Vol.. 11, 1992
In Vitro Activity of Sparfloxacin and Three Other Fluoroquinolones Against Methicillin-Resistant Staphylococcus aureus and
Staphylococcus epidermidis K.R. Smith1,, C.G. C o b b s 2
Fluoroquinolones are variably active against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant StaphylococCus epidermidis (MRSE). The purpose of this Study was to test the in vitro susceptibility of 50 isolates each of MRSA and MRSE to four of the new fluoroquinolones - sparfl0xacin, ciproflOXacin, ofloxacin and norfloxacin - and to see if resistance could be induced in five susceptible Strains of each species by serial passage with inCreasing concentrations of drug. A standard microdilution technique was used to determine minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of each drug for each isolate. Agar dilution plates Were also prepared containing concentrations of drug varying from one-half of the reported MIC to 128 times the reported MIC, and microorganisms persisting were serially passaged. Initially, 98 % of the strains of MRSA were susceptible to the fluoroquinolones. MBCs were essentially identical to MICs. Similarly, 96 % of the strains of MRSE were susceptible. Following exposure to increasing concentrations of each fluoroquinolone, resistance appeared to emerge less rapidly to sparfloxaein and ofloxacin than to norfloxacin and ciprofloxacin.
Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant StaphylococCus epidermidis (MRSE) presently pose serious clinical problems in hospitalized and non-hospitalized patients. A number of the new fluorinated quinolones possess activity against these microorganisms, but resistance has emerged rather quick1
,
.
.
I31vJslonof Infectious Diseases, Department of Medicine, Universityof Alabama at Birmingham, THT 229, Univer2slty Station, Birmingham, Alabama 35294, USA. Medical Services,Veterans Administration Medical Center, 700 South t9th Street, Birmingham, Alabama 35233, USA.
55
ly. Recent reports describe the rapid emergence of resistance to ciprofloxacin among M R S A (1, 2). Sparfloxacin is a new fluoroquinolone that appears to have greater antibacterial activity against gram-positive species than some of the other new fluorinated quinolones such as ciprofloxacin, ofloxacin, norfloxacin and enoxacin (3-5). Sparfloxacin also possesses good antibacterial activity against mycobacteria and chlamydia (4). The purpose of this study was to investigate the activity of sparfloxacin against isolates of M R S A and M R S E obtained from clinical sources and to determine if stepwise resistance develops in vitro.
Materials and Methods. Fifty strains each of MRSA and MRSE were collected from the University of Alabama at Birmingham Medical Center during the time period 1982-1988. The majority of these strains were from patients hospitalized during 1987 and 1988, when significant numbers of patients with clinical M R S A and MRSE disease were encountered. Isolates were stored at -70 °C in skim milk and streaked onto a sheep blood agar plate the day prior to testing. Methicillin resistance was determined using the Kirby-Bauer oxacillin disc method (6). Standard microdilution methods were used to determine minimal inhibitory concentrations (MICs) of sparfloxacin, ciprofloxacin, norfloxacin and ofloxacin (7). For each isolate a final inoculum of 5 x 105 cfu/ml was prepared from an initial suspension, equivalent in turbidity to a 0.5 McFarland standard (-108 cfu/ml), made from 24 h growth on a blood agar plate. This suspension was diluted 1:10 in water, and 5 pl of the 107 inoculum was dispensed into each of 96 wells containing 0.1 ml of an antibiotic dilution or broth alone as the growth control. The broth used in the susceptibility testing was cation-supplemented Mueller-Hinton broth. A 0.001 ml aliquot of the growth control well was plated onto a blood agar plate immediately after inoculation to confirm colony count. The MICs were read after 24 h of incubation at 35 °C as the lowest concentration of drug preventing visible growth. Minimal bactericidal concentrations (MBCs) were determined by subculturing 10/al from each well showing no growth and, among wells with visible growth, from the well containing the highest concentration of each antibiotic. These samples were streaked onto blood agar plates and incubated for 24 h at 35 °C. The MBC was defined as the lowest concentration of an antimicrobial agent that resulted in at least a 99.9 % killing of the original inoculum. Using Pearsons' rejection
56
Eur. J. Clin. Microbiol. Infect. Dis.
