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Streptococcus sinensis may react with Lancefield group F antiserum

Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital, Hong Kong

Correspondence Kwok-yung Yuen hkumicro{at}hkucc.hku.hk

Received May 19, 2004
Accepted July 6, 2004

Lancefield group F streptococci have been found almost exclusively as members of the ‘Streptococcus milleri’ group, although they have been reported very occasionally in some other streptococcal species. Among 302 patients with bacteraemia caused by viridans streptococci over a 6-year period, three cases were caused by Streptococcus sinensis (type strain HKU4^T, HKU5 and HKU6). All three patients had infective endocarditis complicating their underlying chronic rheumatic heart diseases. Gene sequencing showed no base differences between the 16S rRNA gene sequences of HKU5 and HKU6 and that of HKU4^T. All three strains were Gram-positive, non-spore-forming cocci arranged in chains. All grew on sheep blood agar as -haemolytic, grey colonies of 0.5–1 mm in diameter after 24 h incubation at 37 °C in ambient air. Lancefield grouping revealed that HKU5 and HKU6 were Lancefield group F, but HKU4^T was non-groupable with Lancefield groups A, B, C, D, F or G antisera. HKU4^T was identified by the Vitek system (GPI), API system (20 STREP) and ATB system (ID32 STREP) as 99 % Streptococcus intermedius, 51.3 % S. intermedius and 99.9 % Streptococcus anginosus, respectively. Using the same tests, HKU5 was identified as 87 % Streptococcus sanguinis/Streptococcus gordonii, 59 % Streptococcus salivarius and 99.6 % S. anginosus, respectively, and HKU6 as 87 % S. sanguinis/S. gordonii, 77 % Streptococcus pneumoniae and 98.3 % S. anginosus, respectively. The present data revealed that a proportion of Lancefield group F streptococci could be S. sinensis. Lancefield group F streptococci should not be automatically reported as ‘S. milleri'.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Since the discovery of the presence of different Lancefield groups in different species of streptococci and the correlation between the different Lancefield groups and different streptococcal species, Lancefield grouping has been used extensively in clinical microbiology laboratories for the identification of streptococci (Lancefield, 1933). Members of the ‘Streptococcus milleri’ group (genotypically known as the anginosus group; Kawamura et al., 1995) – Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius – can be of Lancefield group A, C, F or G or non-groupable. Lancefield group F streptococci have been found almost exclusively as members of the ‘S. milleri’ group, although they have been reported very occasionally in some other streptococcal species (Facklam, 1977, 2002). In one recent study that evaluated the usefulness of Lancefield grouping and a caramel smell for identification of the ‘S. milleri’ group, it was shown that Lancefield group F alone or accompanied by the caramel smell had a specificity of 100 % in the identification of ‘S. milleri’ (Brogan et al., 1997).

Recently, we reported the discovery of a novel viridans streptococcus, Streptococcus sinensis (type strain HKU4^T), from multiple blood cultures of a 42-year-old Chinese woman with infective endocarditis complicating her chronic rheumatic heart disease (Woo et al., 2002). HKU4^T is non-groupable by Lancefield serogrouping and it possesses no caramel smell. In this study, we report the phenotypic and genotypic characterization of two Lancefield group F S. sinensis isolates recovered from patients with bacteraemia caused by viridans streptococci. The significance of finding Lancefield group F streptococci in Streptococcus species other than members of the ‘S. milleri’ group is discussed.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Bacterial isolates, patients and microbiological methods.

The clinical isolates used in this study were isolates from blood cultures of patients hospitalized at the Queen Mary Hospital in Hong Kong during a 6-year period (July 1995 to June 2001). All viridans streptococci (-haemolytic streptococci other than Streptococcus pneumoniae) isolated from blood cultures were subjected to PCR using S. sinensis-specific primers as described below. All isolates identified as S. sinensis by PCR and 16S rRNA gene sequencing were tested by standard conventional biochemical methods (Murray et al., 2003) and by the Vitek System (GPI), the API system (20 STREP) and the ATB Expression system (ID32 STREP) (all from bioMérieux). Lancefield serogrouping was performed using Streptex (Murex Biotech) according to the manufacturer's instructions. All tests were performed in triplicate with freshly prepared media on separate occasions. Antimicrobial susceptibility was tested by the E-test for penicillin and the Kirby–Bauer disk diffusion method for the other antibiotics, and the results were interpreted according to the National Committee for Clinical Laboratory Standards criteria for -haemolytic streptococci.

