Detection of Inducible Clindamycin Resistance in Beta-Hemolytic Streptococci by Testing

Transcription

1 JCM Accepts, published online ahead of print on 1 April J. Clin. Microbiol. doi:.2/jcm.00- Copyright, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 2 Detection of Inducible Clindamycin Resistance in Beta-Hemolytic Streptococci by Testing Erythromycin-Clindamycin Combinations Using the CLSI Broth Microdilution Test Jason E. Bowling, 1 Aaron E. Owens, 1,2 M. Leticia McElmeel, Letitia C. Fulcher, Monica L. Herrera, Brian L. Wickes, and James H. Jorgensen* 1, Department of Medicine, The University of Texas Health Science Center at San Antonio, 1 Medicine Service, University Hospital, 2 Department of Pathology, and Department of Microbiology, University of Texas Health Science Center, San Antonio, Texas *Corresponding author: James H. Jorgensen, Ph.D. Department of University of Texas Health Science Center 0 Floyd Curl Drive San Antonio, Texas -00 PH 2--0 Fax 2--2 jorgensen@uthscsa.edu 1

2 Abstract This study assessed an erythromycin-clindamycin (ERY + CC) broth test for inducible CC resistance in beta-hemolytic streptococci. 1 isolates of Groups A, B, C, F, and G were tested by the CLSI broth microdilution method. Combinations of or µg/ml ERY + CC detected all inducible isolates. Erythromycin resistance was first noted in staphylococci in 1 and reported for the first time in streptococci in 1 (). There are two main mechanisms of macrolide resistance in the streptococci. One is an active efflux mechanism encoded by the mef A/E genes that affects only macrolides (). The second major mechanism, which causes the majority of resistance in streptococci, is modification of the same or adjacent binding site on the ribosome targeted by macrolides and lincosamides. Proteins encoded by erm (erythromycin ribosomal methylase) genes methylate a single adenine in the large 0S ribosomal subunit that impairs drug binding and leads to cross-resistance to macrolides, lincosamides, and streptogramins B (). This is known as the MLSB phenotype. Expression of MLSB resistance in staphylococci and streptococci can either be constitutive or inducible (). Fourteen- and fifteen-member macrolides are good inducers of the ribosomal conformational change, but lincosamides such as clindamycin are poor inducers (). Thus, inducible resistance is not detected during routine clindamycin disk diffusion or agar or broth dilution tests. Some evidence exists, especially with 2

3 staphylococci that there is a risk of spontaneous conversion from inducible to constitutive resistance phenotype during clindamycin therapy as a result of a single mutation in a promoter region that controls expression of the erm genes (). Thus patients could be at risk of clinical failure if inducible clindamycin resistance as well as constitutive resistance were not detected during routine antimicrobial susceptibility testing of individual patient s isolates (,,). Both constitutive and inducible clindamycin resistance has increased in recent years, especially in Groups A and B streptococci (,,,,,1). The Clinical and Laboratory Standards Institute (CLSI) has described a disk diffusion test that places standard erythromycin and clindamycin susceptibility testing disks in close proximity on the surface of an inoculated agar plate (2). If the erythromycin disk induces expression of the ribosomal methylase among bacterial cells in the area adjacent to the clindamycin disk, there is a flattening of the zone of inhibition around one side of the clindamycin zone that resembles the letter D. This is commonly referred to as the D-zone test. The CLSI has provided recommendations for performing D-zone testing of both staphylococci and beta-hemolytic streptococci (2). While simple to perform and interpret, it represents the need to perform an additional test for laboratories that routinely use a broth-based susceptibility test method such as broth microdilution or an automated instrument. Many clinical laboratories would prefer to have an inducible clindamycin resistance test included in their routine broth dilution test systems (1). For this reason, a single erythromycin-clindamycin combination well broth microdilution screening test for inducible clindamycin resistance in staphylococci has been described by the CLSI (2) based on a multicenter laboratory evaluation by Swenson et al (1). The CLSI has not yet described a single well broth induction method for testing the streptococci. The goal of this

