Streptococcus pneumoniae is the most common cause of lobar pneumonia and an important agent in other infections, including meningitis, otitis media, conjunctivitis, etc. The procedures used for presumptive identification of pneumococci are optochin susceptibility and biles solubility; the serological method (omniserum reaction) is used as the confirmatory test. In 1987, Kontiainen and Sivonen, described two S. pneumoniae isolates that contained an optochinresistant population on primary culture; this was also observed by Phillips et al. (1988) in two pneumococci. During the last two years, among 814 clinical pneumococcal isolates submitted to this Center from different hospital laboratories, we found ten isolates with optochin-resistant variants. Three were detected during 1987, representing 0.8% of the pneumococci studied that year, and seven in 1988, representing 1.5% of referred strains. Mueller-Hinton agar plates supplemented with 5% sheep blood were inoculated with each isolate, and an optochin disk (Bacto Differentiation Disks,
Optochin, Difco Laboratories) was placed onto the inoculated area. Plates were incubated at 37°C for 18 hr in a 5% CO2 atmosphere, as recommended by the manufacturer. Zones of inhibition varying from 22 to 27 mm were observed, but inside the inhibition zone there were optochin-resistant colonies. After several subcultures the optochin-resistant variants remained resistant. The susceptible subpopulation could be isolated, although, in two isolates, when after two subcultures resistant variants reappeared. Minimal inhibitory concentration (MIC) to optochin was determined by adding two-fold dilutions (ranging from 0.37 to 100 ~g/ml) of ethyl hydrocupreine hydrochloride (Sigma, Chemical Company) into Mueller-Hinton blood agar plates. From an 18hr plate culture, a 10s CFU/ml pneumococcal suspension in saline solution was inoculated using a multipoint inoculator (Microtiter AM 80). After an 18-hr of aerobic incubation at 37°C we observed the resistant variants possessing a several-fold higher MIC compared to susceptible subpopulations for each isolate. These results are summarized in Table 1. To characterize these isolates we subjected them to a range of laboratory techniques, such as bile solubility, coagglutination using the Phadebact Pneumococcus Test (Phadebact, Pneumococcal Reagents, Pharmacia Diagnostics, AB Uppsala, Sweden), and Neufeld capsular-swelling test performed with polyvalent and the 46 type/group-specific pneumococcal antisera (Staten Seruminstitut, Copenhagen, Denmark). Three control strains were used in all the assays: S. pneumoniae R6 (derivate of the unencapsulated Rockefeller University strain R36A),
From the Serviciode Bacteriologia,Centro Nacional de Microbiologia, Virologiae InmunologiaSanitarias, Institutode Salud Carlos Ill, Majadahonda,Madrid, Spain. Address reprint requests to: Rosario Mufloz, Ph.D., Serviciode Bacteriologia, Centro Nacional de Microbiologia, Institutode Salud Carlos III, Majadahonda,28220 Madrid, Spain. Received April 12, 1989; revised and accepted September 29, 1989. © 1990 ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/90/$3.50
R. Mufloz et al.
64
TABLE 1.
