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Antimicrobial susceptibility and macrolide resistance genes in Enterococcus faecium with reduced susceptibility to quinupristin–dalfopristin: level of quinupristin–dalfopristin resistance is not dependent on erm(B) attenuator region sequence
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Diagnostic Microbiology and Infectious Disease 66 (2010) 73 – 77 www.elsevier.com/locate/diagmicrobio
Antimicrobial Susceptibility Studies
Antimicrobial susceptibility and macrolide resistance genes in Enterococcus faecium with reduced susceptibility to quinupristin–dalfopristin: level of quinupristin–dalfopristin resistance is not dependent on erm(B) attenuator region sequence Fátima López, Esther Culebras⁎, Carmen Betriú, Iciar Rodríguez-Avial, María Gómez, Juan J. Picazo Department of Microbiology, Hospital Clínico San Carlos, Plaza de Cristo Rey s/n. 28040, Madrid, Spain Received 12 February 2008; accepted 1 June 2008
Abstract Antimicrobial resistance and the mechanisms implicated were studied in 148 clinical Enterococcus faecium isolates with a quinupristin– dalfopristin (Q/D) MIC ≥1 μg/mL. As expected, higher levels of resistance were detected for macrolide antibiotics (94% erythromycin, 100% azithromycin, 100% josamycin). High-level resistance to gentamicin and streptomycin was detected in 18.9% and 66.2% of isolates, respectively, in our series of enterococci. Resistance against tetracycline was found in only 12.8% of the isolates, and 13 isolates were resistant to vancomycin. The dalvabancin MIC90 for these isolates was N16 μg/mL. Polymerase chain reaction screening for the previously described streptogramin resistance determinants erm(A), erm(B), mefA/E, vat(D), and vat(E) was performed to determine the prevalence of streptogramin resistance mechanisms in these clinical enterococcal isolates. The combination of erm(B) and vat(D) genes encoding resistance to streptogramins was detected in 1 Q/D-resistant E. faecium isolate. A total of 131 Q/D-nonsusceptible enterococci only contained the erm(B) gene. The erm(B) promoter sequence reveals no differences between the strains analyzed, regardless of the Q/D MIC value. © 2010 Elsevier Inc. All rights reserved. Keywords: Enterococcus faecium; erm(B) attenuator; Quinupristin–dalfopristin nonsusceptibility
1. Introduction Enterococci are normal constituents of the intestinal flora and are widely distributed in the environment. Enterococcal infections are the 2nd to 3rd most common nosocomial infection. Multidrug resistance among enterococci has increased, and Enterococcus faecium, in particular, possesses a broad spectrum of natural and acquired antibiotic resistance. As a result, therapeutic options are becoming increasingly limited for the treatment of enterococcal infections (Harbarth et al., 2002; Malathum and Murray, 1999). Quinupristin– dalfopristin (Q/D) is a streptogramin that has been approved
⁎ Corresponding author. Tel.: +34-91-330-3269; fax: +34-91-330-3478. E-mail address: [email protected] (E. Culebras). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.06.004
for human treatment. Streptogramins have been considered valuable antimicrobial drugs for the treatment of infections with methicillin-resistant Staphylococcus aureus and multiresistant E. faecium (Soltani et al., 2000). Resistance to Q/D can occur by 1 or more of several mechanisms, including enzymatic modification, active transport of efflux mediated by an adenosine triphosphatebinding protein, and alteration of the target site (Hershberger et al., 2004). Inhibitors of mammalian multidrug efflux, such as the plant alkaloid reserpine, are also active in boosting antibiotic activity by inhibiting bacterial efflux. Q/D is a mixture of streptogramin B and streptogramin A compounds, which act synergistically at different sites of the bacterial ribosome to disrupt peptide elongation. Resistance to the streptogramin A component is associated with acetylases encoded by vat(D) and vat(E), 1st described in
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F. López et al. / Diagnostic Microbiology and Infectious Disease 66 (2010) 73–77