4 study was to develop an analogous broth microdilution screening test for inducible clindamycin resistance in beta-hemolytic streptococci. A group of retained clinical isolates of beta-hemolytic streptococci recovered between 00 and 0 from patients in the University Health System, San Antonio, Texas was used in this study. Standard disk diffusion D-zone testing was performed using erythromycin (1 µg) and clindamycin (2 µg) disks placed mm apart on Mueller-Hinton % sheep blood agar plates incubated at o C in % CO 2 for to 2 hours (2). Broth microdilution panels were prepared according to CLSI guidelines using % lysed horse blood supplemented Mueller-Hinton broth (1). Panels included erythromycin (ERY) from 0.01 to 2 µg/ml, clindamycin (CC) from 0.01 to 1 µg/ml, and combinations of erythromycin and clindamycin of µg/ml, µg/ml, and µg/ml in separate wells. The frozen panels also included nine other antimicrobial agents: penicillin, amoxicillin, ceftriaxone, tetracycline, doxycycline, minocycline, moxifloxacin, vancomycin and linezolid. Panels were inoculated with the standard x CFU/ml density and incubated for 1 to h at o C (1). Growth or no growth in the erythromycin-clindamycin combination wells was compared to the disk diffusion D-zone test results. A combination well was considered to have accurately detected inducible clindamycin resistance if growth was present with an isolate determined to have a positive D-zone test. Streptococcus pneumoniae ATCC 1 was used as the control strain on each day of testing and Staphylococcus aureus ATCC BAA was used for quality assessment of the erythromycin + clindamycin combination wells (2). The mechanisms of both constitutive and inducible clindamycin resistance were determined by performance of PCR using primers for ermtr and for ermb with each clindamycin resistant isolate. Primers for ermtr were -AGA AGT TTA TAA TGA AAC

5 AGA - (). Thermocycling was performed in a Brinkman Eppendorf Mastercycler Gradient. Reactions for were carried out in a total volume of 0µl. Each reaction tube contained 1µl of the template DNA (-ng DNA), µl of x PCR buffer (00mM KCl, mm MgCl 2, 0mM Tris- HCl, ph.), µl of mm mixture of dntp s, 1.µl of 0mM MgCl 2, 1µl of 0pM of each primer and 0.2µl of AmpliTaq DNA polymerase from Applied Biosystems. Thirty-five cycles of amplification were carried out. Each cycle consisted of a 0 s denaturation step at C, a 0 s annealing step at 2 C and a 0 s extension step at 2 C. The first step of denaturation was for minutes at C and the last step of extension was increased by minutes. Amplification products were separated by electrophoresis on 1% agarose gel that incorporated ethidium bromide then visualized with an Electrophoresis Systems Photo Documentation Camera using black and white film. The presence if a 2 bp product following electrophoresis was considered evidence of the presence of the ermtr gene (). Primers for ermb were -CGA GTG AAA AAG TAC TCA ACC- (1). Each reaction tube contained 2µl of the template DNA (-ng DNA), µl of x PCR buffer (00mM KCl, mm MgCl 2, 0mM Tris-HCl, ph.), µl of mm mixture of dntp s, 2µl of 0mM MgCl 2, 1µl of 0pM of each primer and 0.µl of AmpliTaq DNA polymerase from Applied Biosystems. Forty cycles of amplification were carried out. Each cycle consisted of a s denaturation step at C, a 0 s annealing step at C and a 0 s extension step at 2 C. The first step of denaturation was for minutes at C and the last step of extension was increased by minutes. Following agarose gel electrophoresis as described above, the presence of a 1 bp PCR product on electrophoresis was considered evidence of the presence of the ermb gene (1). A total of 0 beta-hemolytic streptococci isolates were tested including Group A, 2 Group B, 1 Group C, Group F, and 2 Group G streptococci. Of these isolates,

