Vol. 11, 1992
13. Edelstein PH, Edelstein MAC: In vitro activity of azithromycin against clinical isolates of Legionella species. Antimicrobial Agents and Chemotherapy 1991, 35: 180-181. 14. Farrell ID, Barker J, Chiodini PL, Hutchison JGP, Geddes AM: The activityof imipenem on Legionella pneumophila with a note on the treatment of two cases. Journal of Antimicrobial Chemotherapy1985, 16: 6165. 15. Fitzgeorge RB, Featherstone ASR, BaskerviUe A: Efficacy of azithromycinin the treatment of guinea pigs infectedwith Legionellapneumophila by aerosol.Journal of Antimicrobial Chemotherapy 1990, 25, Supplement A: 101-108. 16. Pfaller M, Hollis R, Johnson W, Massanari RM, Helms C, WenzeiR, Hall N, Moyer N, July J: The application of molecularand immunologictechniques to study the epidemiologyof Legionelta pnel~wophila sero Group 1. Diagnostic Microbiology and Infectious Disease 1989; 12: 295-302.
In Vitro Antimicrobial Activity of the New Antibiotic Vermisporin N.X. Chin 1, H.C. Neu 1'2.
The antimicrobial activity of vermisporin, a new antibiotic produced by fermentation of the fungus Ophiobolus vermisporis, was tested in vitro. Vermisporin inhibited 90 % of Bacteroides fragiIis and otherBacteroides spp. at I ~g/ml (range 0.251 ~g/ml). Clostridium perfringens were inhibited by 1 gg/mi (range 0.25-2 pg/ml). Vermisporin inhibited 90 % of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, at 0.5 ~g/ml (range 0.12-0.5 ~g/ml). Vermisporin MICs for group A, B, C, F and G streptococci were < 1 ~g/ml when tested in Haemophilus Test Medium but >_8 pg/ml in the presence of blood. Vermisporin MICs for Enterobacteriaceae, Pseudomonas aeruginosa and Haemophilus influenzae exceeded 64 ~tg/ml. Inhibited organisms had MBCs 16- to 32-fold above the MICs.
There is continued need to find new antimicrobial agents that inhibit gram-positive bacteria and anaerobic species. Vermisporin is a new antibiotic i Department of Medicineand 2Departmentof Pharmacology,Collegeof Physicians& Surgeons,ColumbiaUniversity, 630 West i68th Street, New York, New York 10032, USA.
755
produced by the fermentation of the fungus Ophiobolus vermisporis. It contains an N-methyltetramic acid and a dehydrodecaline skeleton with the molecular formula C25H37NO4. In preliminary tests it has been reported to have anti-anaerobic activity (I. Sato et al., 30th ICAAC, 1990, Abstract no. 433). We investigated the activity of vermisporin and the effect of growth conditions on its activity.
Materials and Methods. Vermisporin was provided by Meiji Seika Kaisha, Tokyo, Japan. Bacterial isolates tested were obtained from patients hospitalized at the Columbia-Presbyterian Medical Center in New York City. Isolates were identified by standard methods. Antimicrobial susceptibility was determined by an agar dilution method using Mueller-Hinton agar (Baltimore Biologicals, USA) for staphylococci and anaerobic gram-negative bacteria (1). Susceptibility of streptococci was determined using Mueller-Hinton agar supplemented with 5 % lysed sheep blood and using Haemophilus Test Medium (2). Susceptibility of anaerobes was determined using Wilkins-Chalgren medium (Difco Laboratories, USA) as recommended by the National Committee for Clinical Laboratory Standards (3). A replicating spot device was used to apply inocula prepared from fresh overnight broth cultures at a strength of 104 for aerobic organisms and 105 cfu for anaerobic species. Broth dilutions were carried out with 5 x 105 cfu in 1 ml tubes. Aerobic bacteria were incubated at 35 *C for 18-20 h, and anaerobic bacteria for 48 h. The MIC was defined as the lowest concentration which inhibited visible growth on agar or in tubes. The MBC was determined by plating 0.01 ml from tubes which showed no visible growth onto antibiotic-free agar plates, and then reading the plates after incubation for 24 h at 35 °C. The MBC was defined as the concentration at which there was a 99.9 % reduction in the colony count compared with the original inoculum according to the method of Pearson et al. (4). Control strains in each assay were the respective American Type Culture strains.
