Abstract
Two strains of Streptococcus pneumoniae isolated from sputum and bronchoalveolar samples with high-level resistance to cefotaxime (MIC = 8 to 16 μg/ml) are described. One of them, belonging to serogroup 19, was also highly resistant to penicillin (MIC = 16 μg/ml), while the other, of serogroup 14, was intermediate in its resistance to penicillin (MIC = 0.25 μg/ml). To our knowledge, these are the first two strains to be isolated in Spain with such high levels of resistance to cefotaxime.
Resistance of Streptococcus pneumoniae to penicillin has been of increasing concern during the last decade. Pneumococci with intermediate-level (MIC = 0.1 to 1 μg/ml) and high-level (MIC of ≥2 μg/ml) resistance to penicillin are now encountered in many countries, Spain being one of the most important foci. The resistance, which is due to alterations in the penicillin-binding proteins (PBPs), also affects the other beta-lactams, although not to the same extent. Thus, it is not unusual to find strains for which the MIC of cefotaxime or ceftriaxone is 1 (intermediately resistant) or 2 (resistant) μg/ml (12), MICs that have produced therapeutic failure in the treatment of meningitis (2, 9). Much less frequent are those strains that present higher levels of resistance to these antibiotics: MICs of 8 μg/ml and greater. This phenomenon is of considerable medical concern because of the importance of these cephalosporins in the treatment of severe pneumococcal infections.
We describe two highly cefotaxime-resistant strains of S. pneumoniae isolated in Spain, one with high resistance to penicillin and the other, even more unusually, with only moderate resistance to penicillin.
Case 1.
A 67-year-old patient, with chronic bronchitis, multiple hospital admissions, and numerous courses of antibiotic treatment with oral cephalosporins and ceftazidime, was admitted to the Costa del Sol Hospital (Marbella, Málaga, Spain) due to exacerbation of his cough and usual dyspnea, with whitish sputum and bilateral pleural pain. The culture of his sputum (<10 epithelial cells and >25 leukocytes) revealed growth of numerous colonies of S. pneumoniae (serogroup 14) intermediately resistant to penicillin and resistant to cefotaxime.
Case 2.
A 37-year-old, human immunodeficiency virus-positive patient with multiple hospital admissions, the last two due to lower respiratory tract infection, was admitted to the Virgen de la Arrixaca Hospital (Murcia, Spain). He had been treated with cefotaxime for 10 days and ceftazidime for 8 days during the first episode and ceftazidime for 22 days during the second. Forty-eight hours after discharge, the patient was readmitted due to fever, cough, and purulent sputum. The culture of a sample taken by means of bronchoalveolar lavage revealed pure growth of >105 CFU of S. pneumoniae (serogroup 19), resistant to penicillin and cefotaxime, per ml.
Determination of MICs.
Three different methods were used to determine the MICs: broth microdilution, according to National Committee for Clinical Laboratory Standards criteria (12); diffusion in Mueller-Hinton agar with E-test strips according to the manufacturer’s instructions; and dilution in Mueller-Hinton agar, supplemented with 5% lysed horse blood, and incubation at 35°C in an aerobic atmosphere. S. pneumoniae ATCC 49619 was used for quality control of all test methods.
The first strain, belonging to serogroup 14, revealed intermediate resistance to penicillin (MIC = 0.25 μg/ml); resistance to cefotaxime (MIC = 8 μg/ml); and susceptibility to tetracycline (MIC = 0.5 μg/ml), chloramphenicol (MIC = 2 μg/ml), and erythromycin (MIC = 0.06 μg/ml). The second strain, from serogroup 19, was resistant to penicillin (MIC = 16 μg/ml), cefotaxime (MIC = 16 μg/ml), erythromycin (MIC = 4 μg/ml), and tetracycline (MIC = 64 μg/ml) and susceptible to chloramphenicol (MIC = 2 μg/ml) (Table 1).
TABLE 1.