enterococcal isolates in Europe (Rende-Fournier et al., 1993; Werner and Witte, 1999). Resistance to streptogramin B is mainly due to erm gene products, which methylate an adenine residue at 23S rRNA. Methylation occurs in a region of overlapping binding sites for macrolides and lincosamides and results in the macrolide–lincosamide–streptogramin B (MLS) resistance phenotype. Streptogramin B resistance may also be due to hydrolysis of the ring molecule via the lactonase encoded by vgb genes, which were initially reported in Staphylococcus and are rarely present in Enterococcus (Hershberger et al., 2004). Streptogramin B (and macrolide) resistance has also been linked to the MsrC efflux pump, present in most E. faecium isolates (Portillo et al., 2000; Werner et al., 2001). Many streptogramin-resistant E. faecium isolates harbor none of the described resistance mechanisms (Hayes et al., 2003). We describe the diversity and distribution of MLS resistance as well as the known genetic determinants that contribute to streptogramin resistance among E. faecium clinical isolates. We also characterize the attenuator region of the erm(B) methylase gene to establish a possible relationship between mutations in the erm(B) leader region and Q/D MICs. 2. Materials and methods 2.1. Bacterial strains A total of 148 clinical isolates were selected with a Q/D MIC ≥1 μg/mL: 43 strains were intermediate and 90 were resistant to Q/D. Strains were collected from May 2002 to January 2004. Multiple isolates from the same patient were avoided. The sources of the 148 E. faecium isolates included skin and soft tissues (72 isolates, 48.6%), abdomen (47 isolates, 31.75%), urine (20 isolates, 13.5%), and other sites (9 isolates, 6%). 2.2. Antimicrobials and MIC testing Susceptibilities to rifampicin, chloramphenicol, gentamicin, streptomycin, and dalbavancin were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) (2007). The inoculum was adjusted to 105 and 107 colony-forming units (CFU)/ microwell for dalbavancin to investigate inoculum effects. The MICs were determined by the agar dilution method in Mueller–Hinton agar (Difco, Detroit, MI) according to the procedure recommended by the CLSI (2007). The antibiotics tested were erythromycin, tetracycline, Q/D, vancomycin, teicoplanin, linezolid, tigecycline, minocycline, josamycin, and azithromycin. The plant alkaloid reserpine was used to determine the role of the efflux pump in the Q/D and tetracycline MIC values observed. If reserpine reduces the MIC of antibiotics at least 2-fold, it has been concluded to be an active efflux system. Enterococcus faecalis strain ATCC 29212 and S. aureus strain ATCC 29213 were used as control strains.
2.3. Bactericidal effect of Q/D The bactericidal effect was evaluated in 6 erm(B)-positive isolates and 6 erm(B)-negative isolates. The methods used was as follows: Mueller–Hinton broth (MHB) inoculated with 5 × 105 CFU/mL was supplemented with Q/D. The following concentrations were used: 2×, 3×, and 4× the MIC. After 18 h of incubation at 37 °C, serial dilutions of 0.1 mL samples were subcultured onto agar plates, and the plates were incubated for 24 h before the numbers of CFU were counted. 2.4. Polymerase chain reaction analysis The presence of genes involved in MLS resistance was determined by polymerase chain reaction (PCR) amplification. The presence of erm(B), erm(A), mefA/E, vat(D), and vat(E) was assayed by PCR using oligonucleotide primers and reaction conditions described elsewhere (Culebras et al., 2005; Hayes et al., 2005; Soltani et al., 2000). 2.5. Nucleotide sequence of the erm(B) attenuator The erm(B) upstream region was amplified as previously described (Rosato et al., 1999). PCR products were then purified using Qiagen PCR product purification kit and sequence in an automated ABI PRISM BIO system (PerkinElmer, Waltham, MA). erm(B) upstream sequences of the Tn1545 of Streptococcus pneumoniae (gi:47454) and of the Tn917 of E. faecalis (gi:21492651) were used as the reference wild-type sequence. 3. Results and discussion 3.1. Antimicrobial susceptibility testing The distribution of MIC values for vancomycin, teicoplanin, dalbavancin, tetracycline, minocycline, erythromycin, azithromycin, josamycin, chloramphenicol, linezolid, tigecycline, and Q/D are shown in Table 1. No isolates were resistant to teicoplanin or linezolid. Tigecycline shows very good activity against all the isolates with MIC values ≤0.06 μg/mL. Among the 148 E. faecium clinical isolates, overall resistance was as follows: tetracycline 12.8%, chloramphenicol 31.75%, vancomycin 8.8%, and erythromycin 94%. High-level resistance to streptomycin and gentamicin was detected in 66.2% and 18.9% of the isolates, respectively. There was no link between reduced susceptibility to Q/D and resistance to other classes of antimicrobials. Resistance to tetracycline, minocycline, and chloramphenicol appears in both Q/D-susceptible and Q/D-resistant isolates. Susceptibility to linezolid and teicoplanin and tigecycline MIC also shows similar distribution in both groups of isolates. Our data showed that the Q/D resistance rate is high in vancomycin-susceptible E. faecium (VSEF) isolates (58% of VSEF were resistant to Q/D). These data are consistent with previous data (Oh et al., 2005). However, in our study, all vancomycin-resistant E. faecium isolates (13 isolates) were resistant to Q/D.