6 demonstrated inducible clindamycin resistance by the disk D-zone test and showed constitutive clindamycin resistance. Table 1 indicates the numbers of clindamycin resistant isolates per streptococcal group and the erm genes responsible for either constitutive or inducible resistance. The ermtr mechanism was responsible for 1 of clindamycin resistant isolates, while only isolates possessed ermb. One isolate contained both ermtr and ermb, and four isolates (1 Group A and Group G) did not provide a PCR product with the primers and conditions that we employed. The combination of µg/ml ERY + CC detected / (0%) of D-zone positive isolates, µg/ml ERY + CC detected / (0%), and µg/ml ERY + CC detected / (%) (Table 2). None of the clindamycin susceptible isolates grew in any of the three combination wells (Table 2). These included the erythromycin-susceptible isolates and the erythromycin-resistant, D-zone-negative isolates. Resistance to tetracycline, doxycycline, and minocycline was %, 1%, and % respectively in this isolate collection as indicated in Table. All isolates were susceptible to beta-lactams, vancomycin, linezolid, and moxifloxacin. In this study, combined concentrations of µg/ml and µg/ml of erythromycin and clindamycin combinations in the CLSI broth microdilution test detected all beta-hemolytic streptococci with inducible clindamycin resistance as evidenced by positive agarbased D-zone tests. Occasional case reports have documented clinical failures when clindamycin was used for treatment of S. aureus infections with an inducible clindamycin resistance (). This raises the obvious concern for potential clinical failures in infections caused by beta-hemolytic streptococci that possess the same inducible MLSB phenotype and has led some experts to recommend that inducible clindamycin resistance be detected and reported in the streptococci (,). Clindamycin monotherapy or combinations of penicillin plus clindamycin are

7 sometimes used to treat severe streptococcal soft tissue infections such as necrotizing fasciitis, in which failure of therapy due to emergence of resistance could be limb- or life-threatening. Routine detection and reporting of inducible clindamycin resistance in beta-hemolytic streptococcal skin and soft tissue infections or bacteremia would serve to alert clinicians and might prevent clinical failures due to emergence of resistance during therapy. Broth microdilution testing with a single erythromycin-clindamycin combination well would provide a convenient option to clinical laboratories to facilitate routine detection of inducible clindamycin resistance. It should be noted that Group F beta-hemolytic streptococci were included in this study along with Groups A, B, C, and G isolates. Strictly speaking, Group F beta-hemolytic streptococci are members of the S. anginosus complex, as are small colony types of Groups A, C, and G (1). The CLSI has chosen to recommend that all S. anginosus isolates, whether alpha-, beta-, or non-hemolytic should be considered as members of the viridans group of streptococci. The CLSI testing recommendations and certain drug interpretive breakpoints differ based upon isolates belonging to the beta-hemolytic group as opposed to the viridans group (2). In this study, we included the Group F beta-hemolytic S. anginosus isolates reasoning that some laboratories might test them using the beta-hemolytic group criteria. Notably, none of the Group F isolates was macrolide or clindamycin resistant. Further, multi-laboratory studies are needed to confirm and extend these initial results and to select the optimal drug concentrations for a single erythromycin-clindamycin test well for detection of inducible clindamycin resistance in routine streptococcal test panels. This study identified two potential erythromycin-clindamycin combinations that are worthy of further study. The goal would be to include a single well screening test to detect inducible clindamycin

8 resistance in broth panels used for routine testing of beta-hemolytic streptococci without need for additional agar-based D-zone testing.

9 References 1. Clinical and Laboratory Standards Institute. 0. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard, eighth edition, M0- A. Clinical and Laboratory Standards Institute, Wayne, PA. 2. Clinical and Laboratory Standards Institute. 0. Performance standards for antimicrobial susceptibility testing. Nineteenth informational supplement, M0-S1. Clinical and Laboratory Standards Institute, Wayne, PA. De Mouy, D., J.D. Cavallo, R. Leclercq, and R. Fabre. 01. Antibiotic susceptibility and mechanisms of erythromycin resistance in clinical isolates of Streptococcus agalactiae: French multicenter study. Antimicrob. Agents Chemother. :0-2.. Desjardins, M., K.L. Delgaty, K. Ramotar, C Seetaram, and B. Toye. 0. Prevalence and mechanisms of erythromycin resistance in group A and group B Streptococcus: implications for reporting susceptibility results. J. Clin. Microbiol. 2:-2.. Diaz, M, M.J. Torres Sanchez, and M.J. Aznar. 0. Prevalence and mechanisms of erythromycin and clindamycin resistance in clinical isolates of beta-hemolytic streptococci of Lancefield groups A, B, C, and G in Seville, Spain. Clin Micrbiol. Infect. 1:-.. Leclercq, R. 02. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. Clin. Infect. Dis. :2-2.. Lewis, J. S., II, and J. H. Jorgensen. 0. Inducible clindamycin resistance in staphylococci: should clinicians and microbiologists be concerned? Clin. Infect. Dis. 0: 2.. Michos, A.G., C.G. Bakoula, M. Braoudaki, F.I. Koutouzi, E.S. Roma, A. Pangalis, G. Nikolopoulou, G. kirikou, and V.P. Syriopoulou. 0. Macrolide resistance in Streptococcus pyogenes: prevalence, resistance determinants, and emm types. Diagn. Microbiol. Infect. Dis. :2-2.. Portillo, A., M. Lantero, I. Olarte, F. Ruiz-Larrea, and C. Torres. 01. MLS resistance phenotypes and mechanisms in beta-haemolytic group B, C and G Streptococcus isolates in La Rioja, Spain. J. Antimicrob. Chemother. :-.