value for an initial inoculum size of 5 x 105 cfu/ml, a colony count of < 11 would represent 99.9 % killing (8). Five isolates each of MRSA and MRSE which were initially susceptible were chosen for testing against increasing concentrations of the four quinolones. Overnight growth of each isolate was swabbed onto 100 mm Mueller-Hinton agar plates containing a concentration representing one-half of the MIC of each quinolone for the microorganism being tested. After overnight incubation the surface growth was transferred to Mueller-Hinton agar plates containing double the initial concentration (equal to the original MIC). This process was repeated serially, and bacteria growing from the highest concentration tested were retested. MICs and MBCs to all four agents were determined by the microdilution method as described above. Results and Discussion. Sparfloxacin was the most active of the newer quinolones against strains of MRSA and MRSE (Table 1). The MIC50 of sparfloxacin for MRSA averaged 0.06 /ag/ml and the MBC50 was identical. Against strains of MRSE, sparfloxacin was also the most active agent, both in tests of inhibition and in tests of killing. It is noteworthy that only one of our strains of MRSA was resistant to the quinolones presently available and only two strains of MRSE were resistant. These three strains of staphylococci exhibited resistance to all four of the quinolones.
observed when the isolate was tested with the other agents, even though the strain had not been induced with those agents. However, there was a smaller percentage increase in resistance to sparfloxacin than to the other quinolones (Table 3). For example, a strain of MRSA that was inhibited by 0.25 lag/ml of ciprofloxacin was induced to a susceptibility of 8 ~g/ml to that agent (32x). However, its susceptibility to sparfloxacin only changed from 0.06 to 0.12/ag/ml (2x). Sparfloxacin possesses excellent activity against MRSA and MRSE. Most bacteria were inhibited and killed at concentrations < 0.25/ag/ml. MBCs were equal to or two-fold higher than MICs in 98 % of the strains, and the MBC was never more than four-fold higher than the MIC. Compared to the results with ciprofloxacin and norfloxacin, we were unable to induce resistance to sparfloxacin or ofloxacin by stepwise passage. When resistance to ofloxacin, norfl0xacin or ciprofloxacin was induced, strains also became more resistant to the other quinolones, but the percentage of increased resistance to sparfloxacin was less.
Serial passage of five strains each of MRSA and MRSE onto agar plates with increasing concentrations of these fluoroquinolones resulted in the prompt emergence of resistance to norfloxacin and ciprofloxacin (Table 2).
A number of other investigators have examined the susceptibility of methicillin-resistant staphylococci to both the older and newer fluoroquinolones. Simor et al. (9) included MRSA in a study of sparfloxacin and other antimicrobial agents against respiratory isolates. They examined 160 strains of MRSA and found all were susceptible to
In Vitro Activity of Sparfloxacin and Three Other Fluoroquinolones Against Methicillin-Resistant Staphylococcus aureus and
Staphylococcus epidermidis K.R. Smith1,, C.G. C o b b s 2
Fluoroquinolones are variably active against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant StaphylococCus epidermidis (MRSE). The purpose of this Study was to test the in vitro susceptibility of 50 isolates each of MRSA and MRSE to four of the new fluoroquinolones - sparfl0xacin, ciproflOXacin, ofloxacin and norfloxacin - and to see if resistance could be induced in five susceptible Strains of each species by serial passage with inCreasing concentrations of drug. A standard microdilution technique was used to determine minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of each drug for each isolate. Agar dilution plates Were also prepared containing concentrations of drug varying from one-half of the reported MIC to 128 times the reported MIC, and microorganisms persisting were serially passaged. Initially, 98 % of the strains of MRSA were susceptible to the fluoroquinolones. MBCs were essentially identical to MICs. Similarly, 96 % of the strains of MRSE were susceptible. Following exposure to increasing concentrations of each fluoroquinolone, resistance appeared to emerge less rapidly to sparfloxaein and ofloxacin than to norfloxacin and ciprofloxacin.
Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant StaphylococCus epidermidis (MRSE) presently pose serious clinical problems in hospitalized and non-hospitalized patients. A number of the new fluorinated quinolones possess activity against these microorganisms, but resistance has emerged rather quick1
,
.
.
I31vJslonof Infectious Diseases, Department of Medicine, Universityof Alabama at Birmingham, THT 229, Univer2slty Station, Birmingham, Alabama 35294, USA. Medical Services,Veterans Administration Medical Center, 700 South t9th Street, Birmingham, Alabama 35233, USA.
55
ly. Recent reports describe the rapid emergence of resistance to ciprofloxacin among M R S A (1, 2). Sparfloxacin is a new fluoroquinolone that appears to have greater antibacterial activity against gram-positive species than some of the other new fluorinated quinolones such as ciprofloxacin, ofloxacin, norfloxacin and enoxacin (3-5). Sparfloxacin also possesses good antibacterial activity against mycobacteria and chlamydia (4). The purpose of this study was to investigate the activity of sparfloxacin against isolates of M R S A and M R S E obtained from clinical sources and to determine if stepwise resistance develops in vitro.