F. López et al. / Diagnostic Microbiology and Infectious Disease 66 (2010) 73–77 Table 1 Antibiotic resistance in E. faecium clinical isolates (n = 148) Antimicrobial agent
MIC50 (μg/mL)
MIC90 (μg/mL)
MIC range (μg/mL)
Resistance (%)a
Vancomycin Teicoplanin Dalbavancin (105) Dalbavancin (107) Tetracycline Minocycline Erythromycin Azithromycin Josamycin Chloramphenicol Linezolid Tigecycline Q/D Gentamicinb Streptomycinb
0.5 1 0.03 0.25 b1 b1 256 256 256 8 1 b0.06 4 – –
0.5 1 0.06 0.25 N32 16 256 256 256 N8 1 b0.06 8 – –
0.5 to N32 b0.06 to 16 b0.016 to N16 0.06 to N16 b1 to N32 b1 to N32 0.5 to 256 8 to 256 32 to 256 2 to N8 1 b0.06 1 to 32 – –
8.8 0 Not available Not available 12.8 10.2 94 100 100 31.75 0 Not available 61.5 18.9 66.2
a b
Susceptibility interpretations according to CLSI criteria (CLSI, 2007). High-level resistance to aminoglycosides.
To establish the contribution of the efflux pump to the MIC of tetracycline and Q/D, we determined the MIC values of both antibiotics in the presence and absence of reserpine. The results showed that reserpine affects the MIC of tetracycline and Q/D in 3 (16% tetracycline resistant) and 84 (57% of total) isolates, respectively, indicating that efflux pumps can play a role in resistance to Q/D and, to a lesser extent, in resistance to tetracycline. Q/D efflux probably is due to the presence of Msr-C protein. Msr-C is similar to the ABC proteins of other Grampositive bacteria, and it was found in all human E. faecium isolates (Portillo et al., 2000; Singh et al., 2001). Dalbavancin has been reported to be active against resistant enterococcal species other than vanA (Gales et al., 2005). In our study, the dalbavancin MIC90 value against the tested strain that was susceptible to vancomycin was 0.06 μg/mL. This value is similar to previously described values (Gales et al., 2005; Streit et al., 2005) for Enterococcus spp. and showed higher activity than the other glycopeptide antibiotics available for use in humans. When the inoculum was increased to 107 CFU/microwell, dalbavancin MICs were significantly affected, and both the MIC50 and MIC90 increased 3- and 2-fold, respectively, at the higher inoculum rates. Vancomycin-resistant enterococci (VRE) had dalbavancin MIC results ranging from 0.5 to N16 μg/mL (MIC50 N16 μg/mL), and these were very similar to those described elsewhere (Gales et al., 2005; Oh et al., 2005). 3.2. Distribution of MLS resistance elements among E. faecium PCR screening for MLS resistance determinants revealed the presence of the rRNA methylase gene erm(B) in 89.2% of all isolates. There were 131 of those isolates with only erm(B) gene. For all these strains, the MICs of azithromycin and erythromycin were always N64 μg/mL. One strain with a Q/D MIC of 32 μg/mL was positive for both erm(B) and vat
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(D). No PCR fragment of the expected size was obtained from any of the enterococcal isolates for which erythromycin MICs were b32 μg/mL. In addition, neither of the macrolide resistance determinants was accounted for in 16 (11.5%) isolates that were phenotypically resistant to streptogramins. The different combinations of resistance genes found are shown in Table 2. This result is similar to those reported by other authors (Donabedian et al., 2006; Karanika et al., 2008) where presence of erm(B) gene is also associated with reduced susceptibility to Q/D. Karanika et al. found a high percentage of erm(B)-positive isolates in intermediate Q/D strains without presence of any of the known genes involved in Q/D resistance. They concluded that a nontransferable undetermined mechanism has to be responsible for the expression of low-level Q/D resistance. We were able to distinguish 3 groups of strains among the E. faecium isolates, depending on the Q/D and erythromycin MICs obtained. These groups proved to be homogeneous: all strains that belonged to the same group had the same antibiotic resistance determinants. There was a reduction in the bactericidal effect of Q/D when strains acquired a constitutively expressed erm gene (data not shown). The msrC gene can confer some protection against Q/D in E. faecium isolates. The results obtained with reserpine probably indicate the presence of this gene in our isolates. The msrC gene, which encodes a putative efflux pump of the ABC transporter family, can affect the efflux of antibiotics in the same way as for many other indigenous efflux pump systems (Portillo et al., 2000). Previous reports have indicated that mefE might have an important role in erythromycin resistance in E. faecium. However, we were unable to detect amplification with any of the enterococcal isolates when the mefA/E-specific primers were used in PCR analysis. This result may reflect a different geographic distribution of mef genes in E. faecium (Portillo et al., 2000). 3.3. Analysis of the regulatory region of the erm(B) gene Attenuators of the erm(B) gene of 19 E. faecium isolates with different Q/D MICs were amplified by PCR and sequenced twice in both directions. The sequencing results showed some anomalies in comparison with references, mainly with the erm(B) upstream sequence of E. faecalis Tn917 but also with S.
