ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, OCt. 1975, p. 444-452 Copyright 0 1975 American Society for Microbiology
Vol. 8, No. 4 Printed in U.S.A.
Simplified Method for Antimicrobial Susceptibility Testing of Anaerobic Bacteria ROBERT J. FASS,* RICHARD B. PRIOR,
AND
CAROL A. ROTILIE
Division of Infectious Diseases, Department of Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210
Received for publication 30 May 1975
A simple, abbreviated broth dilution test (tube test) utilizing a commercially available medium and inexpensive disposable materials, and which could be performed entirely in room air, was developed and used to test the susceptibility of 100 strains of anaerobic bacteria to clindamycin, chloramphenicol, ampicillin, and tetracycline. Results are reported in categories of susceptibility: susceptible to concentrations surpassed in vivo with usual dosage, susceptible to concentrations surpassed in vivo with high dosage, and resistant to concentrations achievable in vivo. Results are compared to minimal inhibitory concentrations which were determined simultaneously by using a microdilution method in an anaerobic glove box. Twenty strains of Bacteroides fragilis, 10 strains of Fusobacterium, 20 strains of Clostridium, 10 strains of gram-positive nonsporeforming bacilli, and 30 strains of cocci grew to visible turbidity within 1 day of incubation. Of the 360 antibiotic-organism combinations tested, 98% were in a susceptibility category that corresponded (within one concentration) to the actual minimal inhibitory concentration as determined by the microdilution method. After 2 days of incubation, growth was more abundant, but results often indicated inappropriate degrees of resistance. Variation in inoculum size had little effect on results. Ten strains of B. melaninogenicus did not grow enough for susceptibility to be categorized accurately. The tube test could be used in any clinical microbiology laboratory for a limited number of susceptibility tests on anaerobic bacteria other than B. melaninogenicus without preparation of special media or purchase of special equipment.
Antimicrobial susceptibility testing of anaerobic bacteria is usually done by agar or broth dilution methods (16). Disk diffusion methods (2, 3, 6, 8, 11, 14, 15, 17, 19), analogous to that described by Bauer et al. (1) for rapidly growing aerobic and facultative bacteria, have also been developed in attempts to simplify and standardize the approach to antimicrobial susceptibility testing of anaerobes. These methods have been limited in their usefulness, however, since a significant percentage of clinical isolates did not grow or grew insufficiently for zones of inhibition to be determined. For those that did grow, extreme variations in growth rates required that separate linear regression plots of minimal inhibitory concentrations (MICs) versus zone diameters be used for different organisms. In addition, there was much greater variation about the regression lines than occurs with aerobic tests, and accurate separation into the categories susceptible, intermediate, and resistant, based on zones of inhibition, was not consistently achieved with statistical validity (7). 444
A modified broth dilution method, the "broth-disk" method, has been proposed by Wilkins and Thiel (20), which provides a simple method for testing all anaerobes including slowgrowing and very oxygen-sensitive strains that could not be tested by disk diffusion techniques. A concentration of antibiotic approximately equal to that achievable in blood is put into a tube of broth medium, with commercial filter paper disks as the carrier of the antibiotic. Growth at that concentration is considered to constitute resistance. Ninety-seven percent of the strains tested by Wilkins and Thiel grew sufficiently for readable results, but the method requires the use of a completely anaerobic environment, and there is considerable variation (73 to 148% of the labeled values in their tests) in the actual antibiotic concentrations delivered by the filter paper disks. A modified broth dilution method has also been proposed by Stalons and Thornsberry (12), which simplifies the susceptibility testing of anaerobic bacteria. Selected concentrations of antibiotics are used so that results are divided
VOL. 8, 1975
SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA
into three categories of susceptibility. Although the method utilizes a commercially available medium, Schaedler broth (BBL, Cockeysville, Md.), and a reduced number of tubes for each susceptibility test performed, it requires the use of an anaerobic glove box or incubation in a GasPak (BBL) system. The present report deals with'another modified broth dilution method which will be referred to as the "tube test." As in the methods of Wilkins and Thiel (20) and Stalons and Thornsberry (12), results are reported in categories of susceptibility and are compared with MICs. The tube test is unique for several reasons. Antibiotic dispensing and inoculum preparation are simplified. All materials are inexpensive, disposable and, like the medium, commercially available. All manipulations including incubation are performed in room air so that an anaerobic glove box, GasPak jar, or other special equipment necessary to provide an