Extraction of bacterial DNA for PCR and 16S rRNA gene sequencing.

Bacterial DNA extraction was performed as previously described (Woo et al., 2001, 2003). Briefly, 80 µl 0.05 M NaOH was added to 20 µl bacterial cells suspended in distilled water and the mixture was incubated at 60 °C for 45 min, followed by the addition of 6 µl Tris/HCl (pH 7.0), achieving a final pH of 8.0. The resultant mixture was diluted 100-fold and 5 µl of the diluted extract was used for PCR.

Identification of S. sinensis by PCR.

Bacterial DNA extracts of all viridans streptococci isolated from blood cultures during the 6-year period, a positive control (S. sinensis type strain HKU4^T) and a negative control (distilled water) were amplified using the following primers (0.5 µM): S. sinensis-specific primers LPW303 (5'-TAGTTTACTACACCGTAC-3') and LPW304 (5'-CTTACCATGCAGTAAGAT-3'), which amplify a 128 bp fragment of the 16S rRNA gene of S. sinensis (nt 75–202), and LPW306 (5'-TGAGATGTTGGGTTAAGT-3') and LPW307 (5'-TAGCGATTCCGACTTCAT-3'), internal control primers that amplify a 268 bp fragment of the 16S rRNA gene of all viridans streptococci (nt 1085–1352 of S. sinensis) (Gibco-BRL). The PCR mixture (50 µl) contained bacterial DNA, PCR buffer (10 mM Tris/HCl, pH 8.3, 50 mM KCl, 2 mM MgCl₂ and 0.01 % gelatin), 200 µM each deoxynucleoside triphosphate (dNTP) and 1.0 U Taq polymerase (Boehringer Mannheim). The mixtures were amplified by 40 cycles of 94 °C for 1 min, 50 °C for 1.5 min and 72 °C for 2 min, with a final extension at 72 °C for 10 min, in an automated thermal cycler (Perkin-Elmer Cetus). Ten microlitres of each amplified product was electrophoresed in a 2.0 % (w/v) agarose gel, with a molecular size marker (X174 HaeIII digest; Boehringer Mannheim) in parallel. Electrophoresis in Tris/borate/EDTA buffer was performed at 100 V for 1.5 h. The gel was stained with ethidium bromide (0.5 µg ml^–1) for 15 min, rinsed and photographed under UV light.

16S rRNA gene sequencing.

PCR amplification and DNA sequencing of the 16S rRNA genes were performed on the two strains of viridans streptococci identified as S. sinensis by the PCR described above (HKU5 and HKU6) according to our previous publication (Woo et al., 2002). Briefly, DNase-I-treated distilled water and PCR master mix (containing dNTPs, PCR buffer and Taq polymerase) were used in all PCR reactions by adding 1 U DNase I (Pharmacia) to 40 µl distilled water or PCR master mix, incubating the mixture at 25 °C for 15 min and subsequently at 95 °C for 10 min to inactivate the DNase I. Bacterial DNA extracts and the control were amplified with 0.5 µM primers (LPW55, 5'-AGTTTGATCCTGGCTCAG-3'; and LPW205, 5'-CTTGTTACGACTTCACCC-3'; Gibco-BRL). The PCR mixture (50 µl) contained bacterial DNA, PCR buffer (10 mM Tris/HCl, pH 8.3, 50 mM KCl, 2 mM MgCl₂ and 0.01 % gelatin), 200 µM each dNTP and 1.0 U Taq polymerase. The mixtures were amplified by 40 cycles of 94 °C for 1 min, 55 °C for 1 min and 72 °C for 2 min, with a final extension at 72 °C for 10 min, in an automated thermal cycler (Perkin-Elmer Cetus). DNase-I-treated distilled water was used as the negative control. Ten microlitres of each amplified product was electrophoresed in a 1.0 % (w/v) agarose gel, with a molecular size marker ( DNA AvaII digest; Boehringer Mannheim) in parallel. Electrophoresis in Tris/borate/EDTA buffer was performed at 100 V for 1.5 h. The gel was stained with ethidium bromide (0.5 µg ml^–1) for 15 min, rinsed and photographed under UV light.