Table 2 Distribution of MLS determinants in E. faecium isolates No. of strains
MLS resistance determinant
131 1 16
erm(B) erm(B)+vat(D) Unknown
MICs (μg/mL) Erythromycin
Q/D
Azithromycin
256 256 1–4
1–8 N32 1–2
256 256 32
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Fig. 1. Sequence alignment of Tn1545, Tn917, and 19 E. faecium isolates with different Q/D MIC values. (A) Sequence of 12 isolates; 3 with Q/D MIC = 1 μg/ mL; 3 with Q/D MIC = 2 μg/mL; 2 with Q/D MIC = 4 μg/mL; 3 with Q/D MIC = 8 μg/mL; and 1 with Q/D MIC = 16 μg/mL. (B) Sequence of 7 isolates; 1 with Q/D MIC = 1 μg/mL; 2 with Q/D MIC = 2 μg/mL; 3 with Q/D MIC = 4 μg/mL; 1 with Q/D MIC = 8 μg/mL; and 1 with Q/D MIC ≥32 μg/mL. (This last isolate also possesses vat(D) gene.)
pneumoniae Tn1545. Only 2 of the differences appear in all E. faecium analyzed and none in any of the reference strains: the C at position 11 and the T at position 18 (Fig. 1). The other differences are common with 1 of the reference strains or appear only in some, but not all, of E. faecium studied. Moreover, all the E. faecium tested were similar to each other, regardless of the Q/D MICs value. In conclusion, the results presented here indicate that Q/D nonsusceptibility is mainly associated with the presence of erm(B). Differences in Q/D MIC values in these strains cannot be explained by the structure of erm(B) upstream region. Dalbavancin and tigecycline were the most potent compound against VSEF. Our results indicate that dalbavancin is not a promising option for VRE.
Acknowledgments This work was supported by a grant from the Fondo de Investigación Sanitaria (FISS PI050344), Madrid, Spain.
The authors thank Tom O'Boyle for his correction of the English version of this manuscript. References Clinical and Laboratory Standards Institute (2007) Performance Standards for Antimicrobial Susceptibility Testing. 17th Informational Supplement. Document M100-S17. Wayne, PA: CLSI. Culebras E, Rodriguez-Avial I, Betriu C, Picazo JJ (2005) Differences in the DNA sequence of the translational attenuator of several constitutively expressed erm(A) genes from clinical isolates of Streptococcus agalactiae. J Antimicrob Chemother 56:836–840. Donabedian SM, Perri MB, Vager D, Hershberger E, Malani P, Simjee S, Chow J, Vergis EN, Muder RR, Gay K, Angulo FJ, Bartlett P, Zervos MJ (2006) Quinupristin–dalfopristin resistance in Enterococcus faecium isolates from humans, farm animals, and grocery store meat in the United States. J Clin Microbiol 44:3361–3365. Gales AC, Sader HS, Jones RN (2005) Antimicrobial activity of dalbavancin tested against Gram-positive clinical isolates from Latin American medical centres. Clin Microbiol Infect 11:95100. Harbarth S, Cosgrove S, Carmeli Y (2002) Effects of antibiotics on nosocomial epidemiology of vancomycin-resistant enterococci. Antimicrob Agents Chemother 46:1619–1628.
F. López et al. / Diagnostic Microbiology and Infectious Disease 66 (2010) 73–77 Hayes JR, Wagner DD, English LL, Carr LE, Joseph SW (2003) Relative absence of known streptogramin resistance determinants among Enterococcus faecium from U.S. Poultry Production Environment. Abstract C2-84. 43rd ICAAC American Society for Microbiology: Washington, DC, p. P115. Hayes JR, Wagner DD, English LL, Carr LE, Joseph SW (2005) Distribution of streptogramin resistance determinants among Enterococcus faecium from a poultry production environment of the USA. J Antimicrob Chemother 55:123–126. Hershberger E, Donabedian S, Konstantinou K, Zervos MJ (2004) Quinupristin–dalfopristin resistance in gram-positive bacteria: mechanism of resistance and epidemiology. Clin Infect Dis 38:92–98. Karanika M, Prati A, Kiritsi M, Spiliopoulou I, Neonakis I, Anifantaki M, Petinaki E (2008) Reduced susceptibility to quinupristin/dalfopristin in Enterococcus faecium in Greece without prior exposure to the agent. Int J Antimicrob Agent 31:55–57. Malathum K, Murray BE (1999) Vancomycin-resistant enterococci: recent advances in genetics, epidemiology and therapeutic options. Drug Resist Updat 2:224–243. Oh WS, Ko KS, Song JH, Lee MY, Park S, Peck KR, Lee NY, Kim CK, Lee H, Kim SW, Chang HH, Kim YS, Jung SI, Son JS, Yeom JS, Ki HK, Woo GJ (2005) High rate of resistance to quinupristin–dalfopristin in Enterococcus faecium clinical isolates from Korea. Antimicrob Agents Chemother 49:5176–5178. Portillo A, Ruiz-Larrea F, Zarazaga M, Alonso A, Martinez JL, Torres C (2000) Macrolide resistance genes in Enterococcus spp. Antimicrob Agents Chemother 44:967–971.