anaerobic environment is not required. MATERIALS AND METHODS Bacteria. One hundred strains of anaerobic bacteria were tested. Seventy-eight were clinical isolates from patients in University Hospital, Columbus, Ohio. Twenty-two were stock cultures identified and provided by the Center for Disease Control, Atlanta, Ga. The anaerobes were grouped into the following categories: Bacteroides fragilis (20 strains), B. melaninogenicus (10 strains), Fusobacterium (10 strains), Clostridium (10 strains of C. perfringens and 10 strains of other Clostridium species), gram-positive nonsporeforming bacilli (10 strains including Propionibacterium, Eubacterium, and Bifidobacterium), and cocci (30 strains including Peptococcus, Peptostreptococcus, and Veillonella). Cultures in Schaedler broth (BBL) with 10% glycerol were maintained frozen in liquid nitrogen and were subcultured onto anaerobically stored 5% sheep blood agar plates prior to use. Antibiotics. Commercially available laboratory standards of clindamycin, chloramphenicol, ampicillin, and tetracycline were dissolved in sterile, distilled water. For the microdilution susceptibility tests, working solutions were prepared by dilution to concentrations of 100 ,g/ml. For the tube tests, working solutions were prepared by dilution to concentrations of 306 ug/ml for clindamycin and chloramphenicol, 306 and 1,224 ;g/ml for ampicillin, and 153 ug/ml for tetracycline. The working solutions were stored for a maximum of 1 month at -20 C in sterile, plastic Microdel bottles (Cooke Laboratory Products, Alexandria, Va.); a fresh set of bottles was thawed for each run of tests. Media. Schaedler broth was used for all tests. For the microdilution tests, it was autoclaved and then stored for up to 5 days in an anaerobic glove box (Coy Manufacturing, Ann Arbor, Mich.) that contained 80% nitrogen, 10% hydrogen, and 10% carbon dioxide at 75 to 85% relative humidity. For the tube test, flint
445
glass tubes (13 by 100 mm) with rubber-lined screw caps (Scientific Products, McGaw Park, Ill.), each containing one 6-mm solid glass bead, were filled with Schaedler broth containing 0.0001% resazurin and then autoclaved. The tubes were stored at room temperature for up to 48 h and were not used if the medium turned pink due to oxidation of the resazurin. The final pH of the media was 7.2. For both tests, heat-inactivated (56 C for 0.5 h) horse serum was added to a final concentration of 1% immediately before the testing of B. melaninogenicus, Fusobacterium, and cocci. MICs. MICs were determined in an anerobic glove box by a microdilution method (10). Each of the four antibiotics studied was added to the first two wells of two horizontal rows in the microdilution plate, and serial twofold dilutions were made from the second set of wells. For inoculum preparation, colonies of each test strain were subcultured from 48-h 5% sheep blood agar plates to 5 ml of Schaedler broth and incubated at 35 C in a glove box overnight. For faintly turbid suspensions (optical density approximately 0.1 to 0.3 at 650 nm), a 1:10 dilution was used; for suspensions that were more turbid, a 1:100 dilution was used. The final inocula ranged from 105 to 107 colony-forming units (CFU) per ml. The final antibiotic concentrations ranged from 50 to 0.05 Ag ml. Plates were sealed with cellophane tape (Cooke Laboratory Products, Alexandria, Va.) to prevent evaporation and were incubated at 35 C in the glove box. The humidity in the incubator was kept at 65 to 75% to further reduce evaporation from the plates. MICs were read at 20 to 22 h (1 day) and 44 to 46 h (2 days) as the lowest concentrations of antibiotics that inhibited visible growth as judged visually with the aid of a test-reading mirror (Cooke Laboratory Products, Alexandria, Va.). When the duplicate tests varied by one concentration, the higher concentration was considered to be the MIC. When they varied by more than one concentration the test was repeated. A strain of B. fragilis with known MICs was tested each day and served as a system control. Tube test. The tube test was performed simultaneously with the microdilution test for each organism. In room air, the glass tubes were uncapped. One to eight drops (0.05 ml/drop) of antibiotic were added to tubes with the Microdel bottles so that for each organism being tested there was a tube with a high and a low concentration of each of the four antibiotics. The concentrations in the Microdel bottles were calculated so that: 1 and 4 drops of clindamycin (306 jg/ml) resulted in final tube concentrations of 1.6 and 6.2 jg/ml, respectively; 1 and 8 drops of chloramphenicol (306 jg/ml) resulted in final tube concentrations of 1.6 and 12.5 Mg/ml, respectively; 1 drop of ampicillin (306 Mg/ml) and 4 drops of ampicillin (1,224 Ag/ml) resulted in final tube concentrations of 1.6 and 25 Ag/ml, respectively; and 1 and 8 drops of tetracycline (153 Mg/ml) resulted in final tube concentrations of 0.8 and 6.2 ug/ml, respectively. (These calculations were based on a tube volume of 9.2 [+0.24 standard deviation I ml and took into consideration the - 5.35% [+2.7 standard deviation] error of the Microdel bottles.) For tests requiring the use of horse serum, two drops (0.05 ml/drop, with a -3.86% [±4.5 standard
446
ANTIMICROB. AGENTS CHEMOTHER.