Results and Discussion. The activity of vermisporin against 115 anaerobic organisms is shown in Table 1. Vermisporin inhibited 90 % of the isolates at 1 pg/ml which included various Bacteroides, Clostridium and Propionibacterium species. As Table 2 demonstrates, vermisporin inhibited Staphylococcus aureus including methicillin-resistant isolates, hemolytic streptococci and Streptococcus pneumoniae at < 1 ~tglml. Suscep-
756
Eur. J. Clin. Microbiol. Infect. Dis.
Table 1: In vitro activity of vemaisporin against anaerobic bacteria. Organism
Number tested
MIC (pg/ml) Range
Bacteroidesfraragilis Bacteroides thetaiotaornicron Clostridiurn perfringens Clostridium difficile Peptostreptococci
Bacteroides bivius Bacteroides distasonis Bacteroides melaninogenicus Bacteroides ovatus CIostridium ramosum Clostridium sordellii Clostridium sphenoides Clostridium subterminale Propionibacterium aches Eubacterium spp.
41 11 26 20 14 1 3 4 2
MIC50
0.25-1 0.5 - 2 0.12- 2 0.12- 2 0.25 - 2 0.25 0.06, 0.25,1 0.12, 0.25, 0.25,1 0.25,1
2
1, 1
3
0.25, 0.25;0.5
1
1
1 5 1
0.5 0.12-1 1
MIC90
0.5 1 1 1 1
0.25
Table 2: In vitro activity of vermisporin against gram-positive bacteria. MICs were determined on Haemophilus Test Medium for streptococcal species. Organism
Number tested
MIC (pg/ml) Range
Staphylococcus aureus
MIC50
MIC90
16
0.12-0.5
0.12
0.5
16
0.12- 0.5
0.25
0.5
14
0.25 - 1
0.25
0.5
16
0.12- 0.5
0.25
0.5
Streptococcus pneumoniae
15
0.06 -0.5
0.5
0.5
Streptococcus pyogenes
18
0.25 - 1
1
1
Streptococcus agalactiae
13
0.25-2
1
2
Group C streptococci
10
0.12-1
0.25
1
Group F streptococci
7
1
1
1
Group G streptococci
12
0.25 - 1
0.5
1
methicilIin-susceptible
Staphylococcus aureus methicillin-resistant
Staphylococcus epidermidis methicillin-susceptible
Staphylococcus epidermidis methicillin-resistant
tibility o f the streptococcal species was determined in H a e m o p h i l u s Test Medium. W h e n tests were p e r f o r m e d using Mueller-Hinton agar containing 5 % sheep blood the MICs were 8 }ag/ml, as shown in Table 3. Vermisporin, irrespective of the medium used for the assay, did not inhibit the
aerobic gram-negative species (10 isolates of each) Escherichia coli, Klebsietla pneumoniae,
Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Pseudomonas aeruginosa and Haemophilus influenzae, the MICs being > 64 pg/ml.
