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Journal of Clinical Microbiology, July 2003, p. 2862-2866, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2862-2866.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genotyping by Amplified Fragment Length Polymorphism Analysis Reveals Persistence and Recurrence of Infection with Streptococcus anginosus Group Organisms

Jan A. Jacobs,^1* Jeroen H. T. Tjhie,¹ Monique G. J. Smeets,¹ Corrie S. Schot,² and Leo M. Schouls²

Department of Medical Microbiology, University Hospital Maastricht, 6202 AZ Maastricht,¹ Laboratory for Vaccine-Preventable Diseases, National Institute of Public Health and the Environment, 3720 BA Bilthoven, The Netherlands²

Received 27 December 2002/ Returned for modification 26 February 2003/ Accepted 11 April 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius, commonly referred to as the Streptococcus anginosus group (SAG), are commensal organisms known for their propensity to cause purulent infections which are difficult to eradicate. In this study, we determined the genetic similarities between SAG isolates consecutively recovered from single patients to assess the duration of infection or colonization. A total of 97 SAG isolates recovered from 30 patients were included; 65 (67.0%) of the isolates were abscess related. The isolates were identified by the 16S rRNA reverse line blot hybridization assay as S. anginosus (n = 34), S. constellatus (n = 55), and S. intermedius (n = 8). Amplified fragment length polymorphism (AFLP) analysis of the SAG isolates produced discriminatory and reproducible patterns. Consecutive SAG isolates with identical AFLP types were found in 27 of 30 (90.0%) patients, and consecutive isolates with only a single AFLP type were demonstrated in 21 (70.0%) patients. The median delay between the times of recovery of the first and last isolates of identical AFLP types from each patient was 36 days, and this delay extended for more than 1 year in patients with both colonizing and abscess-related SAG isolates. In six bacteremic patients, paired blood and nonblood SAG isolates showed identical AFLP types.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius are commonly referred to as the Streptococcus milleri group, recently designated the Streptococcus anginosus group (SAG) (10). SAG strains are known for their association with purulent infections that occur after local disruption of the mucosal barrier, such as in cases of ulceration, perforation, inflammation, or surgery (6, 14). These infections often cause significant morbidity and may require repeat drainage procedures (6). In our collection of consecutive clinical SAG isolates, we observed successive isolates from patients with persistent or recurrent infections. Although biochemical and serological data pointed to a close similarity of at least some of the isolates recovered from single patients, information on relatedness at the genetic level was lacking. To study the genetic similarities between these successive SAG isolates, we used amplified fragment length polymorphism (AFLP) analysis (19).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. SAG isolates were consecutively collected from clinical specimens routinely submitted for culture to the Medical Microbiology Department of the University Hospital Maastricht, a 600-bed tertiary-care hospital. Culture, isolation, and identification to the SAG level have been described previously (7). Species identification was performed by the 16S rRNA reverse line blot hybridization assay. The previously described S. intermedius non-exclusive human hemolytic strains were assigned to the species S. constellatus (8), and the isolates within the S. anginosus species with different 16S rRNA groups were grouped together (9). Consecutively obtained isolates for which there were 7 or more days between the dates of first and subsequent recoveries were selected from the collection of isolates recovered from 1993 to 2000. The medical records of the patients from whom these isolates were recovered were examined for information on sex, age, and clinical history.