FASS, PRIOR, AND ROTILIE
deviation J error) were added with a disposable pipette (Cooke Laboratory Products, Alexandria, Va.) at the same time as the antibiotics. The tube caps were replaced and the tube contents were mixed by inversion. The caps were again removed, and 1 drop (0.05 ml) of undiluted inoculum was added with a disposable pipette. This resulted in a final inoculum that was 110% of that used in the microdilution method when a 1:100 dilution was employed and 11% of that used in the microdilution test when a 1:10 dilution was employed. If any space remained in the tubes after the addition of antibiotic, horse serum, and inoculum, Schaedler broth was added to fill the tubes completely and the caps were replaced tightly. During the two brief exposures to room air, the media in the tubes became slightly pink as the resazurin was oxidized. Within 15 min after the caps were retightened, the indicator became colorless as it was reduced in the Schaedler broth. With each set of eight antibioticcontaining tubes, a ninth tube without antibiotic was similarly inoculated with the organism being tested and served as a growth control. The nine inoculated tubes for each test organism and an uninoculated sterility control tube were then incubated in a standard incubator at 35 C. After 20 to 22 h (1 day) and 44 to 46 h (2 days) of incubation, tubes were observed for turbidity and were read as "growth" or "no growth." Inhibition of growth by both concentrations of a given antibiotic was interpreted as indicating susceptibility to concentrations easily surpassed in vivo with "usual" dosage. Inhibition by only "high" concentration of a given antibiotic was interpreted as indicating susceptibility to concentrations surpassed in vivo with the high dosage. Lack of inhibition by both antibiotic concentrations was interpreted as indicating resistance to concentrations achievable in vivo. The breakpoints for the three categories of susceptibility for each of the four antibiotics used are summarized in Table 1. Inoculum effect. The microdilution and tube tests were performed simultaneously with 10-fold variations in inoculum size. One strain each of B. fragilis, C. perfringens, and Veillonella and two strains of B. melaninogenicus were used. For comparison of results obtained with the two tests, a 1:100 dilution of the inoculum for the microdilution test was considered to be equivalent to an undiluted inoculum for the tube test.
RESULTS
Microdilution method. The 2-day MICs of the four antibiotics tested against the 100 anaerobic strains are shown in Fig. 1. Clindamycin inhibited 97% of the strains at concentrations of 1.6 Mg or less per ml and 99% of the strains at concentrations of 6.2 ,g or less per ml. Chloramphenicol inhibited 33% of the strains at concentrations of 1.6 Mg or less per ml and 99% of the strains at concentrations of 12.5 Mg or less per ml. Ampicillin inhibited 99% of the strains other than Bacteroides at concentrations of 1.6 ,Mg or less per ml; concentrations of 25 Ag or less
TABLE 1. Categories of susceptibility as defined by MICsa MIC (yg/ml) Antibiotic
Clindamycin Chloramphenicol Ampicillin Tetracycline
SS
S
R
< 1.6 < 1.6 < 1.6
> 1.6-6.2 > 1.6-12.5 > 1.6-25 >0.8-6.2
>6.2 > 12.5 > 25 >6.2
Vol. 8, No. 4 Printed in U.S.A.
