A study of susceptibility of 100 clinical isolates belonging to the Streptococcus milleri group to 16 cephalosporins

B. Aracil, J. L. Gomez-Garces* and J. I. Alos

Servicio de Microbiologia, Hospital de Mostoles, Instituto Nacional de la Salud, Calle Rio Jucar s/n, 28935 Mostoles, Madrid, Spain


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The Streptococcus milleri group are uniformly susceptible to penicillin G, but their susceptibilities to different cephalosporins vary considerably. The antimicrobial susceptibilities of 100 clinically significant strains of the S. milleri group to 16 cephalosporins were determined by the agar dilution method. The majority of first-generation cephalosporins were highly active. Cefamandole, cefuroxime and cefprozil were the most active second-generation agents examined. Third-generation parenteral cephalosporins exhibited excellent activity, with the exception of ceftazidime. The most active of the oral preparations of this group was cefpodoxime, with cefixime and ceftibuten being considerably less active. MICs of cefepime, the only fourth-generation cephalosporin tested, were higher than those of cefotaxime and ceftriaxone.


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
The Streptococcus milleri group (SMG) is a highly diverse one which includes the three species: Streptococcus anginosus, Streptococcus intermedius and Streptococcus constellatus.1 The group also includes haemolytic streptococci belonging to Lancefield group A, C, F or G (as well as non-groupable {alpha} and non-haemolytic streptococci). Colonial morphology is variable.2

The SMG are an important part of normal microbial flora in humans and animals. They are indigenous to the upper respiratory tract, the female genital tract and all regions of the gastrointestinal tract. They are most prevalent in the oral cavity. This is especially true of S. anginosus, which is present in subgingival plaque. 2 SMG organisms occasionally produce abdominal infections and are frequently found in abscesses in these and other sites. 1234 Pathogenicity in these cases has been demonstrated by the recovery of the species in pure culture, or in association with other anaerobic genera. Concurrent bacteraemia is a frequent feature. 2,3

Most members of the SMG are susceptible to low concentrations of penicillin G. Approximately 80-90% of strains have MICs of<=0.1 mg/L. 3,4 Many other B-lactam antibiotics have in-vitro activity similar to that of penicillin against the SMG, but the susceptibilities to different cephalosporins are quite variable. 5 The aim of the current study was the evaluation of the antimicrobial susceptibilities of 100 clinically significant strains of the SMG to 16 cephalosporins, using the agar dilution method, as described by the NCCLS. 6


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
A total of 100 strains of the SMG were studied. These were recent clinical isolates from patients in this hospital. The sources of isolates were: abdominal abscesses, hepatic abscesses, peritonsillar, pharyngeal and retroauricular abscesses, gluteus and psoas abscesses, ascitic fluid, pleural empyema, surgical wounds and blood cultures.

All strains were identified as members of the SMG as designated by API 20 Strep and Rapid ID32 Strep systems (Bio-Mérieux, Marcy l'Etoile, France). After presumptive identification, they were stored in skim milk at -20°C.

Morphologic characteristics, including haemolysis, were evaluated on Columbia agar plates (Becton Dickinson, Meylan, France), supplemented with 5% sheep blood. Serologic grouping to detect A, C, F and G Lancefield antigens was carried out using a commercial antisera kit (Streptococcal Grouping, Unipath, Basingstoke, UK). Fermentation of inulin, lactose and trealose was tested in brain-heart infusion broth containing the corresponding carbohydrate (1%) and bromocresol purple (0.0015%). Acetoin production (Voges-Proskauer), aesculin and arginine hydrolysis were tested using standard methods.

Antibiotics were obtained from their manufacturers as laboratory powders of defined potency and were reconstituted in recommended diluents to yield stock solutions. These were kept frozen at -40°C at the indicated concentration ranges: penicillin, G 0.03–0.25 mg/L (Sigma, St Louis, MO, USA); cephalothin, 0.25–4 mg/L (Sigma); cefazolin, 0.25–4 mg/L (Sigma); cefaclor, 0.5–8 mg/L (Sigma); cefprozil, 0.5–8 mg/L (Bristol-Myers Squibb, Barcelona, Spain); cefamandole, 0.5–8 mg/L (Sigma); cefuroxime, 0.06–1 mg/L (Glaxo, Aranda de Duero, Spain); cefoxitin, 0.5–8 mg/L (Merck, Sharp & Dohme, Milan, Italy); cefminox, 0.5–8 mg/L (Tedec-Meiji Farma, Madrid, Spain); cefotaxime, 0.06–0.5 mg/L (Hoechst-Marion-Roussel, Romainville, France); ceftriaxone, 0.06–1 mg/L (Roche, Basle, Switzerland); ceftazidime, 0.5–8 mg/L (Glaxo Wellcome, Madrid, Spain); ceftizoxime, 0.06–1 mg/L (SmithKline Beecham, Toledo, Spain); cefixime 0.25–32 mg/L (Merck Farma & Quimica, Barcelona, Spain); cefpodoxime, 0.06–1 mg/L (Hoechst-Marion-Roussel); ceftibuten 1–16 mg/L (Schering-Plough, Kenilworth, NJ, USA); and cefepime, 0.25–32 mg/L (Bristol-Myers Squibb) were tested.

