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Antimicrobial Agents and Chemotherapy, January 2007, p. 386-389, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.01016-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Quinupristin-Dalfopristin Nonsusceptibility in Pneumococci from Sickle Cell Disease Patients{triangledown}

Caroline A. Obert,1,{dagger} Martha L. Miller,1,{dagger},{ddagger} Jeremy Montgomery,1,§ Thomas Adamkiewicz,2 and Elaine I. Tuomanen1*

Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105,1 Department of Family Medicine, Morehouse School of Medicine, 1513 E. Cleveland, No. 100, East Point, Georgia 303442

Received 14 August 2006/ Returned for modification 11 September 2006/ Accepted 16 October 2006


   ABSTRACT
Top
 ABSTRACT
TEXT
 REFERENCES
 
Sickle cell disease (SCD) is a risk factor for fatal pneumococcal infection. Nonsusceptibilty to quinupristin-dalfopristin (Q-D) was absent from 105 non-SCD-associated pneumococcal isolates but was present in 33/148 (22%) SCD-associated isolates. One-third of the isolates harbored a known resistance mechanism. Q-D is not optimal for use for the treatment of pneumococcal infection in SCD patients.


   TEXT
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 ABSTRACT
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REFERENCES
 
Sickle cell disease (SCD) is characterized by a 600-fold increased risk of invasive pneumococcal infection (26). Consequently, these patients receive penicillin prophylaxis and empirical antibacterial therapy, measures which in turn promote drug resistance (24). Thus, alternative antimicrobials are an ongoing need in the SCD population. Quinupristin-dalfopristin (Q-D; Synercid) is a semisynthetic, intravenous streptogramin approved for use in 1999, and the components of this compound display synergistic, bactericidal activities (8, 13). Resistance is rare but has emerged in staphylococci and vancomycin-resistant Enterococcus faecium (21). Resistance in pneumococci is uncommon (≤1.1%) (15, 20), but mutations in ribosomal proteins and 23S rRNA have been reported (13, 15, 20). Equally rare, Q-D nonsusceptibility was previously reported in 97 of 4,626 (2.1%) pneumococcal isolates obtained from the general population by Jones et al. by using Etest for determination of susceptibility (19), although the mechanism of resistance in these strains was not discussed. Here, we report on the Q-D nonsusceptibilities of nasopharyngeal and invasive pneumococci in over 30% of SCD patients.

Testing for susceptibility to Q-D, clindamycin, and erythromycin was performed in triplicate by Etest, according to the manufacturer's instructions, with 68 SCD-associated nasopharyngeal isolates (12) and 80 SCD-associated invasive isolates (1) collected between 1994 and 2002 and, for comparison, with 105 isolates from healthy peers of the infected patients (25, 27). Twenty-five (37%) nasopharyngeal SCD-associated isolates and eight (10%) invasive SCD-associated isolates were found to be Q-D nonsusceptible (Etest MIC range, 1.5 to 2 µg/ml); no high-level resistance was detected. Broth dilution MICs (10) confirmed the nonsusceptibilities of all 33 isolates (MIC range, 1.5 to 2 µg/ml) found by Etest with 100% reproducibility. All non-SCD-associated strains were Q-D sensitive. Nonsusceptibility to Q-D alone occurred in 23 of the 33 (70%) nonsusceptible strains, while 10 Q-D-nonsusceptible strains (15%) were also resistant to erythromycin and an additional 10 Q-D-nonsusceptible strains (15%) were resistant to clindamycin and erythromycin (Table 1), indicating that nonsusceptibility was not commonly due to the macrolide-lincosamide-streptogramin B (MLSB) or the macrolide-streptogramin B (MSB) phenotype.


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  		TABLE 1. Susceptibilities to clindamycin and erythromycin in Q-D-nonsusceptible SCD isolates

 
eBURST analysis (eburst.mlst.net) of the multilocus sequence types (STs) (24) of the 33 Q-D-nonsusceptible isolates indicated the presence of two clonal groups (Fig. 1). Clonal group 1 included five STs (STs 439, 37, 1499, 42, and 2151), where ST439 is the founder group from which the other STs derived. Clonal group 2 included only two STs (ST199 and ST876), and no founder group was present. The remaining 12 STs were singletons: their sequences were not related to those of either clonal group or to the sequences of the other singletons. This analysis indicates that the prevalence of Q-D nonsusceptibility is not due to the spread of a single clone or clonal group.


Figure 1
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  		FIG. 1. eBURST analysis of Q-D-nonsusceptible isolates based on sequence type.

