Diagnostic Microbiology and Infectious Disease 47 (2003) 511–513
www.elsevier.com/locate/diagmicrobio
Case report
Nosocomial superinfections due to linezolid-resistant Enterococcus faecalis: evidence for a gene dosage effect on linezolid MICs Kathleen A. Ruggeroa, Laura K. Schroedera, Paul C. Schreckenbergerb, Alexander S. Mankinc, John P. Quinnd,* a
University of Illinois at Chicago, Department of Internal Medicine, Section of Infectious Disease, 808 S. Wood St., Rm 888, M/C 735, Chicago, IL 60612, USA b University of Illinois at Chicago, Department of Pathology, 840 S. Wood St., Rm 238, CSB, M/C 750, Chicago, IL 60612, USA c University of Illinois at Chicago, Center for Pharmaceutical Biotechnology, 900 S. Ashland Ave, Rm 3056, M/C 870, Chicago, IL 60607, USA d Cook County Hospital/Rush University, 1900 W. Polk Street, Rm 1258, Chicago, IL 60612, USA Received 1 April 2003; accepted 16 June 2003
Abstract Resistance to linezolid among Enterococcus faecium and Enterococcus faecalis isolates has been reported in patients who receive a prolonged course of the drug. We report two cases of linezolid-resistant Enterococcus faecalis that occurred in patients who previously received linezolid for infections with vancomycin-resistant Enterococcus faecium. Both isolates had the G2576U mutation in the 23S rRNA previously reported in isolates of Enterococcus faecium. The number of gene copies mutated in the 23S rRNA correlated with the level of resistance. © 2003 Elsevier Inc. All rights reserved.
1. Introduction Vancomycin-resistant Enterococcus faecium and Enterococcus faecalis are important causes of nosocomial infections. Some of these infections may be treated with linezolid (Pharmacia Corp., Kalamazoo, MI), an oxazolidinone antibiotic approved by the FDA in the spring of 2000 for the treatment of Gram-positive infections, including those due to vancomycin-resistant enterococci (VRE). Linezolid resistance in vancomycin-resistant E. faecium has been described in multiple reports including one from our institution (Gonzales et al., 2001; Jones et al., 2002; Herrero et al., 1999; Zurenko et al., 1999). Among the first 88 patients treated with linezolid in our institution over the past two
Patient data previously reported in oral presentation K1591 at the 42nd annual Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, 2002, and in poster presentation P1197 at the 13th annual European Congress of Clinical Microbiology and Infectious Diseases, Glasgow, Scotland, 2003. * Corresponding author. Tel.: ⫹1-312-633-7955; fax: ⫹1-312-5723523. E-mail address:
[email protected] (J.P. Quinn). 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(03)00153-6
years, resistance has emerged in 11 (12.5%) of VRE isolates. The most common risk factor among patients who develop vancomycin-resistant linezolid-resistant E. faecium is duration of therapy with linezolid (Pai et al., 2002). We now describe two patients with linezolid-resistant E. faecalis infections who had previously received linezolid for linezolid-susceptible E. faecium infections. Both were isolated within a one month interval.