Simplified Method for Antimicrobial Susceptibility Testing of Anaerobic Bacteria ROBERT J. FASS,* RICHARD B. PRIOR,
AND
CAROL A. ROTILIE
Division of Infectious Diseases, Department of Medicine, The Ohio State University College of Medicine, Columbus, Ohio 43210
Received for publication 30 May 1975
A simple, abbreviated broth dilution test (tube test) utilizing a commercially available medium and inexpensive disposable materials, and which could be performed entirely in room air, was developed and used to test the susceptibility of 100 strains of anaerobic bacteria to clindamycin, chloramphenicol, ampicillin, and tetracycline. Results are reported in categories of susceptibility: susceptible to concentrations surpassed in vivo with usual dosage, susceptible to concentrations surpassed in vivo with high dosage, and resistant to concentrations achievable in vivo. Results are compared to minimal inhibitory concentrations which were determined simultaneously by using a microdilution method in an anaerobic glove box. Twenty strains of Bacteroides fragilis, 10 strains of Fusobacterium, 20 strains of Clostridium, 10 strains of gram-positive nonsporeforming bacilli, and 30 strains of cocci grew to visible turbidity within 1 day of incubation. Of the 360 antibiotic-organism combinations tested, 98% were in a susceptibility category that corresponded (within one concentration) to the actual minimal inhibitory concentration as determined by the microdilution method. After 2 days of incubation, growth was more abundant, but results often indicated inappropriate degrees of resistance. Variation in inoculum size had little effect on results. Ten strains of B. melaninogenicus did not grow enough for susceptibility to be categorized accurately. The tube test could be used in any clinical microbiology laboratory for a limited number of susceptibility tests on anaerobic bacteria other than B. melaninogenicus without preparation of special media or purchase of special equipment.
Antimicrobial susceptibility testing of anaerobic bacteria is usually done by agar or broth dilution methods (16). Disk diffusion methods (2, 3, 6, 8, 11, 14, 15, 17, 19), analogous to that described by Bauer et al. (1) for rapidly growing aerobic and facultative bacteria, have also been developed in attempts to simplify and standardize the approach to antimicrobial susceptibility testing of anaerobes. These methods have been limited in their usefulness, however, since a significant percentage of clinical isolates did not grow or grew insufficiently for zones of inhibition to be determined. For those that did grow, extreme variations in growth rates required that separate linear regression plots of minimal inhibitory concentrations (MICs) versus zone diameters be used for different organisms. In addition, there was much greater variation about the regression lines than occurs with aerobic tests, and accurate separation into the categories susceptible, intermediate, and resistant, based on zones of inhibition, was not consistently achieved with statistical validity (7). 444
A modified broth dilution method, the "broth-disk" method, has been proposed by Wilkins and Thiel (20), which provides a simple method for testing all anaerobes including slowgrowing and very oxygen-sensitive strains that could not be tested by disk diffusion techniques. A concentration of antibiotic approximately equal to that achievable in blood is put into a tube of broth medium, with commercial filter paper disks as the carrier of the antibiotic. Growth at that concentration is considered to constitute resistance. Ninety-seven percent of the strains tested by Wilkins and Thiel grew sufficiently for readable results, but the method requires the use of a completely anaerobic environment, and there is considerable variation (73 to 148% of the labeled values in their tests) in the actual antibiotic concentrations delivered by the filter paper disks. A modified broth dilution method has also been proposed by Stalons and Thornsberry (12), which simplifies the susceptibility testing of anaerobic bacteria. Selected concentrations of antibiotics are used so that results are divided
VOL. 8, 1975
SUSCEPTIBILITY TESTING OF ANAEROBIC BACTERIA
into three categories of susceptibility. Although the method utilizes a commercially available medium, Schaedler broth (BBL, Cockeysville, Md.), and a reduced number of tubes for each susceptibility test performed, it requires the use of an anaerobic glove box or incubation in a GasPak (BBL) system. The present report deals with'another modified broth dilution method which will be referred to as the "tube test." As in the methods of Wilkins and Thiel (20) and Stalons and Thornsberry (12), results are reported in categories of susceptibility and are compared with MICs. The tube test is unique for several reasons. Antibiotic dispensing and inoculum preparation are simplified. All materials are inexpensive, disposable and, like the medium, commercially available. All manipulations including incubation are performed in room air so that an anaerobic glove box, GasPak jar, or other special equipment necessary to provide an anaerobic environment is not required. MATERIALS AND METHODS Bacteria. One