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Rende-Fournier R, Leclercq R, Galimand M, Duval J, Courvalin P (1993) Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145. Antimicrob Agents Chemother 37:2119–2125. Rosato A, Vicarini H, Leclercq R (1999) Inducible or constitutive expression of resistance in clinical isolates of streptococci and enterococci crossresistant to erythromycin and lincomycin. J Antimicrob Chemother 43:559–562. Singh KV, Malathum K, Murray BE (2001) Disruption of an Enterococcus faecium species-specific gene, a homologue of acquired macrolide resistance genes of staphylococci, is associated with an increase in macrolide susceptibility. Antimicrob Agents Chemother 45:263–266. Soltani M, Beighton D, Philpott-Howard J, Woodford N (2000) Mechanisms of resistance to quinupristin–dalfopristin among isolates of Enterococcus faecium from animals, raw meat, and hospital patients in Western Europe. Antimicrob Agents Chemother 44:433–436. Streit JM, Sader HS, Fritsche TR, Jones RN (2005) Dalbavancin activity against selected populations of antimicrobial-resistant Gram-positive pathogens. Diagn Microbiol Infect Dis 53:307–310. Werner G, Witte W (1999) Characterization of a new enterococcal gene, satG, encoding a putative acetyltransferase conferring resistance to streptogramin A compounds. Antimicrob Agents Chemother 43:1813–1814. Werner G, Hildebrandt B, Witte W (2001) The newly described msrC gene is not equally distributed among all isolates of Enterococcus faecium. Antimicrob Agents Chemother 45:3672–3673.
Diagnostic Microbiology and Infectious Disease 66 (2010) 73 – 77 www.elsevier.com/locate/diagmicrobio
Antimicrobial Susceptibility Studies
Antimicrobial susceptibility and macrolide resistance genes in Enterococcus faecium with reduced susceptibility to quinupristin–dalfopristin: level of quinupristin–dalfopristin resistance is not dependent on erm(B) attenuator region sequence Fátima López, Esther Culebras⁎, Carmen Betriú, Iciar Rodríguez-Avial, María Gómez, Juan J. Picazo Department of Microbiology, Hospital Clínico San Carlos, Plaza de Cristo Rey s/n. 28040, Madrid, Spain Received 12 February 2008; accepted 1 June 2008
Abstract Antimicrobial resistance and the mechanisms implicated were studied in 148 clinical Enterococcus faecium isolates with a quinupristin– dalfopristin (Q/D) MIC ≥1 μg/mL. As expected, higher levels of resistance were detected for macrolide antibiotics (94% erythromycin, 100% azithromycin, 100% josamycin). High-level resistance to gentamicin and streptomycin was detected in 18.9% and 66.2% of isolates, respectively, in our series of enterococci. Resistance against tetracycline was found in only 12.8% of the isolates, and 13 isolates were resistant to vancomycin. The dalvabancin MIC90 for these isolates was N16 μg/mL. Polymerase chain reaction screening for the previously described streptogramin resistance determinants erm(A), erm(B), mefA/E, vat(D), and vat(E) was performed to determine the prevalence of streptogramin resistance mechanisms in these clinical enterococcal isolates. The combination of erm(B) and vat(D) genes encoding resistance to streptogramins was detected in 1 Q/D-resistant E. faecium isolate. A total of 131 Q/D-nonsusceptible enterococci only contained the erm(B) gene. The erm(B) promoter sequence reveals no differences between the strains analyzed, regardless of the Q/D MIC value. © 2010 Elsevier Inc. All rights reserved. Keywords: Enterococcus faecium; erm(B) attenuator; Quinupristin–dalfopristin nonsusceptibility
1. Introduction Enterococci are normal constituents of the intestinal flora and are widely distributed in the environment. Enterococcal infections are the 2nd to 3rd most common nosocomial infection. Multidrug resistance among enterococci has increased, and Enterococcus faecium, in particular, possesses a broad spectrum of natural and acquired antibiotic resistance. As a result, therapeutic options are becoming increasingly limited for the treatment of enterococcal infections (Harbarth et al., 2002; Malathum and Murray, 1999). Quinupristin– dalfopristin (Q/D) is a streptogramin that has been approved