 
To identify known resistance mechanisms, genomic DNA from all Q-D-nonsusceptible isolates was prepared as described previously (24). By using the primers and parameters described previously (13), the 33 isolates were screened for mutations in the L4 and L22 ribosomal protein genes or four alleles of 23S rRNA. Seventy-four (32%) of the 231 possible genes that could have point mutations were successfully amplified and sequenced. Sequence analysis was performed by using Seqassem software, version 02/2006 (http://www.gwdg.de/~dhepper/), and BioEdit software (16). This low rate may reflect nucleotide substitutions within the area of annealing or variation in allele number (14, 18, 29). Four isolates (isolates DAW6, DAW15, DAW70, and CDC068) had a single nucleotide polymorphism (SNP) in the L4 ribosomal protein, resulting in an amino acid substitution, Ser20-Asn. Two of the four isolates were ST199 and one was ST876; these two STs were part of the same clonal group (Fig. 1). The remaining isolate was ST81 and was not related to the aforementioned clonal group. This SNP has previously been associated with resistance to 16-membered macrolides and streptogramins A and B (13), but in this study, the phenotype was solely associated with Q-D nonsusceptibility (Table 1). The other amplicons did not reveal additional polymorphic sites.

In addition to examination of known Q-D resistance point mutations, all 33 Q-D-nonsusceptible strains were screened for the presence of known resistance genes of erm (7); vatA, vatB, vatC, vatD, and vatE (3, 4, 30); and vgaA, vgaB, and vgaAV (2, 17). An acetyltransferase, vatB, that confers resistance by inactivating streptogramin A (3, 6) was present in two strains (strains DAW22 and DAW57). Both strains exhibited high-level erythromycin and clindamycin resistance. Resistance can also be conferred by the vga genes, which encode ATP-binding proteins involved in active transport (5, 6). vgaA was amplified from four isolates (isolates DAW2, DAW12, DAW75, and CDC018). The first three isolates all had streptogramin nonsusceptibility phenotypes, whereas CDC018 had an MLSB phenotype, with high-level resistance to erythromycin and clindamycin. Finally, a vgaAV fragment was isolated from strain CDC063, which exhibited erythromycin resistance. For the remaining 22 isolates, the remaining genes yielded either nonspecific products or no products. This suggests either that the sequences of the primers designed to detect known resistance genes from enterococcal and staphylococcal isolates are too divergent from the sequences of pneumococcal isolates or that additional uncharacterized genes or mutations are responsible for the intermediate resistance phenotype observed.

Although approval for the therapeutic use of Q-D was not given until 1999, 25 of the 33 nonsusceptible Q-D isolates in this study were collected prior to that time. Resistance to Q-D in isolates collected prior to 1999 has been attributed to the use of virginiamycin (a streptogramin with a structure similar to that of Q-D) in animal husbandry (22, 23, 28). However, this theory was recently challenged (11). Selective pressure by virginiamycin, however, would not explain different Q-D susceptibilities of isolates from SCD versus non-SCD patients.

Despite its absence in the general population, it is evident from these data that nonsusceptibility is prevalent among isolates from the population with SCD. The mechanistic connection between Q-D nonsusceptibility and SCD remains unclear. Patients with SCD are not, in general, treated with Q-D (9). The majority of nonsusceptible isolates in this study were obtained prior to 2003, and therefore, it is unlikely that the patients were exposed to Q-D. Thus, the selective pressure for antibiotic resistance among isolates from this population remains elusive. The increased rate of Q-D nonsusceptibility observed among isolates from SCD patients is concerning, especially given that it was believed that the synergistic properties of this particular antibiotic would allow it to remain efficacious. It is quickly becoming evident that this belief no longer holds true, and as such, this renders Q-D significantly less attractive for the treatment of invasive pneumococcal disease.


   ACKNOWLEDGMENTS
 
We thank William Hanage for help with eBURST software and Jason Rosch for suggestions in editing the manuscript.

This work was supported by the NIH R01 award AI39482, NIH grant U54HL070590, Summer Student Program 111064040, and the American Lebanese Syrian Associated Charities.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 North Lauderdale St., Memphis, TN 38105-2794. Phone: (901) 495-3486. Fax: (901) 495-3099. E-mail: elaine.tuomanen{at}stjude.org. Back

{triangledown} Published ahead of print on 23 October 2006. Back

{dagger} These two authors contributed equally to this project. Back

{ddagger} Present address: Department of Pediatrics, Division of Infectious Diseases, The University of New Mexico, 1 University of New Mexico, Albuquerque, NM 87131. Back

§ Present address: Middle Tennessee State University, 1301 East Main Street, Murfreesboro, TN 37132. Back


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Antimicrobial Agents and Chemotherapy, January 2007, p. 386-389, Vol. 51, No. 1
0066-4804/07/$08.00+0     doi:10.1128/AAC.01016-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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