The SMG strains were taken from frozen stock cultures and grown on blood agar in 5–10% CO2 at 35°C for 24 h. The strains were transferred to tubes containing Muller–Hinton broth (Difco, Detroit, MI, USA) and incubated overnight in 5–10% CO2. All MICs were determined by the agar dilution method using Müller-Hinton agar (Becton Dickinson), supplemented with 5% sheep blood, containing graded concentrations of antibiotics. The inoculum used for the SMG was 104 cfu per spot, applied with a Steers replicator. All plates were incubated in 5–10% CO2 at 35°C for 24 h before being examined.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Results of MIC determinations are shown in Tables I and II. As determined by penicillin MIC breakpoints, recently established by the NCCLS for specific application to Streptococcus spp.,6 93% of isolates may be considered to be susceptible (MIC<=0.12 mg/L) and 7% intermediate (MIC 0.25 mg/L) to penicillin. All first-generation and second-generation cephalosporins were uniformly active, except cefaclor, cefoxitin and cefminox, whose MIC50s were 2 mg/L, 4 mg/mL and 8 mg/L, respectively. These MICs are higher than those of other compounds of the same generation, although breakpoints have not yet been published by the NCCLS. All the strains tested were susceptible to cefotaxime and ceftriaxone. Ceftizoxime showed equally good activity, with MIC50s and MIC90s similar to those of the antibiotics mentioned above. Conversely, ceftazidime, cefixime and ceftibuten (for which breakpoints are not established) showed low levels of activity, with MIC50s of 4 mg/L for cefixime and ceftazidime and >16 mg/L for ceftibuten.6 Cefepime, the only fourth-generation cephalosporin tested, inhibited 98% of strains with concentrations of <=1 mg/L. The two remaining strains had MICs of 2 mg/L.


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Table I. In-vitro activities of penicillin and selected cephalosporins versus 100 isolates of the SMG
 

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Table II. Range, MIC50, MIC90 and percentages of susceptibility of 100 clinical strains of the SMG to 16 cephalosporins
 

    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Most streptococci belonging to the SMG show similar susceptibility patterns to the majority of antibiotics. All are intrinsically resistant to bacitracin (a characteristic differentiating the SMG and strains with the group A Lancefield antigen from Streptococcus pyogenes) and nitrofurantoin.7 They remain uniformly sensitive to vancomycin, teicoplanin, trimethoprim, chloramphenicol and rifampicin. 3,4 Gentamicin, amikacin and all aminoglycosides also have in-vitro activity in all reported series, although alone they are not clinically useful against streptococci.3,8,9 The SMG are not usually resistant to fluoroquinolones, but a large number of strains have MICs that place them in the `intermediate susceptibility' category.3,4 More variable results are obtained with tetracyclines, where rates of resistance in the range 0–37% have been described. 3,4 Resistance to the erythromycin-lincomycin-clindamycin group, although currently rare, is transferable among the streptococci, including the SMG, and can be as high as 12–14%.10 In any case, the B-lactam group provides the antibiotic of choice in infections caused by streptococci, penicillin G being used in a large number of cases. 2 The SMG organisms are almost uniformly sensitive to penicillin, with a few isolates being of intermediate susceptibility (MIC 0.25–2.0 mg/L). 3,6,7 The SMG exhibit variable sensitivities to the cephalosporin group. 5 First-generation cephalosporins have good activity. In this series all strains were susceptible to cephalothin and cefazolin, with MIC50s and MIC90s of 0.50 and 1 mg/L, respectively. Similarly, the SMG were susceptible to some second-generation cephalosporins, although the activity of these compounds was less uniform. Cefamandole and cefuroxime were very active against all the strains tested, with MIC 50s and MIC90s of 0.25 and 0.5 mg/L, respectively. Cefprozil showed similar activity, with an MIC50 of 0.5 mg/L and an MIC90 of 1 mg/L. This was lower than the breakpoint. Cefoxitin and cefaclor needed concentrations of 8 mg/L to inhibit all strains. Cefminox was the least active of the group, with an MIC50 of 8 mg/L. Nearly all strains were considered resistant. Third-generation parenteral cephalosporins had very good activity (with the exception of ceftazidime) and 100% of strains were susceptible. The MIC50s and MIC90s were well below established breakpoints (cefotaxime and ceftriaxone 0.25 and 0.5 mg/L and ceftizoxime 0.5 and 1 mg/L, respectively). Results using ceftazidime, with an MIC50 of 4 mg/L and an MIC 90 of 8 mg/L, were less impressive.