PCR products were gel-purified using the QIAquick PCR purification kit (QIAGEN). Both strands of the PCR products were sequenced twice with an ABI 377 automated sequencer according to the manufacturer's instructions (Perkin-Elmer), using the PCR primers LPW55 and LPW205 and additional sequencing primers LPW304 and LPW306. The sequences of the PCR products were compared with known 16S rRNA gene sequences in GenBank by multiple sequence alignment using the CLUSTAL W program (Thompson et al., 1994).

Phylogenetic characterization.

Phylogenetic relationships among the nucleotide sequences of the 16S rRNA genes of S. sinensis and the other viridans streptococci were determined using the PileUp method with GrowTree (Genetics Computer Group). A total of 1377 nucleotides were included in the analysis.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Identification of S. sinensis by PCR

A total of 302 viridans streptococci were isolated from blood cultures of patients admitted to the Queen Mary Hospital during the 6-year period (July 1995 to June 2001). The 268 bp fragment of the 16S rRNA gene was amplified from all isolates using PCR primers LPW306 and LPW307, whereas the 128 bp fragment could only be amplified from three isolates (including the previously reported HKU4^T; Woo et al., 2002) using S. sinensis-specific primers LPW303 and LPW304 (Fig. 1).

View larger version (56K): [in this window] [in a new window]   Fig. 1. DNA products from PCR of the S. sinensis 16S rRNA gene. Lanes: M, molecular marker X174 DNA HaeIII digest; 1–3, PCR using primers LPW303 and LPW304 (lane 1, HKU4T; lane 2, HKU5; lane 3, negative control containing distilled water); 4–12, PCR using primers LPW303, LPW304, LPW306 and LPW307 (lane 4, negative control containing distilled water; lane 5, HKU4T; lane 6, HKU5; lanes 7–12, six bacteraemic viridans streptococci isolates).

16S rRNA gene sequencing

The 16S rRNA gene sequence of HKU4^T has been reported previously (Woo et al., 2002). PCR of the 16S rRNA genes of strains HKU5 and HKU6 showed bands at 1500 bp. DNA sequencing of the PCR products showed no base differences between the 16S rRNA gene sequences of HKU5 and HKU6 and that of HKU4^T. There was 3.6 % difference between the 16S rRNA gene sequence of S. sinensis and that of Streptococcus gordonii and 3.7 % difference between the 16S rRNA gene sequence of S. sinensis and that of S. intermedius (Fig. 2).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Phylogenetic tree showing the relationships of the nucleotide sequences of 16S rRNA genes of S. sinensis to those of other viridans streptococci of the anginosus and mitis groups. The tree was constructed using the neighbour-joining method with the 16S rRNA gene sequence of Streptococcus mutans as the root. Bootstrap values were calculated from 1000 trees. The scale bar indicates the estimated number of substitutions per 100 bases using the Jukes–Cantor correction. GenBank accession numbers are given.

Patient characteristics

All three patients with S. sinensis bacteraemia had underlying chronic rheumatic heart disease. All had community- acquired monomicrobial S. sinensis bacteraemia. Only one had a history of tooth extraction prior to the infective endocarditis. All had fever and other features of infective endocarditis, such as finger clubbing, heart failure and embolic phenomena. Two of the three patients had echocardiographic evidence of vegetations. All fulfilled Duke's criteria for the diagnosis of infective endocarditis. All responded to treatment with penicillin G/ampicillin and gentamicin.