Vo1.11,1992
757
Table 3: In vitro aetivity of vermisporin against beta-hemolytic streptococci in Mueller-Hinton (MH) blood agar (Mueller-Hinton medium plus 5 % sheep blood) and in Haemophilus Test Medium (HTM) agar. Organism
(n)
Streptococcuspyogenes
Medium
MIC 0ag/ml) Range
MICS0
MIC90
(18)
MH blood agar HTMagar
2- 8 0.25- 1
4 1
8 1
Streptococcusagalactiae (13)
MH blood agar HTM agar
2- 8 0.25 - 2
8 1
8 2
Streptococcus group C (10)
MH blood agar HTMagar
2 - 16 1- 1
Streptococcus group G (12)
MH blood agar HTM agar
0.5 - 16 0.25 - 1
T h e r e was a m a r k e d increase in MBCs for staphylococci, the m e a n vermisporin MIC90 for Staphylococcus aureus being 0.27 lag/ml and the m e a n MBC90 7 ~g/ml. The mean vermisporin MIC90 for Staphylococcus epidermidis was 0.31/ag/ml, and the MBC90 5.3 pg/ml. T h e s e studies demonstrate that the new antibiotic, vermisporin has excellent activity against anaerobic species such as Bacteroides and Clostridium spp., inhibiting the majority of isolates at < 1 lag/ml. Blood inhibits the activity of vermisporin against streptococcal and staphylococcal species, and there is a 16- to 32-fold difference in MICs and MBCs for staphylococci. Further research may reveal whether a c o m p o u n d of this type could b e useful against anaerobic organisms and m a y suggest structural aspects which could be explored in other anti-anaerobic agents. It is conceivable that vermisporin could be used as an oral agent against Clostridiurn difficile and thereby decrease the use of oral vancomycin, which may be contributing to a rise in resistant enterococci.
References 1. National Committee for Clinical Laboratory Standards:Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 2nd edition. Approved Standard MT-A2. NCCLS, Villanova, PA, 1990. 2. Jorgenson JH, Reflding JS, Maher IA, Howell AW: Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. Journal of Clinical Microbiology 1987, 25: 2105-2113.
4 1 2 0.5
8 1
3. National Committee for Clinical Laboratory Standards: Methods for antimicrobial susceptibility testing of anaerobic bacteria, 2nd edition. Approved Standard Mll-A2. NCCLS, Villanova, PA, 1991. 4. PearsonRD, Steigbigel RT, Davis HT, Chapman SW: Method of reliable determination of minimal lethal antibiotic concentrations. Antimierobial Agents and Chemotherapy 1980, 18: 688--708.
m
,lllllllll
In Vitro Activity of L-627 against Anaerobic Bacteria C.E. N o r d 1'2., A . L i n d m a r k 2, I. P e r s s o n 2
The in vitro activity of L-627 against 370 anaerobic bacterial strains including anaerobic cocci, Propionibacterium aches, Clostridlura
perfringens, Clostridium difficile, Bacteroldes fragilis, o t h e r Bacteroides spp. and fusobacteria was determined by the agar dilution method. This activity was compared with that of plperaeillin, cefoxitin, imipenem, meropenem, clindamycin, metronidazole and chloramphenicol. L-627, im. ipenem, meropenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested. L-627 had in vitro activity similar to that of the other carbapenems tested. 1Department of Microbiology, Huddinge University Hospital, Karolinska Institute, 141 86 Huddinge, Sweden. 2National Bacteriological Laboratory, 105 21 Stockholm.
13. Edelstein PH, Edelstein MAC: In vitro activity of azithromycin against clinical isolates of Legionella species. Antimicrobial Agents and Chemotherapy 1991, 35: 180-181. 14. Farrell ID, Barker J, Chiodini PL, Hutchison JGP, Geddes AM: The activityof imipenem on Legionella pneumophila with a note on the treatment of two cases. Journal of Antimicrobial Chemotherapy1985, 16: 6165. 15. Fitzgeorge RB, Featherstone ASR, BaskerviUe A: Efficacy of azithromycinin the treatment of guinea pigs infectedwith Legionellapneumophila by aerosol.Journal of Antimicrobial Chemotherapy 1990, 25, Supplement A: 101-108. 16. Pfaller M, Hollis R, Johnson W, Massanari RM, Helms C, WenzeiR, Hall N, Moyer N, July J: The application of molecularand immunologictechniques to study the epidemiologyof Legionelta pnel~wophila sero Group 1. Diagnostic Microbiology and Infectious Disease 1989; 12: 295-302.