2. Materials and methods Patient 1 was a 74-year-old male who presented to our hospital with a cerebrovascular accident. His hospital course was complicated by acute renal failure, myocardial infarction, pulmonary edema, nosocomial pneumonia and aspiration. He was diagnosed with catheter-related bacteremia due to VRE. This isolate was susceptible to linezolid at 1.0 g/mL by E-test (AB BIODISK, Piscataway, NJ). He was treated with 10 days of i.v. linezolid 600 milligrams every 12 h. His blood cultures became sterile. Ten days after discontinuing linezolid, the patient became clinically septic, and was placed on empiric antibiotics, including linezolid. He received linezolid for this second time for sixteen days,
512
K.A. Ruggero et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 511–513
even though enterococci were not isolated from blood, sputum, or urine cultures during this time. Subsequently, he developed worsening respiratory status, and cultures performed 32 days after his last positive blood culture for VRE revealed a blood culture positive for E.faecalis via Vitek GPI (bioMerieux, Inc., St. Louis, MO) and API 20 Strep (bioMerieux, Inc., St. Louis, MO) identification. This was eight days after linezolid was discontinued. This isolate was susceptible to ampicillin (0.5) and vancomycin (⬍⫽2) via Microscan (Dade Behring, West Sacramento, CA), but resistant to linezolid at ⬎256 g/mL via E-test. Patient 2 was a 53-year-old male who presented with pancreatitis. He subsequently developed multisystem organ failure, requiring intubation. He received vancomycin, imipenem, metronidazole and fluconazole empirically for sepsis syndrome. Surgical debridement was done for necrotizing pancreatitis. Two weeks later, he developed positive blood and urine cultures for vancomycin-resistant, linezolid-susceptible E. faecium. He was given i.v. linezolid for 26 days, at a dose of 600 milligrams every 12 h. His blood cultures and urine cultures became negative. He was off all antibiotics until one month later when a urine culture grew E. faecalis sensitive to ampicillin (2), resistant to vancomycin (⬎16), and resistant to linezolid via E-test at 24 g/mL. Two and a half months later, he had a pancreatic pseudocyst drained that also grew vancomycin-resistant linezolid-resistant Enterococcus faecalis. Using the polymerase chain reaction (PCR) based protocol previously described (Woodford et al., 2002), we performed restriction fragment length polymorphism (RFLP) studies to see if these isolates shared the characteristic G2576U mutation in domain V of the 23S rRNA previously described in linezolid-resistant E. faecium and E. faecalis (Prystowski et al., 2001; Marshall et al., 2002). Enterococcal RNA and DNA were extracted by shaking cells with hot phenol followed by ethanol precipitation. The DNA primers used for amplification were: SfLR2100 (5⬘-CGGTGAAATTTTAGTACCTGTGAAGATG-3⬘) and SfL2660-2 (5⬘-GTCCATCCCGGTCCTCTCG-3⬘) designed to amplify a 662 bp segment from domain V of enterococcal 23S rRNA, encompassing the G2576 position. PCR reaction was performed using rTaq DNA polymerase (Fisher Scientific, Hanover Park, IL). Conditions for amplification were: 94°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min. PCR product was purified using a PCR product purification kit (Qiagen, Valencia, CA) and recovered in 40 L H2O. 5 L DNA was digested at 37°C for 2 h using 5 or 10 units of Nhe1 restriction enzyme (Fermentas, Hanover, MD) under conditions recommended by the enzyme manufacturer. The purified PCR product (5 L) and products of the restriction digest were analyzed on a 1% (w/v) agarose gel. Both isolates were tested for relatedness by pulsed field gel electrophoresis (PFGE) by previously described technique (Matushek et al., 1996).
Fig. 1. Restriction analysis of the PCR fragment amplified from rDNA of linezolid resistant E. faecalis. An rDNA segment corresponding to a portion of domain V of 23S rRNA of E. faecalis was PCR amplified, digested with the excess of a restriction enzyme Nhe1 and the DNA products were resolved by agarose electrophoresis. M, 100 bp DNA size markers (the 700 bp band is indicated); K, control PCR fragment untreated with Nhe1; N, Nhe1-digested PCR fragment.
3. Results The G2576U mutation in 23S rRNA gene generates a new restriction site for the enzyme Nhe1. The PCR fragment amplified using DNA from the E. faecalis isolate from Patient 1 (linezolid MIC ⬎256 g/mL) was completely cut by Nhe1 to produce the 570 bp band and 100 bp band. This indicates that this isolate contains the G2576U mutation in all four of its 23S rRNA gene copies. The PCR product produced using DNA from the E. faecalis isolate from Patient 2 (linezolid MIC ⫽ 24 g/mL) was cut incompletely by Nhe1. Two fold increase in concentration of the restriction enzyme did not increase the extent of cleavage indicating that incomplete restriction digest resulted from heterogeneity of rDNA alleles. The ratio of cut and uncut DNA, as well as allele-specific PCR amplification and RFLP analysis (not shown), indicates that two out of four E. faecalis rRNA operons in this isolate carried the G2576U mutation. (See Fig. 1.) PFGE of chromosomal DNA revealed that these strains were unrelated.