Sensitivity to Optochin in Resistant (R) and Susceptible (S) Variants of the S. pneumoniae Strains Inhibition for optochin
Strain 51450 6921 937 1255 6936 455 936 957 958 959
S R S R S R S R S R S R S R S R S R S R
Diameter (mm)~
MIC (t.tg/ml)b
24 13 26 14 25 6 27 6 22 6 22 6 25 6 24 6 24 6 25 6
1.5 12.5 0.3 12.5 1.5 25 0.7 25 3.1 25 1.5 50 0.3 12.5 0.3 12.5 0.7 25 0.3 25
~Susceptibilityto optochin using disc diffusionmethod. bRange of dilutions varying from 0.37 to 100 ~g/ml. CNMC75862 (capsulated pneumococcus from our collection), and S. oralis (NCTC 11427). Bile solubility was carried out by adding three drops of a 10% desoxycholate solution (DOC) to 1 ml of a light saline suspension (about 10 6 cfu/ml) from an 18-hr plate culture. A tube without DOC was used as a control. The test was read after a 15min incubation at room temperature. All negative tubes were reincubated for 15 min at 37°C. A total clearance of the DOC tube was considered positive. The optochin-susceptible subpopulations and optochin-resistant variants showed identical bile solubility for all isolates. We found only one culture (937 isolate) that showed partial solubility after a 30min exposure to DOC. The coagglutination using the Phadebact and the Quellung reaction with pneumococcal omniserum, produced positive results with eight cultures. The other two isolates (6921 and 1255) had negative results in both the optochin-susceptible culture and its resistant variant. These pneumococci were isolated from conjunctival samples. The isolates belonged to Types 3, 6, 13, 15, 19 and 22. The two strains that failed to agglutinate with omniserum did not react with any type-specific antisera, indicating absence of detectable capsular poly-
saccharide. These characteristics are shown in Table 2. Minimal inhibitory concentrations (MICs) of penicillin, erythromycin, chloramphenicol, tetracycline, and cefotaxime were determined using the method described above for optochin MICs (Table 3). Five pneumococcal strains from our Culture Collection were routinely used as controls in all antimicrobial tests. Optochin-susceptible and -resistant subpopulations of each isolate have identical resistance patterns, except the 937 isolate, which showed different penicillin susceptibility between the two bacterial subpopulations. In 1959, Lund observed that the optochin test was more reliable than the bile test in a differentiating pneumococci, and therefore is often used in laboratories as the primary identification method. Although, the isolation of pneumococci resistant to optochin was observed early (Moore and Chesney, 1917), this phenomenon was rare until 1987, when Kontiainen and Sivonen described two isolates in Finland. Later, Phillips et al., 1988 noticed this situation in the United Kingdom. In both reports the pneumococci were from clinical cultures. Lund (1959) did not find any pneumococci with optochin MICs higher than 2.5 I~g/ml, nor any other streptococcus with optochin MICs lower than 20 ~g/ ml. No culture produced an MIC between these two cited values. He pointed out that this great difference in susceptibility to optochin could be used for differentiating between pneumococcus and other viridans group streptococci. However, in our report we detected pneumococci with MICs as high as 50 ~xg/ml, indicating a decreasing susceptibility of pneumococci to optochin. Most of the isolates possessed other typical pneumococcal characteristics: colonial morphology, bile solubility, Phadebact coagglutination, and reaction with type/group-specific pneumococcal antisera. Two pneumococci isolated from clinical conjunctivitis were negative for the agglutination test and the Quellung reaction. Our observations confirm the results of Shayegani et al. (1982) and Pease et al. (1986), that identified noncapsulated pneumococci from eyes. The pneumococci described in these reports were rough cultures, considered avirulent and incapable of producing a positive Quellung reaction with specific antisera. The capsulate isolates had 3, 6, 13, 15, 19, and 22 capsular polysaccharide type, representing common serotypes found in Spain. Kontiainen and Sivone (1987) found their isolates belonging to types 3 and 6, and Phillips et al. (1988) to types 4 and 6. There is a correlation among the types encountered in this work and those found in previous reports. The results obtained with the susceptibility to other
Notes
65
T A B L E 2.
T e s t C h a r a c t e r i s t i c s of T e n P n e u m o c o c c a l I s o l a t e s
Strain
Source
DOC '~
Phad b
Omni c
Type a
51450 6921 937 1255 6936 455 936 957 958 959
Pharynx Conjunctiva Sputum Conjunctiva Cyst CSF Blood Blood Blood Blood
+ + -*+ + + + + + +
~ ~~+ + + + +
+ + + + + + + +
6 NT" 19 NT ~' 3 6 13 22 15 19
"Solubility in 10@ sodium desoxycholate. bphabebact coagglutination. "Capsular reaction with Omniserum. dCapsular polysaccharide type. "Nontypable strain.
a n t i m i c r o b i a l a g e n t s s e e m to i n d i c a t e little r e l a t i o n s h i p b e t w e e n r e s i s t a n c e to o p t o c h i n a n d r e s i s t a n c e to o t h e r d r u g s . C o n v e r s e l y , K o n t i a i n e n a n d S i v o n e n (1987) o b s e r v e d a c o r r e l a t i o n . S e v e n of t h e t e n o p tochin-resistant isolates were resistant or moderately s u s c e p t i b l e to o n e o r m o r e a n t i m i c r o b i a l a g e n t s . H o w e v e r , this is n o t a n a t y p i c a l f i n d i n g , b e c a u s e p r e v a l e n c e of d r u g - r e s i s t a n t p n e u m o c o c c i in S p a i n is a m o n g t h e h i g h e s t d e s c r i b e d a n y w h e r e in t h e w o r l d
T A B L E 3.
