Tolerance to vancomycin in a multiresistant, Colombian isolate of Streptococcus pneumoniae

Marilyn Hidalgo1,*, Elizabeth Castañeda1 and César A. Arias2

1 Microbiology Group, Instituto Nacional de Salud, Avenida Calle 26 No 51–60, Bogotá; 2 Bacterial Molecular Genetics Unit, Centro de Investigaciones, Universidad El Bosque, Bogotá, D.C., Colombia

Received 20 January 2003; returned 5 May 2003; revised 5 May 2003; accepted 15 May 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The present study was set up to determine vancomycin tolerance in Colombian, multiresistant, invasive, paediatric isolates of Streptococcus pneumoniae. A total of seven clinical isolates of penicillin-resistant S. pneumoniae, serotype 14, corresponding to the Spain9v-3 clone, were studied. Isolates were grown in Todd Hewitt broth at 37°C. Growth (A595) and cell viability (cfu/mL) were monitored hourly for 4 h after addition of vancomycin at 10x MIC. S. pneumoniae R6 was used as a control for growth. For R6, the mean decrease in A595 and cell viability (log10 cfu/mL) after 4 h of vancomycin treatment (in 10 independent experiments) was 95.1% and 4.2 log10, respectively. Six out of seven isolates exhibited similar behaviour. In contrast, for S. pneumoniae E239, the mean decrease in A595 and cell viability was 48.5% and 2.2 log10 after 4 h of vancomycin exposure, which suggested the presence of tolerance to vancomycin. The emergence of vancomycin-tolerant strains is of great concern in Colombia, owing to the alarming increase in resistance to penicillin.

Keywords: antimicrobial tolerance, vancomycin, Colombian isolates, killing curves, Streptococcus pneumoniae


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Streptococcus pneumoniae is a common cause of bacterial meningitis and respiratory tract infections in young children and the elderly.1 Although penicillin has been used successfully for infections caused by this microorganism, in recent years the effectiveness of antibiotic therapy has been compromised by the increasing prevalence of penicillin-resistant pneumococci.1,2 In Colombia, the frequency of invasive isolates with decreased susceptibility to penicillin obtained from children has risen steadily from 10% in 1994 to 36.5% in 2000.3 Currently, vancomycin is one of the last options for the treatment of infections caused by S. pneumoniae. Recently, Henriques et al.4 reported the presence of clinical isolates of S. pneumoniae tolerant to vancomycin: the presence of vancomycin and penicillin tolerance among 116 clinical isolates of S. pneumoniae was 3% and 8%, respectively. Tolerance to vancomycin was found in isolates of serotype 9V, which also had reduced susceptibility to penicillin. Antibiotic tolerance has been implicated in therapeutic failure in experimental models and has been suggested to represent a precursor of resistance.4 The aim of the present study was to investigate vancomycin tolerance in multiresistant, Colombian, invasive isolates of S. pneumoniae.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
For this research, a total of seven clinical isolates of penicillin-resistant S. pneumoniae, serotype 14, corresponding to PFGE patterns in the Spain9v-3 clone, were studied (Table 1).5 The work was based on the report by Henriques et al.,4 in which tolerance was observed in serotype 9V clinical isolates. The isolates were collected between 1995 and 1999 from different patients and had no epidemiological relationship. MICs of vancomycin and other antibiotics were determined for each strain by the broth microdilution method, following NCCLS criteria.6 R6 (a vancomycin-susceptible strain) was used as a growth control and was obtained from the Rockefeller University collection.


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Table 1. Characteristics of seven paediatric clinical isolates of S. pneumoniae serotype 14, genotype Spain9v-3 from Colombia
 
Tolerance to vancomycin was evaluated according to the methodology established by Henriques et al.4 The isolates were grown from frozen stock cultures on blood agar plates for 18–24 h in 5% CO2. Bacteria were resuspended in 5 mL of Todd Hewitt broth modified (THM) with 1% glucose, 1% yeast extract and glutamine (22 µg/mL) and incubated at 37°C for 6 h. Aliquots of 1 mL were added from this culture to 15 mL of THM broth and incubated at 37°C, with gentle agitation (50 rpm) for 8 h. When cultures reached an A595 of 0.2, vancomycin was added at 10x MIC (final concentration between 2.5 and 5.0 mg/L). The A595 and cell viability (determined as log10 cfu/mL) were monitored hourly for 4 h after the addition of the antibiotic. Samples were diluted using saline solution (0.85%), plated on blood agar and incubated at 37°C in 5% CO2 for 24 h. Ten independent experiments were performed for each strain and the control (R6). The thresholds used during this study to define vancomycin tolerance were a mean log10 kill of <2.5 and a mean A595 decrease of <50% after 4 h of incubation.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The percentage decrease in A595 and cell viability after 4 h incubation in the presence of 10x MIC of vancomycin for each isolate is shown in Figure 1. For S. pneumoniae R6, the mean decreases in cell viability and A595 were 4.2 log10 and 95.1 ± 4%, respectively. Six out of seven clinical isolates exhibited behaviour similar to R6 (Table 1). In contrast, for S. pneumoniae E239, the mean decreases in A595 and cell viability were 48.5% and 2.2 log10, respectively (Table 1). This isolate was recovered from a 3-year-old child with pneumonia, and susceptibility tests indicated resistance to penicillin, trimethoprim–sulfamethoxazole and chloramphenicol. The isolate had intermediate susceptibility to ceftriaxone and was susceptible to vancomycin and erythromycin (Table 1).