In vitro susceptibilities of two isolates of S. pneumoniae determined by three different methods
| Antimicrobial | MIC (μg/ml)a
|
|||||
|---|---|---|---|---|---|---|
| Isolate 1
|
Isolate 2
|
|||||
| BMD | AD | E test | BMD | AD | E test | |
| Penicillin | 0.25 | 0.25 | 0.25 | 16 | 8 | 12 |
| Cefotaxime | 8 | 8 | 4 | 16 | 8 | 6 |
| Cefuroxime | 24 | 64 | ||||
| Erythromycin | 0.06 | 0.06 | 4 | 16 | ||
| Tetracycline | 0.5 | 64 | ||||
| Chloramphenicol | 2 | 2 | ||||
BMD, broth microdilution; AD, agar dilution.
Resistance of S. pneumoniae to penicillin is due to changes in high-molecular-weight PBPs, PBP 1a, PBP 2x, and especially PBP 2b, which show reduction in affinity for the antibiotics (5, 8). However, resistance to cephalosporins is simpler than resistance to penicillin, since reduction in the affinity of only two PBPs (1a and 2x) could be involved (1, 11). A serogroup 6 strain with these genetic characteristics in its PBPs for which the MIC of cefotaxime is 4 μg/ml, originating in Spain, was studied by Muñoz et al. (11). Later, Coffey et al. (3) were able to transfer the resistance of two strains, similar to ours, to another, susceptible strain, by means of genetic transformation, confirming that the resistance to cefotaxime was due to changes in PBPs 1a and 2x. These two strains belonged to serogroup 23 and were isolated in the United States. Another strain of serogroup 23, isolated in California, for which the MICs of penicillin and cefotaxime are 0.3 and 2.5 μg/ml, respectively, and studied by Figueiredo et al., presented a marked reduction in the penicillin-binding capacity of PBP 1b (6).
Strains highly resistant to penicillin have been isolated for the last 2 decades, mainly in Eastern European countries (10, 13). More recently, in a European study (7), 25 of 1,131 strains presented high-level resistance to penicillin (MIC of ≥8 μg/ml) and cefotaxime (MIC = 4 to 16 μg/ml), with values similar to those of our second isolate. In the same study, 15 strains for which the MICs of amoxicillin are <0.5 μg/ml were resistant to cefotaxime (MIC of >0.5 μg/ml), six of them highly resistant (MIC of >8 μg/ml), like our first isolate. Some pneumococci with these same characteristics have also been isolated in the United States (1, 14). At present, only limited data are available regarding the worldwide prevalence of S. pneumoniae resistance to broad-spectrum cephalosporins. In Spain, the data collected by the Centro Nacional de Microbiologia in Majadahonda for 1996 were as follows: 16.4% of strains being intermediately resistant and 3.3% being resistant to cefotaxime. In the United States, a multicenter study in 1995, in which 25 hospitals participated, revealed an 88% prevalence of resistance to cefotaxime (15).
This increase in the resistance to broad-spectrum cephalosporins and the increase in the probability that previously susceptible strains may become resistant by transfer in only one step, an easier way than horizontal transfer of high-level resistance to penicillin, which requires the transfer of three PBP genes (4), are sufficient cause for concern regarding the future use of these antibiotics in pneumococcal infections. Thus, the continuous surveillance of the MICs for these organisms, by currently available methods, is more necessary than ever.
We are indebted to Graham C. Arnold for his assistance in the preparation of the manuscript.