(Casal, 1982; P e r e z et al., 1987; L a t o r r e et al., 1988; C a s a l , 1989). T h e s e r e s u l t s s u g g e s t t h a t t h e n u m b e r of o p t o c h i n - r e s i s t a n t p n e u m o c o c c i c o u l d b e h i g h e r t h a n exp e c t e d l e a d i n g to p r o b l e m s in t h e i d e n t i f i c a t i o n of this o r g a n i s m , m a i n l y in cases of r o u g h c o l o n y t y p e s , because they could be considered viridans group streptococci. This error could influence the treatm e n t of t h e p a t i e n t .
S u s c e p t i b i l i t y of t h e I s o l a t e s to Five A n t i m i c r o b i a l A g e n t s MIC (~.g/ml)
Strain 51450 6921 937 1255 6936 455 936 957 958 959
S R S R S R S R S R S R S R S R S R S R
Penicillin
Erythromycin
Chloramphenicol
Tetracycline
Cefotaxime
1 1 1 1 0.06 2a 0.015 0.015 0.007 0.007 0.015 0.015 0.007 0.007 0.015 0.015 0.12 0.12 0.06 0.06
0.25 0.25 4 4 0.5 0.5 0.5 0.5 0.12 0.12 0.015 0.015 0.03 0.03 0.015 0.015 >128 >128 0.015 0.015
8 8 1 1 16 16 4 4 8 8 16 16 2 2 2 2 16 16 4 4
32 32 8 8 64 64 0.25 0.25 0.25 0.25 64 64 32 32 0.5 0.5 64 64 8 8
0.5 0.5 0.25 0.25 0.03 0.03 0.015 0.015 0.007 0.007 0.015 0.015 0.007 0.007 0.015 0.015 0.03 0.03 0.015 0.015
~Only significant variation.
66
R. M u h o z et al.
REFERENCES Casal J (1982) Antimicrobial susceptibility of Streptococcus pneumoniae: Serotype distribution of penicillin-resistant strains in Spain. Antimicrob Agents Chemother 22:222. Casal J (1989) Increase in resistance to penicillin in pneumococci in Spain. Lancet 1:735. Kontiainen S, Sivonen A (1987) Optochin resistance in Streptococcus pneumoniae strains isolated from blood and middle ear fluid. Eur ] Clin Microbiol 6:422. Latorre C, Juncosa T, Sanfeliu I (1988) Antibiotic susceptibility of Streptococcus pneumoniae isolates from paediatric patients. J Antimicrob Chemother 22:659. Lund E (1959) Diagnosis of pneumococci by optochin and bile tests. Acta Pathol Microbiol Scand 47:308. Moore HF, Chesney AM (1917) A study of ethylhydro-
cuprein (optochin) in the treatment of acute lobar pneumonia Arch Intern Med 19:611. Pease AA, Douglas CWI, Spencer RC (1986) Identifying non-capsulated strains of Streptococcus pneurnoniae isolated from eyes. ] Clin Pathol 39:871. Perez JL, Lif~ares J, Bosch J, Lopez de Goicoechea MJ, Martin R (1987) Antibiotic resistance of Streptococcus pneumoniae in childhood carriers. J Antimicrob Chemother 19:278. Phillips G, Barker R, Brogan O (1988) Optochin-resistant Streptococcus pneumoniae. Lancet 2:281. Shayegani M, Malmberg L, Gibbons WE, Campbell D (1982) Characterization of nontypable Streptococcus pneumoniae-like organisms isolated from outbreaks of conjunctivitis. J Clin Microbiol 16:8.
63
NOTES
Optochin-Resistant Variants of
Streptococcus pneumoniae Rosario Mufioz, Asunci6n Fenoll, Dolores Vicioso, and Julio Casal
Ten Streptococcus pneumoniae clinical isolates, possessing physiologically typical pneumococcal characteristics, showed optochin-susceptible and optochin-resistant colonies around the
optochin disk, when tested for optochin susceptibility. Equivocal optochin disk test results should be confirmed by bile solubility, agglutination tests, or both.