4. Discussion Both of these patients had an infection with vancomycinresistant E. faecium, and were treated with linezolid for a prolonged course. Subsequently, they had cultures positive for linezolid-resistant E. faecalis. In in vitro experiments, E. faecalis developed resistance to linezolid more quickly than E. faecium when exposed to increasing concentrations of linezolid (Prystowsky et al., 2001). In addition, MICs to linezolid were higher for E. faecalis than for E. faecium (Prystowsky et al., 2001). Previous investigation showed that the level of resistance
K.A. Ruggero et al. / Diagnostic Microbiology and Infectious Disease 47 (2003) 511–513
of E. faecium to linezolid correlates with the number of 23S rRNA gene copies that carry the G2576U mutation (Marshall et al., 2002). This observation was extended here to isolates of E. faecalis. E. faecalis contains four copies of rRNA operons in its chromosome (Marshall et al., 2002). The mutation G2576U generates a new site for the restriction enzyme Nhe1 in the mutant copies of rRNA operon, while this site is absent in the wild type sequence. All four copies of rRNA genes in the isolate from patient one (referred to as Ent 1 in the figure) carried the G2576U mutation: the PCR fragment encompassing the position 2576 was completely digested with Nhe1 (see Fig. 1). In contrast, only a portion of the PCR fragment amplified from DNA of the isolate from patient two (referred to as Ent 2 in the figure) could be digested with Nhe1 indicating that some of the rRNA operon did not carry the G2576U mutation. RFLP analysis of individual rRNA alleles in this strain showed that two out of four operons carried the G2576U mutation (data not shown). Our results do not exclude the presence of other mutations in any of the two isolates. However, a clear correlation between the level of resistance and the number of mutated operons indicate that the G2576U mutation is the primary cause of the resistance.
Acknowledgments The authors wish to thank the Chicago Infectious Disease Research Institute, for their assistance in this project. This project was supported by a research grant from Pharmacia, Inc.
513
References Gonzales, R., Schreckenberger, P., Graham, M. B., Kelkar, S., DenBesten, K., & Quinn, J. P. (2001). Infections due to vancomycin-resistant Enterococcus faecium resistant to linezolid. Lancet 357, 1179. Herrero, I., Issa, N., & Patel, R. (2002). Nosocomial spread of linezolidresistant, vancomycin-resistant Enterococcus faecium. N Engl J Med 346, 867– 869. Jones, R., Della-Latta, P., Lee, L., & Biedenbach, D. (2002). Linezolidresistant Enterococcus faecium isolated from a patient without prior exposure to an oxazolidinone: Report from the SENTRY Antimicrobial Surveillance Program. Diag Microbiol Infect Dis 42, 137–139. Marshall, S., Donskey, C., Hutton-Thomas, R., Salata, A., & Rice, L. (2002). Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis. Antimicrob Agents Chemother 46, 3334 – 3336. Matushek, M., Bonten, M., & Hayden, M. (1996). Rapid preparation of bacterial DNA for pulsed-field gel electrophoresis. J Clin Microbiol 34, 2598 –2600. Pai, M., Rodvold, K., Schreckenberger, P., Gonzales, R., Petrolatti, J., & Quinn, J. (2002). Risk factors associated with the development of linezolid- and vancomycin-resistant Enterococcus faecium. Clin Infect Dis 35, 1269 –1272. Prystowsky, J., Siddiqui, F., Chosay, J., Shinabarger, D., Millicap, J., Peterson, L., & Noskin, G. (2001). Resistance to linezolid: characterization of mutations in rRNA and comparison of their occurrences in vancomycin-resistant enterococci. Antimicrob Agents Chemother 45, 2154 –2156. Woodford, N., Tysall, L., Auckland, C., Stockdale, M., Lawson, A., Walker, R., & Livermore, D. (2002). Detection of oxazolidinoneresistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis. J Clin Microbiol 40, 4298 – 4300. Zurenko, G., Todd, W., Hafkin, B., Myers, B., Kaffman, C., Bock, J. (1999). Development of linezolid-resistant Enterococcus faecium in two compassionate use program patients treated with linezolid. Proceedings of the 39th Annual Interscience Conference of Antimicrobial Agents and Chemotherapy. San Francisco, CA, 1999.