10 . Raney, P.M., F.C. Tenover, R.B. Carey, J.E. McGowan, Jr., and J.B. Patel. 0. Investigation of inducible clindamycin and telithromycin resistance in isolates of beta-hemolytic streptococci. Diagn. Microbiol. Infect. Dis. :-.. Reig, M., J..C. Galan, F. Baquero, and J.C. Perez-Diaz. 01. Macrolide resistance in Peptostreptococcus spp. mediated by ermtr: a possible source of macrolide-lincosamidestreptogramin B resistance in Streptococcus pyogenes. Antimicrob. Agents Chemother. :0-2.. Richter, S.S., K.P. Heilmann, S.E. Beekmann, N.J. Miller, A.L. Miller, C.L. Rice, C.D. Doern, S.A. Reid, and G.V. Doern. 0. Macrolide-resistant Streptococcus pyogenes in the United States, Clin. Infec. Dis. 1: Shortridge, R. Skov, M. P. Weinstein, B. L. Zimmer, and J. B. Patel. 0. Detection of Inducible Clindamycin Resistance in Staphylococci by Broth Microdilution Using Erythromycin-Clindamycin Combination Wells. J. Clin. Microbiol. :-. 1. Spellerberg, B. and C. Brandt. 0. Streptococcus, pp. -2. In Manual of Clinical Microbiology, th ed. (Eds.) Murray, P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaller, and R.H. Yolken. American Society for Microbiology, Washington, D.C. 1. Swenson, J. M., W. B. Brasso, M. J. Ferraro, D. J. Hardy, C. C. Knapp, L. K. McDougal, L. B. Reller, H. S. Sader, D. Shortridge, R. Skov, M.P. Weinstein, B.L. Zimmer, and J.B. Patel. 0. Detection of inducible clindamycin resistance in staphylococci by broth microdilution using erythromycin-clindamycin combination wells. J. Clin. Microbiol. :- 1. York, M.K., L. Gibbs, F. Perdreau-Remington, and G.F. Brooks. 1. Characterization of antimicrobial resistance in Streptococcus pyogenes isolates from the San Francisco Bay area of northern California. J. Clin. Microbiol. :12-11.

11 Table 1. Beta-hemolytic streptococcal isolates included in the study and their erythromycin and clindamycin susceptibilities and resistance determinants Group No. Erythromycin Constitutive Resistance Determinant Inducible Resistance Determinant Isolates Resistant Clindamycin ermtr ermb Clindamycin ermtr ermb Neither Resistance Resistance found A B C NA NA F 0 0 NA NA 0 NA NA NA G NA = not applicable

12 Table 2. Growth in the wells containing erythromycin clindamycin combinations with 0 streptococcal isolates µg/ml µg/ml µg/ml Clindamycin susceptible strains 0/ 0/ 0/ Clindamycin resistant (inducible strains) / / / Clindamycin resistant (constitutive strains) / / /

13 Table. Erythromycin and clindamycin MICs when tested separately with 0 streptococcal isolates Drug: MIC 0 MIC 0 MIC range % Resistant Erythromycin 2 > >1 Clindamycin <= (susceptible strains) Clindamycin (inducible strains) Clindamycin > > All > 0 (constitutive strains) 1