INTRODUCTION

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Nontypeable Streptococcus pneumoniae is a frequent cause of epidemic bacterial conjunctivitis (9, 12). We have previously utilized BOX-PCR fingerprinting to identify clones of nontypeable pneumococcus responsible for several outbreaks of conjunctivitis (1). In this study, we applied this technique to nontypeable pneumococci implicated in sporadic cases of conjunctivitis in a single city during a 6-month period. To our knowledge, this is the first attempt to determine whether sporadic nontypeable pneumococci that cause conjunctivitis are genetically homogeneous and whether they are related to epidemic strains.

	MATERIALS AND METHODS

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Cases. In order to characterize the antibiotic susceptibility of S. pneumoniae, the Clinical Microbiology Laboratory of the Louisiana State University Medical Center in Shreveport, La. (LSUMC), stored 94 consecutive isolates of this organism obtained from all culture sources between 2 February 1995 and 31 July 1995.

Specimen collection and initial laboratory processing. All eye specimens, a throat specimen (sample 19), and an abdominal drainage specimen (sample 20) were collected with sterile swabs. Swabs were placed in transport medium (Amies with charcoal; Remel, Inc., Lenexa, Kans.) and sent to the Clinical Microbiology Laboratory at LSUMC. Isolates were stored at 70°C in Trypticase soy broth containing 15% glycerol.

Identification and serotyping. Isolates were identified as S. pneumoniae on the basis of typical alpha-hemolysis on Trypticase soy agar plates and by optochin sensitivity and bile solubility. For serotyping, colonies were harvested and suspended in phosphate-buffered saline with 2% formalin. Samples were tested against antiserum pools A, B, C, D, E, F, G, H, I, P, Q, R, S, and T by a method adapted (11) from that of Kronvall (8). Typeable specimens were those that agglutinated within 2 min. Typing within serogroups was performed with specific factor sera to produce the capsular swelling (Quellung) reaction. All antisera were obtained from Statens Seruminstitut (Copenhagen, Denmark). Autoagglutinated samples were noted to have formed clumps in the formalin solution, and when they were placed on glass plates for the subsequent addition of antisera, they had the appearance of a positive serotyping reaction. Consequently, serotyping could not be completed. These isolates were identified as nontypeable. The identities of these isolates were verified by detecting rRNA unique to S. pneumoniae with an Accuprobe S. pneumoniae identification kit (Gen-Probe, Inc., San Diego, Calif.).

BOX-PCR. Strains were grown on Trypticase soy agar overnight in a candle jar at 37°C, and colonies were removed with bacterial loops, suspended in distilled water, washed, and resuspended to a standard turbidity corresponding to 10⁷ to 10⁸ CFU ml¹. BOX-PCR with the BOXA1R primer (7) was then performed by a procedure modified from that of Ertugrul et al. (1). An aliquot of 7.4 µl of each PCR product was mixed with 6× loading buffer (Life Technologies, Grand Island, N.Y.) and then subjected to electrophoresis in a 1% agarose gel at 2.5 V cm¹ for 3 h. Gels were stained in 5 µg of ethidium bromide ml¹ and viewed and photographed under UV light. Because BOX-PCR produces bands of varied intensity, strains were considered identical if they differed only by the absence of one band. Gel banding patterns were also analyzed with RFLPscan software (Scanalytics, Billerica, Mass.) with a tolerance of 2%. A database was created by the "bin" method, which records the presence or absence of bands at given molecular weights. Dendrograms were then produced from these data with the TreeCon algorithm (Yves Van de Peer, University of Antwerp).

	RESULTS

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Clinical characteristics of patients and serotypes. The 18 eye isolates available for study were obtained from unrelated patients at different clinics in the absence of an apparent outbreak of conjunctivitis. Clinical data are summarized in Table 1. One isolate (sample 6) was from a 23-year-old adult; the others were from young children (mean age, 37 months; median age, 19 months; range, 2 months to 14 years). Of the 18 strains implicated in sporadic conjunctivitis, 8 were typeable and 10 were nontypeable (autoagglutinated).

PCR. A gel was run with the 10 nontypeable isolates from LSUMC and two nontypeable isolates from the conjunctivitis outbreak in Illinois (Fig. 1). Four distinct clones were identified among the LSUMC isolates (samples 1 to 5, 6, 7, and 8 to 10); none of these clones was identical to the Illinois outbreak strain, although the clone represented by isolates 1 to 5 was very similar to it.

View larger version (84K): [in this window] [in a new window]   FIG. 1.   BOX-PCR fingerprint of 10 strains of nontypeable pneumococcus that caused sporadic conjunctivitis and of 2 isolates from an outbreak in Illinois.

View larger version (27K): [in this window] [in a new window]   FIG. 2.   Dendrogram of the 24 pneumococcal strains. AA, autoagglutinating strains; CSF, cerebrospinal fluid.

	DISCUSSION

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Shayageni et al. (9) used an array of biochemical, immunologic, and microbiological tests to demonstrate that a closely related strain of nontypeable S. pneumoniae was responsible for outbreaks of conjunctivitis in New York, Illinois, and California in 1980 and 1981. Since then, advances in molecular biology have enhanced our ability to discriminate among isolates implicated in a variety of infections caused by many pathogens, including S. pneumoniae (4). Random amplification by PCR with the BOX sequence has compared well with other more established methods of molecular typing such as restriction fragment end labeling and pulsed-field gel electrophoresis (13). We (1) have previously used this technique to confirm the clonality of the strain studied by Shayageni et al. and to document its similarity to isolates of nontypeable S. pneumoniae implicated in outbreaks in Illinois in 1996.

In this study, pneumococci causing sporadic cases, even the nontypeable strains, were diverse. The preservation of an outbreak clone in the context of the diversity noted in this study is consistent with the epidemic population structure of pneumococci as defined by Hall et al. (5): a diverse collection of freely recombining strains with occasional well-conserved and virulent clones. It should be noted that previously studied outbreaks have been among adults but that our sporadic cases occurred in a much younger population. Differences in the susceptibility to infection between adults and children may explain how the pneumococcal conjunctivitis strains fit into such a population structure. Young patients are probably more susceptible to otitis media and upper respiratory tract infections due to their naive immune systems and unique anatomies. In adults, who are not as susceptible, pneumococci able to cause an outbreak would require a more specific armamentarium of virulence factors and, hence, might be clonal. It is interesting that a clone similar to the virulent outbreak strain was responsible for one-half of the nontypeable conjunctivitis seen in this study.

Nontypeable pneumococci rarely cause invasive infection (2, 3), but for unclear reasons, they are frequently implicated in cases of pneumococcal conjunctivitis (1, 9, 10, 12). While it is still uncertain whether the nontypeable strains are always unencapsulated, recent studies of phase variation in pneumococci suggest that decreased capsule production may facilitate bacterial persistence in certain tissues. Kim and Weiser and Weiser et al. (6, 15) have observed that colonies with decreased amounts of polysaccharide capsule are adept at nasopharyngeal colonization but that variants with greater amounts of polysaccharide capsule are adept at invasion. While there is no evidence that the conjunctival strains studied here undergo phase changes, these studies suggest possible tissue-specific advantages of decreased or absent capsule production. We have found (8a) that transposon-induced unencapsulated mutants of S. pneumoniae types 3 and 14 adhere far more avidly to mammalian cells in tissue culture than do encapsulated wild-type strains. While capsule is important to the ability of pneumococci to cause invasive disease (14), it may be irrelevant to, or even an impediment to, conjunctival infection.