In Vitro Antimicrobial Activity of the New Antibiotic Vermisporin N.X. Chin 1, H.C. Neu 1'2.
The antimicrobial activity of vermisporin, a new antibiotic produced by fermentation of the fungus Ophiobolus vermisporis, was tested in vitro. Vermisporin inhibited 90 % of Bacteroides fragiIis and otherBacteroides spp. at I ~g/ml (range 0.251 ~g/ml). Clostridium perfringens were inhibited by 1 gg/mi (range 0.25-2 pg/ml). Vermisporin inhibited 90 % of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, at 0.5 ~g/ml (range 0.12-0.5 ~g/ml). Vermisporin MICs for group A, B, C, F and G streptococci were < 1 ~g/ml when tested in Haemophilus Test Medium but >_8 pg/ml in the presence of blood. Vermisporin MICs for Enterobacteriaceae, Pseudomonas aeruginosa and Haemophilus influenzae exceeded 64 ~tg/ml. Inhibited organisms had MBCs 16- to 32-fold above the MICs.
There is continued need to find new antimicrobial agents that inhibit gram-positive bacteria and anaerobic species. Vermisporin is a new antibiotic i Department of Medicineand 2Departmentof Pharmacology,Collegeof Physicians& Surgeons,ColumbiaUniversity, 630 West i68th Street, New York, New York 10032, USA.
755
produced by the fermentation of the fungus Ophiobolus vermisporis. It contains an N-methyltetramic acid and a dehydrodecaline skeleton with the molecular formula C25H37NO4. In preliminary tests it has been reported to have anti-anaerobic activity (I. Sato et al., 30th ICAAC, 1990, Abstract no. 433). We investigated the activity of vermisporin and the effect of growth conditions on its activity.
Materials and Methods. Vermisporin was provided by Meiji Seika Kaisha, Tokyo, Japan. Bacterial isolates tested were obtained from patients hospitalized at the Columbia-Presbyterian Medical Center in New York City. Isolates were identified by standard methods. Antimicrobial susceptibility was determined by an agar dilution method using Mueller-Hinton agar (Baltimore Biologicals, USA) for staphylococci and anaerobic gram-negative bacteria (1). Susceptibility of streptococci was determined using Mueller-Hinton agar supplemented with 5 % lysed sheep blood and using Haemophilus Test Medium (2). Susceptibility of anaerobes was determined using Wilkins-Chalgren medium (Difco Laboratories, USA) as recommended by the National Committee for Clinical Laboratory Standards (3). A replicating spot device was used to apply inocula prepared from fresh overnight broth cultures at a strength of 104 for aerobic organisms and 105 cfu for anaerobic species. Broth dilutions were carried out with 5 x 105 cfu in 1 ml tubes. Aerobic bacteria were incubated at 35 *C for 18-20 h, and anaerobic bacteria for 48 h. The MIC was defined as the lowest concentration which inhibited visible growth on agar or in tubes. The MBC was determined by plating 0.01 ml from tubes which showed no visible growth onto antibiotic-free agar plates, and then reading the plates after incubation for 24 h at 35 °C. The MBC was defined as the concentration at which there was a 99.9 % reduction in the colony count compared with the original inoculum according to the method of Pearson et al. (4). Control strains in each assay were the respective American Type Culture strains.
Results and Discussion. The activity of vermisporin against 115 anaerobic organisms is shown in Table 1. Vermisporin inhibited 90 % of the isolates at 1 pg/ml which included various Bacteroides, Clostridium and Propionibacterium species. As Table 2 demonstrates, vermisporin inhibited Staphylococcus aureus including methicillin-resistant isolates, hemolytic streptococci and Streptococcus pneumoniae at < 1 ~tglml. Suscep-
756
Eur. J. Clin. Microbiol. Infect. Dis.