⁎ Corresponding author. Tel.: +34-91-330-3269; fax: +34-91-330-3478. E-mail address: [email protected] (E. Culebras). 0732-8893/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.06.004
for human treatment. Streptogramins have been considered valuable antimicrobial drugs for the treatment of infections with methicillin-resistant Staphylococcus aureus and multiresistant E. faecium (Soltani et al., 2000). Resistance to Q/D can occur by 1 or more of several mechanisms, including enzymatic modification, active transport of efflux mediated by an adenosine triphosphatebinding protein, and alteration of the target site (Hershberger et al., 2004). Inhibitors of mammalian multidrug efflux, such as the plant alkaloid reserpine, are also active in boosting antibiotic activity by inhibiting bacterial efflux. Q/D is a mixture of streptogramin B and streptogramin A compounds, which act synergistically at different sites of the bacterial ribosome to disrupt peptide elongation. Resistance to the streptogramin A component is associated with acetylases encoded by vat(D) and vat(E), 1st described in
74
F. López et al. / Diagnostic Microbiology and Infectious Disease 66 (2010) 73–77
enterococcal isolates in Europe (Rende-Fournier et al., 1993; Werner and Witte, 1999). Resistance to streptogramin B is mainly due to erm gene products, which methylate an adenine residue at 23S rRNA. Methylation occurs in a region of overlapping binding sites for macrolides and lincosamides and results in the macrolide–lincosamide–streptogramin B (MLS) resistance phenotype. Streptogramin B resistance may also be due to hydrolysis of the ring molecule via the lactonase encoded by vgb genes, which were initially reported in Staphylococcus and are rarely present in Enterococcus (Hershberger et al., 2004). Streptogramin B (and macrolide) resistance has also been linked to the MsrC efflux pump, present in most E. faecium isolates (Portillo et al., 2000; Werner et al., 2001). Many streptogramin-resistant E. faecium isolates harbor none of the described resistance mechanisms (Hayes et al., 2003). We describe the diversity and distribution of MLS resistance as well as the known genetic determinants that contribute to streptogramin resistance among E. faecium clinical isolates. We also characterize the attenuator region of the erm(B) methylase gene to establish a possible relationship between mutations in the erm(B) leader region and Q/D MICs. 2. Materials and methods 2.1. Bacterial strains A total of 148 clinical isolates were selected with a Q/D MIC ≥1 μg/mL: 43 strains were intermediate and 90 were resistant to Q/D. Strains were collected from May 2002 to January 2004. Multiple isolates from the same patient were avoided. The sources of the 148 E. faecium isolates included skin and soft tissues (72 isolates, 48.6%), abdomen (47 isolates, 31.75%), urine (20 isolates, 13.5%), and other sites (9 isolates, 6%). 2.2. Antimicrobials and MIC testing Susceptibilities to rifampicin, chloramphenicol, gentamicin, streptomycin, and dalbavancin were determined by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) (2007). The inoculum was adjusted to 105 and 107 colony-forming units (CFU)/ microwell for dalbavancin to investigate inoculum effects. The MICs were determined by the agar dilution method in Mueller–Hinton agar (Difco, Detroit, MI) according to the procedure recommended by the CLSI (2007). The antibiotics tested were erythromycin, tetracycline, Q/D, vancomycin, teicoplanin, linezolid, tigecycline, minocycline, josamycin, and azithromycin. The plant alkaloid reserpine was used to determine the role of the efflux pump in the Q/D and tetracycline MIC values observed. If reserpine reduces the MIC of antibiotics at least 2-fold, it has been concluded to be an active efflux system. Enterococcus faecalis strain ATCC 29212 and S. aureus strain ATCC 29213 were used as control strains.
