Streptococcus pseudopneumoniae: an emerging respiratory tract pathogen

Streptococcus pseudopneumoniae is a recently described member of the Streptococcus mitis/oralis group of viridans Streptococci that shares some characteristics with S. pneumoniae1. Key features of S. pseudopneumoniae are the absence of pneumococcal capsule, insolubility in bile, resistance or indeterminate susceptibility to optochin when incubated in 5 per cent CO₂ but susceptibility to optochin when incubated in ambient air, positive reactions in DNA probe hybridization and antigen detection tests12. Currently, most clinical laboratories in India depend only on the optochin susceptibility test done in 5 per cent CO₂ but not in ambient air for S. pneumoniae identification. As a result, S. pseudopneumoniae may be missed out or wrongly identified.

The clinical relevance of S. pseudopneumoniae has not yet been fully established, although it is found to be associated with chronic obstructive pulmonary disease2. Very few reports are available on potential pathogenic effect and drug resistance of S. pseudopneumoniae3–5. Until now, perhaps there are no published reports on the isolation of S. pseudopneumoniae from clinical samples in India. Therefore, an attempt was made to isolate S. pseudopneumoniae from sputum samples collected from patients suspected to have lower respiratory tract infection (LRTI) and to study their antibiogram.

Material & Methods

Collection of samples: Sputum samples (n=200) of patients, clinically suspected of having LRTI, submitted to the microbiology department of Kasturba Medical College, Mangalore, Karnataka, India, during March 2010 to August 2010 for routine culture and sensitivity testing were included in the study by following convenient sampling method.

Microscopic examination: Gram stain of sputum samples (n=150) showing >25 leukocytes and <10 squamous epithelial cells/×100 microscopic field along with large number of Gram positive cocci in pairs were included in the study and processed further for culture. Remaining sputum samples (n=50) showing >10 epithelial cells/x100 microscopic field were excluded from the study.

Culture and identification of the isolates: Samples were inoculated onto 5 per cent sheep blood agar and chocolate agar plates and incubated at 37°C for 24 h. Alpha haemolytic colonies on the culture plates, that were Gram positive cocci in pairs by staining, were identified as S. pseudopneumoniae and differentiated from S. pneumoniae on the basis of the tests for pneumococcal capsule, bile solubility and optochin susceptibility2.

Bile solubility test was performed according to standard procedures5. Capsules were detected by observing a halo around Pneumococci with India ink at × 400 magnification3.

Optochin susceptibility test: Colonies from overnight cultures were inoculated onto 5 per cent sheep blood agar plates in duplicate. Optochin disks of 6 mm diameter containing 5 μg antibiotic [Hi-media Laboratories (P) Ltd., Mumbai] were placed on both the inoculated blood agar plates. One plate was incubated in 5 per cent CO ₂and another in ambient air (O₂ atmosphere) at 37°C for 24 h. Zones of inhibition around the disk were measured and interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines36. S. pneumoniae strain ATCC 49619 procured from Himedia Laboratories (P) Ltd., Mumbai, was used as control. Isolates were considered to be resistant to optochin when the inhibition zones were smaller than 14 mm.

Antibiotic susceptibility test: Biochemically confirmed S. pseudopneumoniae were subjected to antibiotic susceptibility testing on Mueller Hinton blood agar plates by Kirby Bauer's disk diffusion method7 using antibiotics like cefepime, oxacillin, cefotaxime, ciprofloxacin, chloramphenicol, erythromycin, meropenem, tetracycline, teicoplanin, trimethoprim-sulphamethoxazole and vancomycin. Plates were incubated at 37°C for 24 h. The result was interpreted as either sensitive, intermediate or resistant as per CLSI guidelines6. S.pneumoniae strain ATCC 49619 was used as control.

Results & Discussion

Of the 150 sputum samples cultured, 25 showed the growth of α haemolytic streptococci. Among these, six isolates lacked capsules, were insoluble in 10 per cent bile, showed resistance to optochin (zone size <14 mm diameter when incubated in 5% CO₂) but were susceptible to optochin (zone sizes between 18 to 25 mm diameter) when incubated in ambient air. Hence, these six isolates were presumptively identified as S. pseudopneumoniae biochemically. S. pseudopneumoniae colonies grown on 5 per cent sheep blood agar were typically small (up to 1 mm in diameter after 24 h of incubation), smooth, shiny, and domed, with entire edges. Occasional colonies had depressed centers, appearing as a smaller version of the draftsmen colonies of S. pneumoniae.