AFLP analysis. AFLP analysis was performed as described previously, with slight modifications (15, 19). DNA isolation was performed as described by Boom et al. (3), except that the pellet of bacteria was resuspended in lysis buffer heated for 10 min at 80°C. Restriction and ligation were performed with aliquots of 20 ng of isolated DNA and RNA in one reaction for 4 h at 37°C instead of in two different reactions. For the selective amplification, several different primer combinations were tested. The combination EcoRI+AA (cyanine 5-GACTCGCTACCAATTC+AA) and Mse-Ad1+0 (GATGAGTCCTGAGTAAC) gave the most discriminative patterns and was used in the rest of the study. After electrophoresis of the PCR products (ALFexpress; Amersham Pharmacia Biotech, Biosciences Europe GmbH, Roosendaal, The Netherlands), the patterns were visualized with the DAN fragment analyzer (Amersham Pharmacia), converted into a tagged image file format, and processed with Imagemaster 1D Elite and Database software (Amersham Pharmacia Biotech). The sizes of the fragments were estimated by comparison with phage DNA processed by the same AFLP protocol with primers EcoRI and Mse-Ad1 without the selective bases (15). Similarities between fingerprints were calculated by use of both the Dice and the Pearson product moment correlation coefficients (r). Cluster analysis was performed by the unweighted pair method with average linkages.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reproducibility and discriminatory index by AFLP analysis. To assess the reproducibility of the AFLP procedure, subculture, DNA extraction, and AFLP analysis were performed five times with 24 epidemiologically unrelated SAG strains. Although the intensities of the larger bands occasionally varied, the repeat assays yielded identical banding profiles. The discriminatory power of AFLP analysis was calculated by using the discriminatory index described by Hunter (5). Nineteen epidemiologically unrelated SAG isolates, S. anginosus ATCC 33397, S. constellatus ATCC 27823, S. intermedius ATCC 27335, and a single isolate from each patient were included. Use of a similarity level of 90% as the cutoff value yielded a discriminatory index of 0.9973. This resulted in the assignment of 49 distinct AFLP types to the 52 epidemiologically unrelated SAG strains. There were two small clusters with identical AFLP types, one comprising two isolates and the other comprising three isolates. One cluster comprised abscess-related S. intermedius strains from patients 3 and 13, and the other cluster contained the strains from patients 27 and 30 together with strain 1007, which are all three so-called motile S. anginosus stains (9).

Patient characteristics and species identification of SAG isolates. Table 1 shows the characteristics of the patients, the sites and times of recovery of the SAG isolates, and the species identifications and AFLP types. A total of 97 SAG isolates were recovered from the 30 patients included in this study. The ratio of males to females for this group of patients was 1.5:1, and the mean age for the patients was 49.9 ± 24.1 years (age range, 1 to 82 years). The majority of the patients had been hospitalized, and the mean hospital stay for the 19 patients with abscess formation was 72.9 ± 24.5 days. All but one of these patients underwent multiple surgical or percutaneous drainage procedures, and four (21.5%) of them died during hospitalization.

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TABLE 1. Patients' clinical histories and results of AFLP typing and species identification of the consecutive SAG isolates

The isolates were identified by the 16S rRNA reverse line blot hybridization assay as S. anginosus (n = 34), S. constellatus (n = 55), and S. intermedius (n = 8). Sixty-five (67.0%) isolates from 19 (63.3%) patients were associated with clear abscess formation and originated from the abdominal cavity (patients 1 to 16), the skin or soft tissues (patients 17 and 19), or the pleural cavity (patient 18). The two urinary tract isolates from patient 27 were considered infection related, as they grew in pure culture at quantities of >10⁵ CFU/ml and were associated with pyuria. The remaining 30 (30.1%) isolates from 11 patients grew as part of colonizing flora from the respiratory tract (patients 19 and 21 to 26), the skin (patient 20), and the urinary tract (patients 28 to 30). Both infection- and colonization-related isolates were recovered from patient 19.

Data on the antimicrobial therapy administered after the initial infectious event were retrieved for 13 of 19 patients with abscesses. Antimicrobial agents active against SAG organisms were given for at least 10 days to eight patients (patients 1, 3, 4, 6, 8, 12, 16, and 18), for 7 days to three patients (patients 2, 5, and 10), and for less than 7 days to two patients (patients 9 and 15).

AFLP types of SAG isolates consecutively recovered from each patient. Table 1 lists the AFLP types of the SAG isolates recovered from each patient. Successive isolates with identical AFLP types and species identifications were recovered from 27 (90.0%) patients. Only a single AFLP type and SAG species were found in 21 (70.0%) patients, and either different species or AFLP types were found in the other 9 patients.