2.3. Bactericidal effect of Q/D The bactericidal effect was evaluated in 6 erm(B)-positive isolates and 6 erm(B)-negative isolates. The methods used was as follows: Mueller–Hinton broth (MHB) inoculated with 5 × 105 CFU/mL was supplemented with Q/D. The following concentrations were used: 2×, 3×, and 4× the MIC. After 18 h of incubation at 37 °C, serial dilutions of 0.1 mL samples were subcultured onto agar plates, and the plates were incubated for 24 h before the numbers of CFU were counted. 2.4. Polymerase chain reaction analysis The presence of genes involved in MLS resistance was determined by polymerase chain reaction (PCR) amplification. The presence of erm(B), erm(A), mefA/E, vat(D), and vat(E) was assayed by PCR using oligonucleotide primers and reaction conditions described elsewhere (Culebras et al., 2005; Hayes et al., 2005; Soltani et al., 2000). 2.5. Nucleotide sequence of the erm(B) attenuator The erm(B) upstream region was amplified as previously described (Rosato et al., 1999). PCR products were then purified using Qiagen PCR product purification kit and sequence in an automated ABI PRISM BIO system (PerkinElmer, Waltham, MA). erm(B) upstream sequences of the Tn1545 of Streptococcus pneumoniae (gi:47454) and of the Tn917 of E. faecalis (gi:21492651) were used as the reference wild-type sequence. 3. Results and discussion 3.1. Antimicrobial susceptibility testing The distribution of MIC values for vancomycin, teicoplanin, dalbavancin, tetracycline, minocycline, erythromycin, azithromycin, josamycin, chloramphenicol, linezolid, tigecycline, and Q/D are shown in Table 1. No isolates were resistant to teicoplanin or linezolid. Tigecycline shows very good activity against all the isolates with MIC values ≤0.06 μg/mL. Among the 148 E. faecium clinical isolates, overall resistance was as follows: tetracycline 12.8%, chloramphenicol 31.75%, vancomycin 8.8%, and erythromycin 94%. High-level resistance to streptomycin and gentamicin was detected in 66.2% and 18.9% of the isolates, respectively. There was no link between reduced susceptibility to Q/D and resistance to other classes of antimicrobials. Resistance to tetracycline, minocycline, and chloramphenicol appears in both Q/D-susceptible and Q/D-resistant isolates. Susceptibility to linezolid and teicoplanin and tigecycline MIC also shows similar distribution in both groups of isolates. Our data showed that the Q/D resistance rate is high in vancomycin-susceptible E. faecium (VSEF) isolates (58% of VSEF were resistant to Q/D). These data are consistent with previous data (Oh et al., 2005). However, in our study, all vancomycin-resistant E. faecium isolates (13 isolates) were resistant to Q/D.
F. López et al. / Diagnostic Microbiology and Infectious Disease 66 (2010) 73–77 Table 1 Antibiotic resistance in E. faecium clinical isolates (n = 148) Antimicrobial agent
MIC50 (μg/mL)
MIC90 (μg/mL)
MIC range (μg/mL)
Resistance (%)a
Vancomycin Teicoplanin Dalbavancin (105) Dalbavancin (107) Tetracycline Minocycline Erythromycin Azithromycin Josamycin Chloramphenicol Linezolid Tigecycline Q/D Gentamicinb Streptomycinb
0.5 1 0.03 0.25 b1 b1 256 256 256 8 1 b0.06 4 – –
0.5 1 0.06 0.25 N32 16 256 256 256 N8 1 b0.06 8 – –
0.5 to N32 b0.06 to 16 b0.016 to N16 0.06 to N16 b1 to N32 b1 to N32 0.5 to 256 8 to 256 32 to 256 2 to N8 1 b0.06 1 to 32 – –
8.8 0 Not available Not available 12.8 10.2 94 100 100 31.75 0 Not available 61.5 18.9 66.2
a b
Susceptibility interpretations according to CLSI criteria (CLSI, 2007). High-level resistance to aminoglycosides.