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Figure 1 Mean decrease in A595 and log10 kill at 4 h of incubation after addition of vancomycin (10x MIC), in 10 independent experiments on S. pneumoniae.

 
The phenomenon of tolerance to vancomycin has been described recently in S. pneumoniae, but the molecular basis for this phenomenon is still unclear.7,8 However, tolerance to penicillin has been observed for more than 10 years, and this phenotype presumably results from alterations in the sequence of events following antibiotic exposure that leads to decreased bacterial killing.9 A defective death response has been observed in which a defective autolysin, or an altered signal transduction cascade involved in the activation of the autolysin, are likely to be responsible.4

The presence of vancomycin-tolerant clinical isolates of S. pneumoniae has been reported previously in three isolates of serotype 9V (out of 116 studied):4,9,10 two from the nasopharynx and one from an invasive bloodstream infection. However, in Spain (where most isolates of S. pneumoniae with diminished susceptibility to penicillin have been characterized), the phenomenon of vancomycin tolerance was not detected in 120 isolates studied.2 In Colombia, the rampant increase of resistance to penicillin is of great concern.3 We have identified one isolate (out of seven studied) which expresses phenotypical characteristics of tolerance to vancomycin. S. pneumoniae E239 was isolated from a paediatric patient and exhibited high-level resistance to penicillin and other antibiotics. This alarming finding (albeit in a small number of isolates) indicates that multiresistant strains of S. pneumoniae tolerant to vancomycin could be encountered frequently in clinical settings in our country, which further undermines alternative therapeutic treatments. In 1999, Novak et al.10 reported that loss of function of the VncS histidine kinase of a two-component regulator system resulted in tolerance to vancomycin and several other classes of antibiotics. However, this finding has not been confirmed in other laboratories.7 Recently, Robertson et al.8 demonstrated that the loss of VncS function alone does not result in tolerance to vancomycin.


    Acknowledgements
 
We are grateful to P. Reynolds for reading the manuscript and for helpful discussions. This work was funded in part by an International Development Award from the Wellcome Trust.


    Footnotes
 
* Corresponding author. Tel: +571-2227700 ext 446; Fax: +571-2221093; E-mail: mhidalgo{at}ins.gov.co Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Schrag, S., Beall, B. & Dowell, S. (2000). Limiting the spread of resistant pneumococci: biological and epidemiological evidence for the effectiveness of alternative interventions. Clinical Microbiology Reviews 13, 588–601.[Abstract/Free Full Text]

2 . Antón, N., Blásquez, R., Gómez-Garcés, J. L. et al. (2001). Study of vancomycin tolerance in 120 strains of Streptococcus pneumoniae isolated in 1999 in Madrid, Spain. Journal of Antimicrobial Chemotherapy 47, 902–3.[Free Full Text]

3 . Agudelo, C. I., Sanabria, O. M., Ovalle, M. V. et al. (2001). Vigilancia por el laboratorio de Streptococcus pneumoniae, aislado de procesos invasores en niños menores de 5 años. Actualización de los datos 1994–2000. Informe Quincenal Epidemiológico Nacional 7, 97–112.

4 . Henriques, B., Novak, R., Örtqvist, A. et al. (2001). Clinical isolates of Streptococcus pneumoniae that exhibit tolerance of vancomycin. Clinical Infectious Diseases 32, 552–8.[CrossRef][ISI][Medline]

5 . Vela, M. C., Fonseca, N., Di Fabio, J. L. et al. (2001). Presence of international multiresistant clones of Streptococcus pneumoniae in Colombia. Microbial Drug Resistance 7, 153–64.[CrossRef][ISI][Medline]

6 . National Committee for Clinical Laboratory Standards. (2002). Performance Standards for Antimicrobial Susceptibility Testing; Twelfth Informational Supplement M100-S12. NCCLS, Wayne, PA, USA.

7 . Grinius, L. L., Coleman,W., Desal, B. et al. (2001). Lack of an antibiotic tolerance phenotype in Vncs deletion mutants of Streptococcus pneumoniae. In Programs and Abstracts of the Forty-first Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2001. Abstract C1-1509, p. 96. American Society for Microbiology, Washington, DC, USA.

8 . Robertson, G. T., Zhao, J., Desai, B. V. et al. (2002). Vancomycin tolerance induced by erythromycin but not by loss of vncRS, vex3, or pep27 function in Streptococcus pneumoniae. Journal of Bacteriology 184, 6987–7000.[Abstract/Free Full Text]

9 . Handwerger, S. & Tomasz, A. (1985). Antibiotic tolerance among clinical isolates of bacteria. Reviews of Infectious Diseases 7, 368–82.[ISI][Medline]

10 . Novak, R., Henriques, B., Carpentier, E. et al. (1999). Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399, 590–3.[CrossRef][ISI][Medline]