Streptococcus pneumoniae is the most common cause of lobar pneumonia and an important agent in other infections, including meningitis, otitis media, conjunctivitis, etc. The procedures used for presumptive identification of pneumococci are optochin susceptibility and biles solubility; the serological method (omniserum reaction) is used as the confirmatory test. In 1987, Kontiainen and Sivonen, described two S. pneumoniae isolates that contained an optochinresistant population on primary culture; this was also observed by Phillips et al. (1988) in two pneumococci. During the last two years, among 814 clinical pneumococcal isolates submitted to this Center from different hospital laboratories, we found ten isolates with optochin-resistant variants. Three were detected during 1987, representing 0.8% of the pneumococci studied that year, and seven in 1988, representing 1.5% of referred strains. Mueller-Hinton agar plates supplemented with 5% sheep blood were inoculated with each isolate, and an optochin disk (Bacto Differentiation Disks,
Optochin, Difco Laboratories) was placed onto the inoculated area. Plates were incubated at 37°C for 18 hr in a 5% CO2 atmosphere, as recommended by the manufacturer. Zones of inhibition varying from 22 to 27 mm were observed, but inside the inhibition zone there were optochin-resistant colonies. After several subcultures the optochin-resistant variants remained resistant. The susceptible subpopulation could be isolated, although, in two isolates, when after two subcultures resistant variants reappeared. Minimal inhibitory concentration (MIC) to optochin was determined by adding two-fold dilutions (ranging from 0.37 to 100 ~g/ml) of ethyl hydrocupreine hydrochloride (Sigma, Chemical Company) into Mueller-Hinton blood agar plates. From an 18hr plate culture, a 10s CFU/ml pneumococcal suspension in saline solution was inoculated using a multipoint inoculator (Microtiter AM 80). After an 18-hr of aerobic incubation at 37°C we observed the resistant variants possessing a several-fold higher MIC compared to susceptible subpopulations for each isolate. These results are summarized in Table 1. To characterize these isolates we subjected them to a range of laboratory techniques, such as bile solubility, coagglutination using the Phadebact Pneumococcus Test (Phadebact, Pneumococcal Reagents, Pharmacia Diagnostics, AB Uppsala, Sweden), and Neufeld capsular-swelling test performed with polyvalent and the 46 type/group-specific pneumococcal antisera (Staten Seruminstitut, Copenhagen, Denmark). Three control strains were used in all the assays: S. pneumoniae R6 (derivate of the unencapsulated Rockefeller University strain R36A),
From the Serviciode Bacteriologia,Centro Nacional de Microbiologia, Virologiae InmunologiaSanitarias, Institutode Salud Carlos Ill, Majadahonda,Madrid, Spain. Address reprint requests to: Rosario Mufloz, Ph.D., Serviciode Bacteriologia, Centro Nacional de Microbiologia, Institutode Salud Carlos III, Majadahonda,28220 Madrid, Spain. Received April 12, 1989; revised and accepted September 29, 1989. © 1990 ElsevierScience PublishingCo., Inc. 655 Avenue of the Americas, New York, NY 10010 0732-8893/90/$3.50
R. Mufloz et al.
64
TABLE 1.