Third-generation oral cephalosporins were the most varied group. Cefpodoxime had excellent activity, with MIC50s and MIC90s of 0.25 and 0.5 mg/L, respectively, for all strains. Cefixime was less active, with only 8% of strains having MICs <=2 mg/L. Ceftibuten was the least active, all strains having MICs >16 mg/L.

Finally, cefepime, the sole representative of the fourth-generation cephalosporins, showed very good activity, with 98% of strains being susceptible (MIC50 0.5 mg/L and MIC90 1 mg/L) and only two strains having MICs>=2 mg/L.

Although breakpoints for some cephalosporins for the SMG are not included in the most recently published NCCLS tables,6 making the interpretation of their values difficult, it is interesting to observe that some cephalosporins with similar clinical uses have very different antibacterial activities against these organisms. Therefore the selection of one of these cephalosporins, when initiating treatment of an SMG infection, should be made with knowledge of previously ascertained activity against these organisms, which are assuming increasing importance in the aetiology of human infections.


    Acknowledgments
 
Part of the results reported in this paper was presented at the 8th European Congress of Clinical Microbiology and Infectious Diseases, Lausanne, 25-28 May 1997.


    Notes
 
* Corresponding author. Tel: +34-1-6243009; Fax: +34-1-6476371; E-mail: jlgarces{at}microb.net Back


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
1 . Whiley, R. A. & Beighton, D. (1991). Emended descriptions and recognition of Streptococcus constellatus , Streptococcus intermedius and Streptococcus anginosus as distinct species. International Journal of Systematic Bacteriology 41, 1-5.[Abstract]

2 . Ruoff, K. L. (1988). Streptococcus anginosus (` Streptococcus milleri' ): the unrecognized pathogen. Clinical Microbiology Reviews 1, 102-8.[Medline]

3 . Gómez-Garcés, J.-L., Alós, J. I. & Cogollos, R. (1994). Bacteriologic characteristics and antimicrobial susceptibility of 70 clinically significant isolates of Streptococcus milleri group. Diagnostic Microbiology and Infectious Diseases 19, 69-73.[ISI][Medline]

4 . Doern, G. V., Ferraro, M. J., Brueggemann, A. B. & Ruoff, K. L. (1996). Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States. Antimicrobial Agents and Chemotherapy 40, 891-4.[Abstract]

5 . Alcaide, F., Liñares, J., Pallarés, R., Carratala, J., Benitez, M. A., Gudiol, F. et al. (1995). In vitro activities of 22 ß -lactam antibiotics against penicillin-resistant and penicillin-susceptible viridans group streptococci isolated from blood. Antimicrobial Agents and Chemotherapy 39, 2243-7.[Abstract]

6 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically -Fourth Edition: Approved Standard M7-A4 (and Sixth Informational Supplement M100-S7). NCCLS, Wayne, PA.

7 . Poole, P. M. & Wilson, G. (1979). Occurrence and cultural features of Streptococcus milleri in various body sites. Journal of Clinical Pathology 32 , 764-8.[Abstract]

8 . Piscitelli, S. C., Shwed, J., Schreckenberger, P. & Danziger, L. H. (1992). Streptococcus milleri group: renewed interest in an elusive pathogen. European Journal of Clinical Microbiology and Infectious Diseases 11, 491-8.[ISI][Medline]

9 . Francioli, P., Moreillon, P. & Glauser, M. P. (1985). Comparison of single doses of amoxicillin or amoxicillin-gentamicin for the prevention of endocarditis caused by Streptococcus faecalis and by viridans streptococci. Journal of Infectious Diseases 152, 83-9.[ISI][Medline]

10 . Horodniceanu, T., Bougueleret, L. & Bieth, G. (1981). Conjugative transfer of multiple-antibiotic resistance markers in ß -hemolytic group A, B, F and G streptococci in the absence of extrachromosomal deoxyribonucleic acid. Plasmids 5, 127-37.

Received 11 May 1998; returned 29 June 1998; revised 15 July 1998; accepted 9 September 1998