hundred strains of anaerobic bacteria were tested. Seventy-eight were clinical isolates from patients in University Hospital, Columbus, Ohio. Twenty-two were stock cultures identified and provided by the Center for Disease Control, Atlanta, Ga. The anaerobes were grouped into the following categories: Bacteroides fragilis (20 strains), B. melaninogenicus (10 strains), Fusobacterium (10 strains), Clostridium (10 strains of C. perfringens and 10 strains of other Clostridium species), gram-positive nonsporeforming bacilli (10 strains including Propionibacterium, Eubacterium, and Bifidobacterium), and cocci (30 strains including Peptococcus, Peptostreptococcus, and Veillonella). Cultures in Schaedler broth (BBL) with 10% glycerol were maintained frozen in liquid nitrogen and were subcultured onto anaerobically stored 5% sheep blood agar plates prior to use. Antibiotics. Commercially available laboratory standards of clindamycin, chloramphenicol, ampicillin, and tetracycline were dissolved in sterile, distilled water. For the microdilution susceptibility tests, working solutions were prepared by dilution to concentrations of 100 ,g/ml. For the tube tests, working solutions were prepared by dilution to concentrations of 306 ug/ml for clindamycin and chloramphenicol, 306 and 1,224 ;g/ml for ampicillin, and 153 ug/ml for tetracycline. The working solutions were stored for a maximum of 1 month at -20 C in sterile, plastic Microdel bottles (Cooke Laboratory Products, Alexandria, Va.); a fresh set of bottles was thawed for each run of tests. Media. Schaedler broth was used for all tests. For the microdilution tests, it was autoclaved and then stored for up to 5 days in an anaerobic glove box (Coy Manufacturing, Ann Arbor, Mich.) that contained 80% nitrogen, 10% hydrogen, and 10% carbon dioxide at 75 to 85% relative humidity. For the tube test, flint
445
glass tubes (13 by 100 mm) with rubber-lined screw caps (Scientific Products, McGaw Park, Ill.), each containing one 6-mm solid glass bead, were filled with Schaedler broth containing 0.0001% resazurin and then autoclaved. The tubes were stored at room temperature for up to 48 h and were not used if the medium turned pink due to oxidation of the resazurin. The final pH of the media was 7.2. For both tests, heat-inactivated (56 C for 0.5 h) horse serum was added to a final concentration of 1% immediately before the testing of B. melaninogenicus, Fusobacterium, and cocci. MICs. MICs were determined in an anerobic glove box by a microdilution method (10). Each of the four antibiotics studied was added to the first two wells of two horizontal rows in the microdilution plate, and serial twofold dilutions were made from the second set of wells. For inoculum preparation, colonies of each test strain were subcultured from 48-h 5% sheep blood agar plates to 5 ml of Schaedler broth and incubated at 35 C in a glove box overnight. For faintly turbid suspensions (optical density approximately 0.1 to 0.3 at 650 nm), a 1:10 dilution was used; for suspensions that were more turbid, a 1:100 dilution was used. The final inocula ranged from 105 to 107 colony-forming units (CFU) per ml. The final antibiotic concentrations ranged from 50 to 0.05 Ag ml. Plates were sealed with cellophane tape (Cooke Laboratory Products, Alexandria, Va.) to prevent evaporation and were incubated at 35 C in the glove box. The humidity in the incubator was kept at 65 to 75% to further reduce evaporation from the plates. MICs were read at 20 to 22 h (1 day) and 44 to 46 h (2 days) as the lowest concentrations of antibiotics that inhibited visible growth as judged visually with the aid of a test-reading mirror (Cooke Laboratory Products, Alexandria, Va.). When the duplicate tests varied by one concentration, the higher concentration was considered to be the MIC. When they varied by more than one concentration the test was repeated. A strain of B. fragilis with known MICs was tested each day and served as a system control. Tube test. The tube test was performed simultaneously with the microdilution test for each organism. In room air, the glass tubes were uncapped. One to eight drops (0.05 ml/drop) of antibiotic were added to tubes with the Microdel bottles so that for each organism being tested there was a tube with a high and a low concentration of each of the four antibiotics. The concentrations in the Microdel bottles were calculated so that: 1 and 4 drops of clindamycin (306 jg/ml) resulted in final tube concentrations of 1.6 and 6.2 jg/ml, respectively; 1 and 8 drops of chloramphenicol (306 jg/ml) resulted in final tube concentrations of 1.6 and 12.5 Mg/ml, respectively; 1 drop of ampicillin (306 Mg/ml) and 4 drops of ampicillin (1,224 Ag/ml) resulted in final tube concentrations of 1.6 and 25 Ag/ml, respectively; and 1 and 8 drops of tetracycline (153 Mg/ml) resulted in final tube concentrations of 0.8 and 6.2 ug/ml, respectively. (These calculations were based on a tube volume of 9.2 [+0.24 standard deviation I ml and took into consideration the - 5.35% [+2.7 standard deviation] error of the Microdel bottles.) For tests requiring the use of horse serum, two drops (0.05 ml/drop, with a -3.86% [±4.5 standard
446
ANTIMICROB. AGENTS CHEMOTHER.