Sensitivity to Optochin in Resistant (R) and Susceptible (S) Variants of the S. pneumoniae Strains Inhibition for optochin
Strain 51450 6921 937 1255 6936 455 936 957 958 959
S R S R S R S R S R S R S R S R S R S R
Diameter (mm)~
MIC (t.tg/ml)b
24 13 26 14 25 6 27 6 22 6 22 6 25 6 24 6 24 6 25 6
1.5 12.5 0.3 12.5 1.5 25 0.7 25 3.1 25 1.5 50 0.3 12.5 0.3 12.5 0.7 25 0.3 25
~Susceptibilityto optochin using disc diffusionmethod. bRange of dilutions varying from 0.37 to 100 ~g/ml. CNMC75862 (capsulated pneumococcus from our collection), and S. oralis (NCTC 11427). Bile solubility was carried out by adding three drops of a 10% desoxycholate solution (DOC) to 1 ml of a light saline suspension (about 10 6 cfu/ml) from an 18-hr plate culture. A tube without DOC was used as a control. The test was read after a 15min incubation at room temperature. All negative tubes were reincubated for 15 min at 37°C. A total clearance of the DOC tube was considered positive. The optochin-susceptible subpopulations and optochin-resistant variants showed identical bile solubility for all isolates. We found only one culture (937 isolate) that showed partial solubility after a 30min exposure to DOC. The coagglutination using the Phadebact and the Quellung reaction with pneumococcal omniserum, produced positive results with eight cultures. The other two isolates (6921 and 1255) had negative results in both the optochin-susceptible culture and its resistant variant. These pneumococci were isolated from conjunctival samples. The isolates belonged to Types 3, 6, 13, 15, 19 and 22. The two strains that failed to agglutinate with omniserum did not react with any type-specific antisera, indicating absence of detectable capsular poly-
saccharide. These characteristics are shown in Table 2. Minimal inhibitory concentrations (MICs) of penicillin, erythromycin, chloramphenicol, tetracycline, and cefotaxime were determined using the method described above for optochin MICs (Table 3). Five pneumococcal strains from our Culture Collection were routinely used as controls in all antimicrobial tests. Optochin-susceptible and -resistant subpopulations of each isolate have identical resistance patterns, except the 937 isolate, which showed different penicillin susceptibility between the two bacterial subpopulations. In 1959, Lund observed that the optochin test was more reliable than the bile test in a differentiating pneumococci, and therefore is often used in laboratories as the primary identification method. Although, the isolation of pneumococci resistant to optochin was observed early (Moore and Chesney, 1917), this phenomenon was rare until 1987, when Kontiainen and Sivonen described two isolates in Finland. Later, Phillips et al., 1988 noticed this situation in the United Kingdom. In both reports the pneumococci were from clinical cultures. Lund (1959) did not find any pneumococci with optochin MICs higher than 2.5 I~g/ml, nor any other streptococcus with optochin MICs lower than 20 ~g/ ml. No culture produced an MIC between these two cited values. He pointed out that this great difference in susceptibility to optochin could be used for differentiating between pneumococcus and other viridans group streptococci. However, in our report we detected pneumococci with MICs as high as 50 ~xg/ml, indicating a decreasing susceptibility of pneumococci to optochin. Most of the isolates possessed other typical pneumococcal characteristics: colonial morphology, bile solubility, Phadebact coagglutination, and reaction with type/group-specific pneumococcal antisera. Two pneumococci isolated from clinical conjunctivitis were negative for the agglutination test and the Quellung reaction. Our observations confirm the results of Shayegani et al. (1982) and Pease et al. (1986), that identified noncapsulated pneumococci from eyes. The pneumococci described in these reports were rough cultures, considered avirulent and incapable of producing a positive Quellung reaction with specific antisera. The capsulate isolates had 3, 6, 13, 15, 19, and 22 capsular polysaccharide type, representing common serotypes found in Spain. Kontiainen and Sivone (1987) found their isolates belonging to types 3 and 6, and Phillips et al. (1988) to types 4 and 6. There is a correlation among the types encountered in this work and those found in previous reports. The results obtained with the susceptibility to other
Notes
65
T A B L E 2.
T e s t C h a r a c t e r i s t i c s of T e n P n e u m o c o c c a l I s o l a t e s
Strain
Source
DOC '~
Phad b
Omni c
Type a
51450 6921 937 1255 6936 455 936 957 958 959
Pharynx Conjunctiva Sputum Conjunctiva Cyst CSF Blood Blood Blood Blood
+ + -*+ + + + + + +
~ ~~+ + + + +
+ + + + + + + +
6 NT" 19 NT ~' 3 6 13 22 15 19
"Solubility in 10@ sodium desoxycholate. bphabebact coagglutination. "Capsular reaction with Omniserum. dCapsular polysaccharide type. "Nontypable strain.