The remaining 19 α-haemolytic Streptococci were identified as S. pneumoniae due to the presence of capsule, bile soluble and sensitive to optochin at 5 per cent CO₂ and generally poor growth or no growth in ambient air. The colonies had depressed centers typical of S. pneumoniae. Remaining 125 samples, showed heavy growth of various pathogens like Moraxella catarrhalis (n=21), Klebsiella spp. (n=8), Enterococcus spp. (n=6), Pseudomonas spp. (n=5), Haemophilus influenzae (n=6), Staphylococcus aureus (n=4) and Candida albicans (n=10). Some of the samples (n=35) also showed scanty growth of other Streptococcus spp. (Streptococci which were biochemically neither S. pneumoniae nor S. pseudopneumoniae) and mixed scanty growth of oropharyngeal flora (n=30).

All six S. pseudopneumoniae isolates, were sensitive to teicoplanin, vancomycin, cefotaxime, meropenem, cefepime and showed resistance to erythromycin. S. pseudopneumoniae isolates sensitive to ciprofloxacin and tetracycline were found to be four and three, respectively. Resistance to penicillin (as determined by oxacillin screening test) and co-trimoxazole was 66 per cent. All S. pneumoniae isolates were sensitive to teicoplanin (Table).

Table Antibiotic susceptibility pattern of S. pseudopneumoniae and S. pneumoniae

Our findings indicated the presence of S. pseudopneumoniae in this region which could be isolated from a small proportion of sputum samples. All six isolates of S. pseudopneumoniae were the predominant or only microorganism from good-quality purulent sputum samples obtained from patients with symptomatic LRTI and not from URTI. Further, in all six cases, the sputum Gram stain results indicated the presence of Gram positive cocci in pairs as the predominant bacteria along with pus cells. These findings provide supporting evidence of a potential pathogenic role of S. pseudopneumoniae.

All six S. pseudopneumoniae isolates were found to be resistant to erythromycin, four were resistant to penicillin (by oxacillin screening test) and co-trimoxazole, three were resistant to tetracycline and two to ciprofloxacin. Our findings corroborated with those reported from New Zealand and North America24. We could not find any reported cases of isolation of S. pseudopneumoniae from India to compare our results. However, antibiotic resistance in these isolates needs to be confirmed by detecting the minimum inhibitory concentration (MIC) for the resistant isolates.

High prevalence of antimicrobial resistance in major respiratory pathogens has led to serious concern in the selection of an appropriate antimicrobial agent for the empirical treatment of respiratory tract infections worldwide. Increasing penicillin and multidrug resistance in S. pneumoniae has important clinical implications. In the present study, resistance to penicillin as determined by oxacillin screening test was found to be 31 per cent and that of tetracycline and co-trimoxazole were found to range from 42 to 47 per cent for S. pneumoniae. These results are in agreement with the previous findings8. However, resistance to erythromycin and ciprofloxacin was also found.

Currently, most clinical laboratories depend only on the optochin susceptibility test by incubation in 5 per cent CO₂ for identification of S. pneumoniae. If the clinical sample has S. pseudopneumoniae it will show resistance to optochin when incubated only in 5 per cent CO₂ and such isolates are likely to be reported as oropharyngeal flora. Though S. pseudopneumoniae have been isolated in this region from sputum samples of patients with LRTI, further studies are necessary to prove the potential pathogenic role of these bacteria in lower respiratory tract infection or in chronic obstructive pulmonary disease (COPD). Since the presumptive identification of S. pseudopneumoniae was done by biochemical reaction, further characterization of these isolates by molecular methods is necessary. Testing of large number of samples, increased awareness to look for the presence of S. pseudopneumoniae and characterization to the genetic level will help to better determine its prevalence and clinical importance. Preliminary data from this study should prompt further research to characterize the role of S. pseudopneumoniae in COPD and LRTI.