The median delay between the time of recovery of the first SAG isolate and the last SAG isolate of the same AFLP type was 36 days, and the delay extended for more than 1 year in three patients (patients 23, 26, and 30) with colonizing SAG isolates and in two patients (patients 17 and 27) with infection- or abscess-related SAG isolates. For patients 27 and 30, S. anginosus could still be isolated from the urogenital tracts after 1 and 5 years, respectively. Other cases of long-term persistence were detected for patient 17 (with a soft tissue abscess) and patients 23 and 24 (with colonization of the respiratory tract).

In all six patients with blood- and abscess-related strains (patients 2 to 4, 6, 10, and 15), the paired blood- and abscess-related strains from each patient were of identical AFLP types. The date of isolation of these isolates from abscesses either preceded or coincided with the date of isolation from blood.

The observation of identical or distinct AFLP types in single patients was supported by identification to the 16S rRNA ribogroup level (8, 9) and by phenotypic characteristics such as the pattern of hemolysis and Lancefield group (results not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the report presented here, we describe the genotypic similarities of multiple SAG isolates consecutively recovered from 30 patients. The samples from the majority of the patients contained only a single SAG AFLP type. However, in some cases coinfection with other SAG species or AFLP types was found. Many of the patients suffered from prolonged persistence of SAG in abscesses or were carrying SAG isolates as part of their colonizing flora. In some of them, the persistence lasted for more than a year. In addition, in all bacteremic patients, the blood- and abscess-derived SAG isolates from the same patient had identical AFLP types.

Bartie and coworkers (1) demonstrated extensive genetic diversity between epidemiologically unrelated SAG isolates using pulsed-field gel electrophoresis (PFGE). Those investigators hypothesized that diversity is essential for colonization and survival of the SAG isolates within the host. In view of this diversity, the genotypic similarities of the consecutive isolates from the patients described here highlight the ability of SAG isolates to persist as both infecting and colonizing flora. The persistence of SAG in deep-seated infections is in line with the need for repeated wound drainage procedures and the protracted hospital stays of the affected patients. Among the factors that might explain the tendency for abscess formation, the cytotoxic intermedilysin has been demonstrated to be a significant contributing factor (12). Intermedilysin production is confined to the species S. intermedius, which is more frequently associated with abscess formation than S. anginosus and S. constellatus (4, 7, 8). However, in our study the majority of SAG isolates persisting in abscesses were identified as S. anginosus and S. constellatus. This may indicate that other factors are important in abscess formation, too, or that our collection may have been influenced by selection bias, e.g., the surgeon's decision to submit or not submit specimens for culture.

In the present study, the persistence of SAG isolates was also demonstrated for non-infection-related colonizing flora. As for abscess-related strains, a prospective study is required to elucidate the genotypic and phenotypic properties of colonizing SAG isolates. Nevertheless, in line with the rates of recovery of single isolates (4, 6, 20), our study confirmed the predilection of S. constellatus for the respiratory tract and demonstrated long-term colonization with this species. Among the isolates recovered from the urinary tract, the so-called motile S. anginosus ribogroup accounted for two cases of persistence extending for more than 1 year. These motile strains exhibit a gliding type of motility, caused by the production of an extracellular glycocalyx, which might aid in the colonization of the mucosal surfaces of host tissues (2).

Blood- and abscess-related SAG isolates were demonstrated to have identical AFLP types, confirming that the deep-seated abscesses were the portal for bacteremia. Episodes such as that in patient 3 showed that SAG bacteremia may develop even long after initial mucosal damage and infection have occurred. Similarly, identical ribotypes were found for pairs of oral and blood isolates of viridans group streptococci recovered from neutropenic patients (13). Also, identical PFGE patterns for blood and nonblood isolates were demonstrated for multiple pairs of Staphylococcus aureus isolates, but not for pairs of Pseudomonas aeruginosa isolates (11).