a n t i m i c r o b i a l a g e n t s s e e m to i n d i c a t e little r e l a t i o n s h i p b e t w e e n r e s i s t a n c e to o p t o c h i n a n d r e s i s t a n c e to o t h e r d r u g s . C o n v e r s e l y , K o n t i a i n e n a n d S i v o n e n (1987) o b s e r v e d a c o r r e l a t i o n . S e v e n of t h e t e n o p tochin-resistant isolates were resistant or moderately s u s c e p t i b l e to o n e o r m o r e a n t i m i c r o b i a l a g e n t s . H o w e v e r , this is n o t a n a t y p i c a l f i n d i n g , b e c a u s e p r e v a l e n c e of d r u g - r e s i s t a n t p n e u m o c o c c i in S p a i n is a m o n g t h e h i g h e s t d e s c r i b e d a n y w h e r e in t h e w o r l d
T A B L E 3.
(Casal, 1982; P e r e z et al., 1987; L a t o r r e et al., 1988; C a s a l , 1989). T h e s e r e s u l t s s u g g e s t t h a t t h e n u m b e r of o p t o c h i n - r e s i s t a n t p n e u m o c o c c i c o u l d b e h i g h e r t h a n exp e c t e d l e a d i n g to p r o b l e m s in t h e i d e n t i f i c a t i o n of this o r g a n i s m , m a i n l y in cases of r o u g h c o l o n y t y p e s , because they could be considered viridans group streptococci. This error could influence the treatm e n t of t h e p a t i e n t .
S u s c e p t i b i l i t y of t h e I s o l a t e s to Five A n t i m i c r o b i a l A g e n t s MIC (~.g/ml)
Strain 51450 6921 937 1255 6936 455 936 957 958 959
S R S R S R S R S R S R S R S R S R S R
Penicillin
Erythromycin
Chloramphenicol
Tetracycline
Cefotaxime
1 1 1 1 0.06 2a 0.015 0.015 0.007 0.007 0.015 0.015 0.007 0.007 0.015 0.015 0.12 0.12 0.06 0.06
0.25 0.25 4 4 0.5 0.5 0.5 0.5 0.12 0.12 0.015 0.015 0.03 0.03 0.015 0.015 >128 >128 0.015 0.015
8 8 1 1 16 16 4 4 8 8 16 16 2 2 2 2 16 16 4 4
32 32 8 8 64 64 0.25 0.25 0.25 0.25 64 64 32 32 0.5 0.5 64 64 8 8
0.5 0.5 0.25 0.25 0.03 0.03 0.015 0.015 0.007 0.007 0.015 0.015 0.007 0.007 0.015 0.015 0.03 0.03 0.015 0.015
~Only significant variation.
66
R. M u h o z et al.
REFERENCES Casal J (1982) Antimicrobial susceptibility of Streptococcus pneumoniae: Serotype distribution of penicillin-resistant strains in Spain. Antimicrob Agents Chemother 22:222. Casal J (1989) Increase in resistance to penicillin in pneumococci in Spain. Lancet 1:735. Kontiainen S, Sivonen A (1987) Optochin resistance in Streptococcus pneumoniae strains isolated from blood and middle ear fluid. Eur ] Clin Microbiol 6:422. Latorre C, Juncosa T, Sanfeliu I (1988) Antibiotic susceptibility of Streptococcus pneumoniae isolates from paediatric patients. J Antimicrob Chemother 22:659. Lund E (1959) Diagnosis of pneumococci by optochin and bile tests. Acta Pathol Microbiol Scand 47:308. Moore HF, Chesney AM (1917) A study of ethylhydro-
cuprein (optochin) in the treatment of acute lobar pneumonia Arch Intern Med 19:611. Pease AA, Douglas CWI, Spencer RC (1986) Identifying non-capsulated strains of Streptococcus pneurnoniae isolated from eyes. ] Clin Pathol 39:871. Perez JL, Lif~ares J, Bosch J, Lopez de Goicoechea MJ, Martin R (1987) Antibiotic resistance of Streptococcus pneumoniae in childhood carriers. J Antimicrob Chemother 19:278. Phillips G, Barker R, Brogan O (1988) Optochin-resistant Streptococcus pneumoniae. Lancet 2:281. Shayegani M, Malmberg L, Gibbons WE, Campbell D (1982) Characterization of nontypable Streptococcus pneumoniae-like organisms isolated from outbreaks of conjunctivitis. J Clin Microbiol 16:8.