Materials and Methods. Fifty strains each of MRSA and MRSE were collected from the University of Alabama at Birmingham Medical Center during the time period 1982-1988. The majority of these strains were from patients hospitalized during 1987 and 1988, when significant numbers of patients with clinical M R S A and MRSE disease were encountered. Isolates were stored at -70 °C in skim milk and streaked onto a sheep blood agar plate the day prior to testing. Methicillin resistance was determined using the Kirby-Bauer oxacillin disc method (6). Standard microdilution methods were used to determine minimal inhibitory concentrations (MICs) of sparfloxacin, ciprofloxacin, norfloxacin and ofloxacin (7). For each isolate a final inoculum of 5 x 105 cfu/ml was prepared from an initial suspension, equivalent in turbidity to a 0.5 McFarland standard (-108 cfu/ml), made from 24 h growth on a blood agar plate. This suspension was diluted 1:10 in water, and 5 pl of the 107 inoculum was dispensed into each of 96 wells containing 0.1 ml of an antibiotic dilution or broth alone as the growth control. The broth used in the susceptibility testing was cation-supplemented Mueller-Hinton broth. A 0.001 ml aliquot of the growth control well was plated onto a blood agar plate immediately after inoculation to confirm colony count. The MICs were read after 24 h of incubation at 35 °C as the lowest concentration of drug preventing visible growth. Minimal bactericidal concentrations (MBCs) were determined by subculturing 10/al from each well showing no growth and, among wells with visible growth, from the well containing the highest concentration of each antibiotic. These samples were streaked onto blood agar plates and incubated for 24 h at 35 °C. The MBC was defined as the lowest concentration of an antimicrobial agent that resulted in at least a 99.9 % killing of the original inoculum. Using Pearsons' rejection
56
Eur. J. Clin. Microbiol. Infect. Dis.
value for an initial inoculum size of 5 x 105 cfu/ml, a colony count of < 11 would represent 99.9 % killing (8). Five isolates each of MRSA and MRSE which were initially susceptible were chosen for testing against increasing concentrations of the four quinolones. Overnight growth of each isolate was swabbed onto 100 mm Mueller-Hinton agar plates containing a concentration representing one-half of the MIC of each quinolone for the microorganism being tested. After overnight incubation the surface growth was transferred to Mueller-Hinton agar plates containing double the initial concentration (equal to the original MIC). This process was repeated serially, and bacteria growing from the highest concentration tested were retested. MICs and MBCs to all four agents were determined by the microdilution method as described above. Results and Discussion. Sparfloxacin was the most active of the newer quinolones against strains of MRSA and MRSE (Table 1). The MIC50 of sparfloxacin for MRSA averaged 0.06 /ag/ml and the MBC50 was identical. Against strains of MRSE, sparfloxacin was also the most active agent, both in tests of inhibition and in tests of killing. It is noteworthy that only one of our strains of MRSA was resistant to the quinolones presently available and only two strains of MRSE were resistant. These three strains of staphylococci exhibited resistance to all four of the quinolones.
observed when the isolate was tested with the other agents, even though the strain had not been induced with those agents. However, there was a smaller percentage increase in resistance to sparfloxacin than to the other quinolones (Table 3). For example, a strain of MRSA that was inhibited by 0.25 lag/ml of ciprofloxacin was induced to a susceptibility of 8 ~g/ml to that agent (32x). However, its susceptibility to sparfloxacin only changed from 0.06 to 0.12/ag/ml (2x). Sparfloxacin possesses excellent activity against MRSA and MRSE. Most bacteria were inhibited and killed at concentrations < 0.25/ag/ml. MBCs were equal to or two-fold higher than MICs in 98 % of the strains, and the MBC was never more than four-fold higher than the MIC. Compared to the results with ciprofloxacin and norfloxacin, we were unable to induce resistance to sparfloxacin or ofloxacin by stepwise passage. When resistance to ofloxacin, norfl0xacin or ciprofloxacin was induced, strains also became more resistant to the other quinolones, but the percentage of increased resistance to sparfloxacin was less.
Serial passage of five strains each of MRSA and MRSE onto agar plates with increasing concentrations of these fluoroquinolones resulted in the prompt emergence of resistance to norfloxacin and ciprofloxacin (Table 2).
A number of other investigators have examined the susceptibility of methicillin-resistant staphylococci to both the older and newer fluoroquinolones. Simor et al. (9) included MRSA in a study of sparfloxacin and other antimicrobial agents against respiratory isolates. They examined 160 strains of MRSA and found all were susceptible to