AFLP analysis of the SAG isolates produced reliable, discriminatory, and reproducible typing results; and the existence of distinct AFLP types in single patients was supported by differences in 16S rRNA (sub)species identification. Although PFGE has been successfully used to type SAG isolates (1), we chose the AFLP technique. The reason for our preference for a PCR-based fingerprinting technique was that some of the isolates exhibited fastidious growth, and there were significant variations in the yields of isolated DNA. AFLP analysis is more robust than other PCR-based fingerprinting methods such as random amplified polymorphism PCR, as the latter method is subject to many technical factors affecting its reproducibility (17). Like other molecular typing methods, AFLP analysis can be used to trace the transmission routes of pathogens (18) or to study phylogenetic relationships between organisms (16). It should be emphasized that we did not focus on these issues in the present study. Like other viridans group streptococci, the SAG species are regarded as commensal organisms with a low probability of cross-transmission (21), and a prospective study to assess patient-to-patient transmission would require inclusion of commensal SAG isolates from numerous patients at risk. Likewise, an AFLP study of the possible phylogenetic relationships between SAG isolates should encompass a more extended sample of SAG isolates, with equal representation of the different SAG species and 16S rRNA subtypes.

In conclusion, AFLP analysis proved to be a suitable technique for determination of the genetic similarities of consecutive SAG isolates. The technique can be used for further studies on the pathogenesis of SAG infections. SAG isolates may persist for long periods as part of both colonizing and abscess-related flora.

 
We thank D. Jansen and K. Spee for expert technical assistance.

 
* Corresponding author. Mailing address: Department of Medical Microbiology, University Hospital Maastricht, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands. Phone: 31 43 387 66 65. Fax: 31 43 387 66 43. E-mail: jja{at}lmib.azm.nl.

Top Abstract Introduction Materials and Methods Results Discussion References

 