FASS, PRIOR, AND ROTILIE
deviation J error) were added with a disposable pipette (Cooke Laboratory Products, Alexandria, Va.) at the same time as the antibiotics. The tube caps were replaced and the tube contents were mixed by inversion. The caps were again removed, and 1 drop (0.05 ml) of undiluted inoculum was added with a disposable pipette. This resulted in a final inoculum that was 110% of that used in the microdilution method when a 1:100 dilution was employed and 11% of that used in the microdilution test when a 1:10 dilution was employed. If any space remained in the tubes after the addition of antibiotic, horse serum, and inoculum, Schaedler broth was added to fill the tubes completely and the caps were replaced tightly. During the two brief exposures to room air, the media in the tubes became slightly pink as the resazurin was oxidized. Within 15 min after the caps were retightened, the indicator became colorless as it was reduced in the Schaedler broth. With each set of eight antibioticcontaining tubes, a ninth tube without antibiotic was similarly inoculated with the organism being tested and served as a growth control. The nine inoculated tubes for each test organism and an uninoculated sterility control tube were then incubated in a standard incubator at 35 C. After 20 to 22 h (1 day) and 44 to 46 h (2 days) of incubation, tubes were observed for turbidity and were read as "growth" or "no growth." Inhibition of growth by both concentrations of a given antibiotic was interpreted as indicating susceptibility to concentrations easily surpassed in vivo with "usual" dosage. Inhibition by only "high" concentration of a given antibiotic was interpreted as indicating susceptibility to concentrations surpassed in vivo with the high dosage. Lack of inhibition by both antibiotic concentrations was interpreted as indicating resistance to concentrations achievable in vivo. The breakpoints for the three categories of susceptibility for each of the four antibiotics used are summarized in Table 1. Inoculum effect. The microdilution and tube tests were performed simultaneously with 10-fold variations in inoculum size. One strain each of B. fragilis, C. perfringens, and Veillonella and two strains of B. melaninogenicus were used. For comparison of results obtained with the two tests, a 1:100 dilution of the inoculum for the microdilution test was considered to be equivalent to an undiluted inoculum for the tube test.
RESULTS
Microdilution method. The 2-day MICs of the four antibiotics tested against the 100 anaerobic strains are shown in Fig. 1. Clindamycin inhibited 97% of the strains at concentrations of 1.6 Mg or less per ml and 99% of the strains at concentrations of 6.2 ,g or less per ml. Chloramphenicol inhibited 33% of the strains at concentrations of 1.6 Mg or less per ml and 99% of the strains at concentrations of 12.5 Mg or less per ml. Ampicillin inhibited 99% of the strains other than Bacteroides at concentrations of 1.6 ,Mg or less per ml; concentrations of 25 Ag or less
TABLE 1. Categories of susceptibility as defined by MICsa MIC (yg/ml) Antibiotic
Clindamycin Chloramphenicol Ampicillin Tetracycline
SS
S
R
< 1.6 < 1.6 < 1.6
> 1.6-6.2 > 1.6-12.5 > 1.6-25 >0.8-6.2
>6.2 > 12.5 > 25 >6.2