To establish the contribution of the efflux pump to the MIC of tetracycline and Q/D, we determined the MIC values of both antibiotics in the presence and absence of reserpine. The results showed that reserpine affects the MIC of tetracycline and Q/D in 3 (16% tetracycline resistant) and 84 (57% of total) isolates, respectively, indicating that efflux pumps can play a role in resistance to Q/D and, to a lesser extent, in resistance to tetracycline. Q/D efflux probably is due to the presence of Msr-C protein. Msr-C is similar to the ABC proteins of other Grampositive bacteria, and it was found in all human E. faecium isolates (Portillo et al., 2000; Singh et al., 2001). Dalbavancin has been reported to be active against resistant enterococcal species other than vanA (Gales et al., 2005). In our study, the dalbavancin MIC90 value against the tested strain that was susceptible to vancomycin was 0.06 μg/mL. This value is similar to previously described values (Gales et al., 2005; Streit et al., 2005) for Enterococcus spp. and showed higher activity than the other glycopeptide antibiotics available for use in humans. When the inoculum was increased to 107 CFU/microwell, dalbavancin MICs were significantly affected, and both the MIC50 and MIC90 increased 3- and 2-fold, respectively, at the higher inoculum rates. Vancomycin-resistant enterococci (VRE) had dalbavancin MIC results ranging from 0.5 to N16 μg/mL (MIC50 N16 μg/mL), and these were very similar to those described elsewhere (Gales et al., 2005; Oh et al., 2005). 3.2. Distribution of MLS resistance elements among E. faecium PCR screening for MLS resistance determinants revealed the presence of the rRNA methylase gene erm(B) in 89.2% of all isolates. There were 131 of those isolates with only erm(B) gene. For all these strains, the MICs of azithromycin and erythromycin were always N64 μg/mL. One strain with a Q/D MIC of 32 μg/mL was positive for both erm(B) and vat
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(D). No PCR fragment of the expected size was obtained from any of the enterococcal isolates for which erythromycin MICs were b32 μg/mL. In addition, neither of the macrolide resistance determinants was accounted for in 16 (11.5%) isolates that were phenotypically resistant to streptogramins. The different combinations of resistance genes found are shown in Table 2. This result is similar to those reported by other authors (Donabedian et al., 2006; Karanika et al., 2008) where presence of erm(B) gene is also associated with reduced susceptibility to Q/D. Karanika et al. found a high percentage of erm(B)-positive isolates in intermediate Q/D strains without presence of any of the known genes involved in Q/D resistance. They concluded that a nontransferable undetermined mechanism has to be responsible for the expression of low-level Q/D resistance. We were able to distinguish 3 groups of strains among the E. faecium isolates, depending on the Q/D and erythromycin MICs obtained. These groups proved to be homogeneous: all strains that belonged to the same group had the same antibiotic resistance determinants. There was a reduction in the bactericidal effect of Q/D when strains acquired a constitutively expressed erm gene (data not shown). The msrC gene can confer some protection against Q/D in E. faecium isolates. The results obtained with reserpine probably indicate the presence of this gene in our isolates. The msrC gene, which encodes a putative efflux pump of the ABC transporter family, can affect the efflux of antibiotics in the same way as for many other indigenous efflux pump systems (Portillo et al., 2000). Previous reports have indicated that mefE might have an important role in erythromycin resistance in E. faecium. However, we were unable to detect amplification with any of the enterococcal isolates when the mefA/E-specific primers were used in PCR analysis. This result may reflect a different geographic distribution of mef genes in E. faecium (Portillo et al., 2000). 3.3. Analysis of the regulatory region of the erm(B) gene Attenuators of the erm(B) gene of 19 E. faecium isolates with different Q/D MICs were amplified by PCR and sequenced twice in both directions. The sequencing results showed some anomalies in comparison with references, mainly with the erm(B) upstream sequence of E. faecalis Tn917 but also with S.
Table 2 Distribution of MLS determinants in E. faecium isolates No. of strains
MLS resistance determinant
131 1 16
erm(B) erm(B)+vat(D) Unknown
MICs (μg/mL) Erythromycin
Q/D
Azithromycin
256 256 1–4
1–8 N32 1–2
256 256 32
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Fig. 1. Sequence alignment of Tn1545, Tn917, and 19 E. faecium isolates with different Q/D MIC values. (A) Sequence of 12 isolates; 3 with Q/D MIC = 1 μg/ mL; 3 with Q/D MIC = 2 μg/mL; 2 with Q/D MIC = 4 μg/mL; 3 with Q/D MIC = 8 μg/mL; and 1 with Q/D MIC = 16 μg/mL. (B) Sequence of 7 isolates; 1 with Q/D MIC = 1 μg/mL; 2 with Q/D MIC = 2 μg/mL; 3 with Q/D MIC = 4 μg/mL; 1 with Q/D MIC = 8 μg/mL; and 1 with Q/D MIC ≥32 μg/mL. (This last isolate also possesses vat(D) gene.)
pneumoniae Tn1545. Only 2 of the differences appear in all E. faecium analyzed and none in any of the reference strains: the C at position 11 and the T at position 18 (Fig. 1). The other differences are common with 1 of the reference strains or appear only in some, but not all, of E. faecium studied. Moreover, all the E. faecium tested were similar to each other, regardless of the Q/D MICs value. In conclusion, the results presented here indicate that Q/D nonsusceptibility is mainly associated with the presence of erm(B). Differences in Q/D MIC values in these strains cannot be explained by the structure of erm(B) upstream region. Dalbavancin and tigecycline were the most potent compound against VSEF. Our results indicate that dalbavancin is not a promising option for VRE.
Acknowledgments This work was supported by a grant from the Fondo de Investigación Sanitaria (FISS PI050344), Madrid, Spain.
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