1
1. Bartie, K. L., M. J. Wilson, D. W. Williams, and M. A. O. Lewis. 2000. Macrorestriction fingerprinting of "Streptococcus milleri" group bacteria by pulsed-field gel electrophoresis. J. Clin. Microbiol. 38:2141-2149.[Abstract/Free Full Text]
2
2. Bergman, S., M. Selig, M. D. Collins, J. A. E. Farrow, E. J. Baron, G. R. Dickersin, and K. L. Ruoff. 1995. "Streptococcus milleri" strains displaying a gliding type of motility. Int. J. Syst. Bacteriol. 45:235-239.[Abstract/Free Full Text]
3
3. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503.[Abstract/Free Full Text]
4
4. Clarridge, J. E., III, S. Attori, D. M. Musher, J. Hebert, and S. Dunbar. 2001. Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus ("Streptococcus milleri group") are of different clinical importance and are not equally associated with abscess. Clin. Infect. Dis. 32:1511-1515.[CrossRef][Medline]
5
5. Hunter, P. R. 1990. Reproducibility and indices of discriminatory power of microbial typing methods. J. Clin. Microbiol. 28:1903-1905.[Abstract/Free Full Text]
6
6. Jacobs, J. A., H. G. Pietersen, E. E. Stobberingh, and P. B. Soeters. 1994. Bacteremia due to the "Streptococcus milleri" group: analysis of 19 cases. Clin. Infect. Dis. 19:704-713.[Medline]
7
7. Jacobs, J. A., H. G. Pietersen, E. E. Stobberingh, and P. B. Soeters. 1995. Streptococcus anginosus, Streptococcus constellatus and Streptococcus intermedius: clinical relevance, hemolytic and serologic characteristics. Am. J. Clin. Pathol. 104:547-553.[Medline]
8
8. Jacobs, J. A., C. S. Schot, and L. M. Schouls. 2000. Haemolytic activity of the "Streptococcus milleri group" and relation between haemolysis restricted to human red blood cells and pathogenicity in S. intermedius. J. Med. Microbiol. 49:55-62.[Abstract/Free Full Text]
9
9. Jacobs, J. A., C. S. Schot, and L. M. Schouls. 2000. The Streptococcus anginosus species comprises five 16S rRNA ribogroups with different phenotypic characteristics and clinical relevance. Int. J. Syst. Evol. Microbiol. 50:1073-1079.[Abstract]
10
10. Kawamura, Y., X. G. Hou, F. Sultana, H. Miura, and T. Ezaki. 1995. Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int. J. Syst. Bacteriol. 45:406-408.[Abstract/Free Full Text]
11
11. Matsuda, J., Y. Hirakata, F. Iori, C. Mochida, Y. Ozaki, M. Nakano, K. Izumikawa, T. Yamaguchi, R. Yoshida, Y. Miyazaki, S. Maesaki, K. Tomono, Y. Yamada, S. Kohno, and S. Kamihira. 1998. Genetic relationship between blood and nonblood isolates from bacteremic patients determined by pulsed-field gel electrophoresis. J. Clin. Microbiol. 36:3081-3084.[Abstract/Free Full Text]
12
12. Nagamune, H., R. Whiley, T. Gtoto, Y. Inai, T. Maeda, J. M. Hardie, and H. Kourai. 2000. Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius. J. Clin. Microbiol. 38:220-226.[Abstract/Free Full Text]
13
13. Richard, P., G. A. Del Valle, P. Moreau, N. Milpied, M. P. Felice, T. Daeschler, J. L. Harousseau, and H. Richet. 1995. Viridans streptococcal bacteraemia in patients with neutropenia. Lancet 345:1607-1609.[CrossRef][Medline]
14
14. Ruoff, K. L. 1988. Streptococcus anginosus ("Streptococcus milleri"): the unrecognized pathogen. Clin. Microbiol. Rev. 1:102-108.[Abstract/Free Full Text]
15
15. Speijer, H., P. H. M. Savelkoul, M. J. Bonten, E. E. Stobberingh, and J. H. T. Tjhie. 1999. Application of different genotyping methods for Pseudomonas aeruginosa in a setting of endemicity in an intensive care unit. J. Clin. Microbiol. 37:3654-3661.[Abstract/Free Full Text]
16
16. Torriani, S., F. Clementi, M. Vancanneyt, B. Hoste, F. Dellaglio, and K. Kersters. 2001. Differentiation of Lactobacillus plantarum, L. pentosus and L. paraplantarum species by RAPD-PCR and AFLP. Syst. Appl. Microbiol. 24:554-560.[CrossRef][Medline]
17
17. Tyler, K. D., G. Wang, D. Tyler., and M. Johnson. 1997. Factors affecting the reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. J. Clin. Microbiol. 35:339-346.[Medline]
18
18. van der Zwet, W. C., G. A. Parlevliet, P. H. Savelkoul, J. Stoof, A. M. Kaiser, J. Koeleman, and C. M. Vandenbroucke-Grauls. 1999. Nosocomial outbreak of gentamicin-resistant Klebsiella pneumoniae in a neonatal intensive care unit controlled by a change in antibiotic therapy. J. Hosp. Infect. 42:295-302.[CrossRef][Medline]
19
19. Vos, P., R. Hogers, M. Bleeker, M. Reijans, T. van der Lee, M. Hornes, A. Frijters, J. Pot, J. Peleman, M. Kuiper, and M. Zabeau. 1995. AFLP: a new method for DNA fingerprinting. Nucleic Acids Res. 23:4407-4414.[Abstract/Free Full Text]
20
20. Whiley, R. A., D. Beighton, T. G. Winstanley, H. Y. Frazer, and J. M. Hardie. 1992. Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus (the Streptococcus milleri group): association with different body sites and clinical infections. J. Clin. Microbiol. 30:243-244.[Abstract/Free Full Text]
21
21. Wisplinghoff, H., R. R. Reinert, O. Cornely, and H. Seifert. 1999. Molecular relationships and antimicrobial susceptibilities of viridans group streptococci isolated from blood of neutropenic cancer patients. J. Clin. Microbiol. 37:1876-1880.[Abstract/Free Full Text]

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Journal of Clinical Microbiology, July 2003, p. 2862-2866, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2862-2866.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jacobs, J. A. Articles by Schouls, L. M. Search for Related Content PubMed PubMed Citation Articles by Jacobs, J. A. Articles by Schouls, L. M.