Phenotypic characteristics

The phenotypic characteristics of HKU4^T, HKU5 and HKU6 are summarized in Table 1. All three strains were Gram-positive, non-spore-forming cocci arranged in chains. All grew on sheep blood agar as -haemolytic, grey colonies of 0.5–1 mm in diameter after 24 h incubation at 37 °C in ambient air. No enhancement of growth was observed in 5 % CO₂. All grew in microaerophilic or anaerobic environments, but did not grow in 6 % NaCl. HKU4^T was non-groupable with Lancefield groups A, B, C, D, F or G antisera, but HKU5 and HKU6 were Lancefield group F. All were resistant to optochin and bacitracin, and were non-motile at both 25 and 37 °C. All were Voges–Proskauer-test positive. All produced leucine arylamidase and ß-glucosidase, but not catalase, urease, lysine decarboxylase or ornithine decarboxylase. All hydrolysed aesculin and arginine. All utilized glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, mannose, maltose and starch. HKU4^T was identified by the Vitek system (GPI), API system (20 STREP) and ATB system (ID32 STREP) as 99 % S. intermedius, 51.3 % S. intermedius and 99.9 % S. anginosus, respectively. HKU5 was identified using the same systems as 87 % Streptococcus sanguinis/S. gordonii, 59 % Streptococcus salivarius and 99.6 % S. anginosus, respectively, and HKU6 as 87 % S. sanguinis/S. gordonii, 77 % S. pneumoniae and 98.3 % S. anginosus, respectively. All were sensitive to penicillin (MICs of HKU4^T, HKU5 and HKU6 were 0.064, 0.032 and 0.064 µg ml^–1, respectively), ceftriaxone, cefepime, clindamycin, erythromycin, ofloxacin and vancomycin. HKU4^T and HKU6 were sensitive to, but HKU5 was resistant to, tetracycline.

Similar to the other viridans streptococci, S. sinensis is a definite cause of community-acquired infective endocarditis. In the present study, the clinical significance of S. sinensis in all three patients was evident by its isolation from blood cultures from patients with infective endocarditis, fulfilling Duke's criteria (Durack et al., 1994). Furthermore, besides isolating S. sinensis from our patients with infective endocarditis, a 16S rRNA gene sequence with only three base differences from our isolates has been detected in the aortic valve of a patient with infective endocarditis (GenBank accession no. AY049738). This shows that S. sinensis is probably not simply a pathogen of local importance. Instead, its presence in both Asia and Europe implies that S. sinensis may be a bacterium of global importance.

The discovery of Lancefield group F streptococci in Streptococcus species other than members of the ‘S. milleri’ group implies that a proportion of ‘S. milleri’ infections (such as ‘S. milleri’ endocarditis) reported in the literature might actually be cases of S. sinensis infections. In contrast to the other viridans streptococci, members of the ‘S. milleri’ group, especially S. intermedius and S. constellatus, rarely cause infective endocarditis (Woo et al., 2004). In a recently published review on 51 cases of ‘S. milleri’ bacteraemia, none of the patients had endocarditis (Bert et al., 1998). On the other hand, members of the ‘S. milleri’ group, especially S. intermedius and S. constellatus, are major causes of abscess formation, including cases of myocardial abscess complicating ‘S. milleri’ endocarditis (Hurle et al., 1996; Levandowski, 1985). Since S. sinensis and members of the ‘S. milleri’ group are phenotypically similar and Lancefield group F has been found almost exclusively in members of ‘S. milleri’ (Brogan et al., 1997), Lancefield group F streptococci would be reported as ‘S. milleri’ in most clinical microbiology laboratories. The present data imply that a proportion of viridans streptococci that were identified as ‘S. milleri’ in the past may actually be S. sinensis. 16S rRNA gene sequencing should be performed on these strains, especially when they are associated with an atypical diagnosis such as infective endocarditis, to ascertain the identity of the species and hence the epidemiology of the corresponding infections. Lancefield group F streptococci should not automatically be reported as ‘S. milleri'.

We speculate that S. sinensis is part of the human oral flora. Most viridans streptococci are natural inhabitants of the human oral cavity and transient bacteraemia may occur during tooth extraction. This may result in infective endocarditis in patients with pre-existing heart diseases. The fact that one of the patients had a history of tooth extraction 2 months before the illness is in line with this speculation. Further studies on the isolation of S. sinensis from other clinical specimens may add weight to this hypothesis and determine whether S. sinensis is also an important cause of aspiration pneumonia and lung, brain, liver and oral abscesses.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This work is partly supported by the University Development Fund, University Research Grant Council and the Committee for Research and Conference Grant, The University of Hong Kong.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES