Detection of Inducible Clindamycin Resistance in Beta-Hemolytic Streptococci by Using the CLSI Broth Microdilution Test and Erythromycin-Clindamycin Combinations

Abstract

This study assessed an erythromycin-clindamycin (ERY-CC) broth test for inducible CC resistance in beta-hemolytic streptococci. One hundred one isolates of groups A, B, C, F, and G were tested by the CLSI broth microdilution method. Combinations of 1 and 0.25 μg/ml or 0.5 and 0.25 μg/ml of ERY and CC, respectively, detected all inducible isolates.

Erythromycin (ERY) resistance was first noted in staphylococci in 1956 and reported for the first time in streptococci in 1959 (6). There are two main mechanisms of macrolide resistance in the streptococci. One is an active efflux mechanism encoded by the mefA and -E genes that affects only macrolides (6). 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 50S ribosomal subunit that impairs drug binding and leads to cross-resistance to macrolides, lincosamides, and streptogramin B (6). This is known as the MLSB phenotype. Expression of MLSB resistance in staphylococci and streptococci can be either constitutive or inducible (6). Fourteen- and 15-member macrolides are good inducers of the ribosomal conformational change, but lincosamides, such as clindamycin (CC), are poor inducers (6). Thus, inducible resistance is not detected during routine CC disk diffusion or agar or broth dilution tests. Some evidence exists, especially with staphylococci, that there is a risk of spontaneous conversion from inducible to constitutive resistance phenotype during CC therapy as a result of a single mutation in a promoter region that controls expression of the erm genes (7). Thus, patients could be at risk of clinical failure if inducible CC resistance as well as constitutive resistance is not detected during routine antimicrobial susceptibility testing of an individual patient's isolate (4, 6, 7). Both constitutive and inducible CC resistances have increased in recent years, especially in group A and B streptococci (3, 5, 8, 9, 12, 15).

The Clinical and Laboratory Standards Institute (CLSI) has described a disk diffusion test that places standard ERY and CC susceptibility testing disks in close proximity on the surface of an inoculated agar plate (2). If the ERY disk induces expression of the ribosomal methylase among bacterial cells in the area adjacent to the CC disk, there is a flattening of the zone of inhibition around one side of the CC 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 CC resistance test included in their routine broth dilution test systems (14). For this reason, a broth microdilution screening test for inducible CC resistance in staphylococci by the use of a single ERY-CC combination well has been described by the CLSI (2) based on a multicenter laboratory evaluation by Swenson et al. (14). The CLSI has not yet described a single-well broth induction method for testing the streptococci. The goal of this study was to develop an analogous broth microdilution screening test for inducible CC resistance in beta-hemolytic streptococci.

A group of retained clinical isolates of beta-hemolytic streptococci recovered between 2000 and 2009 from patients of the University Health System, San Antonio, TX, was used in this study. Standard disk diffusion D-zone testing was performed using ERY (15 μg) and CC (2 μg) disks placed 12 mm apart on Mueller-Hinton 5% sheep blood agar plates incubated at 35°C in 5% CO₂ for 20 to 24 h (2). Broth microdilution panels were prepared according to CLSI guidelines by using 3% lysed horse blood-supplemented Mueller-Hinton broth (1). Panels included 0.015 to 32 μg/ml ERY, 0.015 to 16 μg/ml CC, and combinations of ERY and CC of 0.5 and 0.25 μg/ml, 1 and 0.25 μg/ml, and 1 and 0.5 μg/ml, respectively, 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 5 × 10⁵-CFU/ml density and incubated for 16 to 20 h at 35°C (1). Growth or no growth in the ERY-CC combination wells was compared to the disk diffusion D-zone test results. A combination well was considered to have accurately detected inducible CC resistance if growth was present with an isolate determined to have a positive D-zone test. Streptococcus pneumoniae ATCC 49619 was used as the control strain on each day of testing, and Staphylococcus aureus ATCC BAA977 was used for quality assessment of the ERY-CC combination wells (2).

The mechanisms of both constitutive and inducible CC resistance were determined by the performance of PCR using primers for ermTR and for ermB with each CC-resistant isolate. The primer for ermTR was 5′-AGA AGT TTA TAA TGA AAC AGA-3′ (12). Thermocycling was performed in a Brinkman Eppendorf Mastercycler gradient. Reactions were carried out in a total volume of 50 μl. Each reaction tube contained 1 μl of the template DNA (5 to 10 ng DNA), 5 μl of 10× PCR buffer (500 mM KCl, 20 mM MgCl_2, 100 mM Tris-HCl [pH 8.3]), 4 μl of 10 mM mixture of deoxynucleoside triphosphates (dNTPs), 1.5 μl of 50 mM MgCl₂, 1 μl of 50 pM of each primer, and 0.25 μl of AmpliTaq DNA polymerase from Applied Biosystems. Thirty-five cycles of amplification were carried out. Each cycle consisted of a 30-s denaturation step at 94°C, a 60-s annealing step at 42°C, and a 90-s extension step at 72°C. The first step of denaturation was for 10 min at 95°C, and the last step of extension was increased by 5 min.

Amplification products were separated by electrophoresis on 1% agarose gel that incorporated ethidium bromide and then visualized with an electrophoresis system photo documentation camera using black and white film. The presence of a 212-bp product following electrophoresis was considered evidence of the presence of ermTR (10, 11).

The primer for ermB was 5′-CGA GTG AAA AAG TAC TCA ACC-3′ (14). Each reaction tube contained 2 μl of the template DNA (5 to 10 ng DNA), 5 μl of 10× PCR buffer (500 mM KCl, 20 mM MgCl_2, 100 mM Tris-HCl [pH 8.3]), 4 μl of 10 mM mixture of dNTPs, 2 μl of 50 mM MgCl₂, 1 μl of 50 pM of each primer, and 0.5 μl of AmpliTaq DNA polymerase from Applied Biosystems. Forty cycles of amplification were carried out. Each cycle consisted of a 45-s denaturation step at 94°C, a 60-s annealing step at 58°C, and a 60-s extension step at 72°C. The first step of denaturation was for 10 min at 95°C, and the last step of extension was increased by 5 min. Following agarose gel electrophoresis as described above, the presence of a 616-bp PCR product on electrophoresis was considered evidence of the presence of the ermB gene (14).

A total of 100 beta-hemolytic streptococcus isolates were tested, including 20 group A, 32 group B, 15 group C, 10 group F, and 23 group G streptococci. Of these isolates, 46 demonstrated inducible CC resistance by the disk D-zone test, and 20 showed constitutive CC resistance. Table 1 indicates the number of CC-resistant isolates per streptococcal group and the erm genes responsible for either constitutive or inducible resistance. The ermTR mechanism was responsible for 51 of 59 CC-resistant isolates, while only 9 isolates possessed ermB. One isolate contained both ermTR and ermB, and four isolates (1 group A and 3 group G) did not provide a PCR product with the primers and conditions that we employed.

TABLE 1.

Beta-hemolytic streptococcal isolates included in the study and their ERY and CC susceptibilities and resistance determinants^a

Group	Total no. of isolates	No. of ERY-resistant isolates	No. of constitutively CC-resistant isolates	No. of constitutively CC-resistant isolates with indicated resistance determinant		No. of inducibly CC-resistant isolates	No. of inducibly CC-resistant isolates with indicated resistance determinant		No. of CC-resistant isolates with neither resistance determinant
				ermTR	ermB		ermTR	ermB
A	20	12	2	2	0	10	9	0	1
B	32	32	16	8	8	16	13	0	0
C	15	1	0	NA	NA	1	1	0	0
F	10	0	0	NA	NA	0	NA	NA	NA
G	23	21	2	2	0	19	16	1	3

^aNA, not applicable.

The combination of 1 and 0.25 μg/ml ERY and CC detected 46/46 (100%) D-zone-positive isolates, 1 and 0.5 μg/ml ERY and CC detected 46/46 (100%), and 0.5 and 0.25 μg/ml ERY and CC detected 39/46 (85%) (Table 2). None of the 34 CC-susceptible isolates grew in any of the three combination wells (Table 2). These included the ERY-susceptible isolates and the ERY-resistant, D-zone-negative isolates. The ERY and CC susceptibilities of the constitutively and inducibly CC-resistant strains are indicated in Table 3. Resistance to tetracycline, doxycycline, and minocycline was 64%, 61%, and 58%, respectively, in this isolate collection (data not further depicted). All isolates were susceptible to β-lactams, vancomycin, linezolid, and moxifloxacin.

TABLE 2.

Growth in the wells containing ERY-CC combinations with 100 streptococcal isolates

Strains	No. of isolates with growth in indicated ERY-CC combination (μg/ml)/total no. of isolates
	0.5 and 0.25	1 and 0.25	1 and 0.5
CC-susceptible strains	0/34	0/34	0/34
CC-resistant (inducible) strains	39/46	46/46	46/46
CC-resistant (constitutive) strains	20/20	20/20	20/20

TABLE 3.

ERY and CC MICs when tested separately with 100 streptococcal isolates

Drug	MIC value(s) (μg/ml)			% Resistance
	MIC50	MIC90	MIC range
ERY	2	>16	0.03->16	68
CC in:
Susceptible strains	0.06	0.12	≤0.015-0.25	0
Inducible strains	0.06	0.12	0.06-0.25	0
Constitutive strains	>4	>4	>4	100

In this study, combined concentrations of 1- and 0.25-μg/ml and 1- and 0.5-μg/ml ERY-CC combinations in the CLSI broth microdilution test detected all 46 beta-hemolytic streptococci with inducible CC resistance, as evidenced by positive agar-based D-zone tests. Occasional case reports have documented clinical failures when CC was used for treatment of S. aureus infections with an inducible CC resistance (7). 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 CC resistance be detected and reported in the streptococci (4, 6). CC monotherapy or combinations of penicillin plus CC are 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 CC 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 ERY-CC combination well would provide a convenient option to clinical laboratories to facilitate routine detection of inducible CC resistance.

It should be noted that group F beta-hemolytic streptococci were included in this study, along with group A, B, C, and G isolates. Strictly speaking, group F beta-hemolytic streptococci are members of the Streptococcus anginosus complex, as are small-colony types of groups A, C, and G (13). The CLSI has chosen to recommend that all S. anginosus isolates, whether alpha-, beta-, or nonhemolytic, should be considered 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 CC resistant.

Furthermore, multilaboratory studies are needed to confirm and extend these initial results and to select the optimal drug concentrations for a single ERY-CC test well for the detection of inducible CC resistance in routine streptococcal test panels. This study identified two potential ERY-CC combinations that are worthy of further study. The goal would be to include a single-well screening test to detect inducible CC resistance in broth panels used for routine testing of beta-hemolytic streptococci without need for additional agar-based D-zone testing.