REFERENCES
- 1.Bradley J J, Connor J D. Ceftriaxone failure in meningitis caused by Streptococcus pneumoniae with reduced susceptibility to β-lactam antibiotics. Pediatr Infect Dis. 1991;10:871–873. [PubMed] [Google Scholar]
- 2.Catalán M J, Fernández J M, Vázquez A, Valera de Seijas E, Suárez A, Bernaldo de Quirós C. Failure of cefotaxime in the treatment of meningitis due to relatively resistant Streptococcus pneumoniae. Clin Infect Dis. 1994;18:766–769. doi: 10.1093/clinids/18.5.766. [DOI] [PubMed] [Google Scholar]
- 3.Coffey T J, Daniels M, McDougal L K, Dowson C G, Tenover F C, Spratt B G. Genetic analysis of clinical isolates of Streptococcus pneumoniae with high-level resistance to expanded-spectrum cephalosporins. Antimicrob Agents Chemother. 1995;39:1306–1313. doi: 10.1128/aac.39.6.1306. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Coffey T J, Dowson C G, Daniels M, Spratt B G. Genetics and molecular biology of β-lactam-resistant pneumococci. Microb Drug Resist. 1995;1:29–34. doi: 10.1089/mdr.1995.1.29. [DOI] [PubMed] [Google Scholar]
- 5.Dowson C G, Coffey T J, Spratt B G. Origin and molecular epidemiology of penicillin-binding protein-mediated resistance to β-lactam antibiotics. Trends Microbiol. 1994;2:361–366. doi: 10.1016/0966-842x(94)90612-2. [DOI] [PubMed] [Google Scholar]
- 6.Figueiredo A M, Connor J D, Severin A, Vaz Pato M V, Tomasz A. A pneumococcal clinical isolate with high-level resistance to cefotaxime and ceftriaxone. Antimicrob Agents Chemother. 1992;36:886–889. doi: 10.1128/aac.36.4.886. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Goldstein F W, Liñares J, Melo Cristino J, Malafiiej E, Marcova M, Marton A, Murphy J, Schito G, Trupl Y, Urbascova P, Vanhoof R the European Study Group Hospital Saint Joseph, Paris. Program and abstracts of the First European Congress of Antimicrobial Chemotherapy, Glasgow, Scotland. 1996. Comparison of the intrinsic activity of 9 betalactams (BL) against 1131 strains of Streptococcus pneumoniae (S.p.) isolated in the 11 European countries between 1993 and 1995, abstr. T148. [Google Scholar]
- 8.Hakenbeck R, Tarpay M, Tomasz A. Multiple changes of penicillin-binding proteins in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother. 1980;17:364–371. doi: 10.1128/aac.17.3.364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Lonks J R, Durkin M R, Meyerhoff A N, Medeiros A A. Meningitis due to ceftriaxone-resistant Streptococcus pneumoniae. N Engl J Med. 1995;332:893–894. doi: 10.1056/NEJM199503303321317. [DOI] [PubMed] [Google Scholar]
- 10.Marton A, Gulyas M, Muñoz R, Tomasz A. Extremely high incidence of antibiotic resistance in clinical isolates of Streptococcus pneumoniae in Hungary. J Infect Dis. 1991;163:542–548. doi: 10.1093/infdis/163.3.542. [DOI] [PubMed] [Google Scholar]
- 11.Muñoz R, Dowson C G, Daniels M, Coffey T J, Martin C, Hakenbeck R, Spratt B G. Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae. Mol Microbiol. 1992;6:2461–2465. doi: 10.1111/j.1365-2958.1992.tb01422.x. [DOI] [PubMed] [Google Scholar]
- 12.National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 4th ed. Approved standard M7-A4. Wayne, Pa: National Committee for Clinical Laboratory Standards; 1997. [Google Scholar]
- 13.Reichler M R, Rakovsky J, Sobotová A, Sláciková M, Hlavácová B, Hill B, Krajcíkova L, Tarina P, Facklam R R, Breiman R F. Multiple antimicrobial resistance of pneumococci in children with otitis media, bacteremia, and meningitis in Slovakia. J Infect Dis. 1995;171:1491–1496. doi: 10.1093/infdis/171.6.1491. [DOI] [PubMed] [Google Scholar]
- 14.Sloas M M, Barrett F F, Chesney P J, English B K, Hill B C, Tenover F C, Leggiadro R J. Cephalosporin treatment failure in penicillin-resistant and cephalosporin-resistant Streptococcus pneumoniae meningitis. Pediatr Infect Dis J. 1992;11:662–666. [PubMed] [Google Scholar]
- 15.Thornsberry C, Burton P H, Vanderhoof B H. Program addendum of the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, D.C: American Society for Microbiology; 1995. Increasing resistance to third-generation cephalosporins in Streptococcus pneumoniae: a 1995 surveillance study in the United States, abstr. LB-24. [Google Scholar]