Table 1: In vitro activity of vemaisporin against anaerobic bacteria. Organism
Number tested
MIC (pg/ml) Range
Bacteroidesfraragilis Bacteroides thetaiotaornicron Clostridiurn perfringens Clostridium difficile Peptostreptococci
Bacteroides bivius Bacteroides distasonis Bacteroides melaninogenicus Bacteroides ovatus CIostridium ramosum Clostridium sordellii Clostridium sphenoides Clostridium subterminale Propionibacterium aches Eubacterium spp.
41 11 26 20 14 1 3 4 2
MIC50
0.25-1 0.5 - 2 0.12- 2 0.12- 2 0.25 - 2 0.25 0.06, 0.25,1 0.12, 0.25, 0.25,1 0.25,1
2
1, 1
3
0.25, 0.25;0.5
1
1
1 5 1
0.5 0.12-1 1
MIC90
0.5 1 1 1 1
0.25
Table 2: In vitro activity of vermisporin against gram-positive bacteria. MICs were determined on Haemophilus Test Medium for streptococcal species. Organism
Number tested
MIC (pg/ml) Range
Staphylococcus aureus
MIC50
MIC90
16
0.12-0.5
0.12
0.5
16
0.12- 0.5
0.25
0.5
14
0.25 - 1
0.25
0.5
16
0.12- 0.5
0.25
0.5
Streptococcus pneumoniae
15
0.06 -0.5
0.5
0.5
Streptococcus pyogenes
18
0.25 - 1
1
1
Streptococcus agalactiae
13
0.25-2
1
2
Group C streptococci
10
0.12-1
0.25
1
Group F streptococci
7
1
1
1
Group G streptococci
12
0.25 - 1
0.5
1
methicilIin-susceptible
Staphylococcus aureus methicillin-resistant
Staphylococcus epidermidis methicillin-susceptible
Staphylococcus epidermidis methicillin-resistant
tibility o f the streptococcal species was determined in H a e m o p h i l u s Test Medium. W h e n tests were p e r f o r m e d using Mueller-Hinton agar containing 5 % sheep blood the MICs were 8 }ag/ml, as shown in Table 3. Vermisporin, irrespective of the medium used for the assay, did not inhibit the
aerobic gram-negative species (10 isolates of each) Escherichia coli, Klebsietla pneumoniae,
Enterobacter cloacae, Citrobacter freundii, Serratia marcescens, Pseudomonas aeruginosa and Haemophilus influenzae, the MICs being > 64 pg/ml.
Vo1.11,1992
757
Table 3: In vitro aetivity of vermisporin against beta-hemolytic streptococci in Mueller-Hinton (MH) blood agar (Mueller-Hinton medium plus 5 % sheep blood) and in Haemophilus Test Medium (HTM) agar. Organism
(n)
Streptococcuspyogenes
Medium
MIC 0ag/ml) Range
MICS0
MIC90
(18)
MH blood agar HTMagar
2- 8 0.25- 1
4 1
8 1
Streptococcusagalactiae (13)
MH blood agar HTM agar
2- 8 0.25 - 2
8 1
8 2
Streptococcus group C (10)
MH blood agar HTMagar
2 - 16 1- 1
Streptococcus group G (12)
MH blood agar HTM agar
0.5 - 16 0.25 - 1
T h e r e was a m a r k e d increase in MBCs for staphylococci, the m e a n vermisporin MIC90 for Staphylococcus aureus being 0.27 lag/ml and the m e a n MBC90 7 ~g/ml. The mean vermisporin MIC90 for Staphylococcus epidermidis was 0.31/ag/ml, and the MBC90 5.3 pg/ml. T h e s e studies demonstrate that the new antibiotic, vermisporin has excellent activity against anaerobic species such as Bacteroides and Clostridium spp., inhibiting the majority of isolates at < 1 lag/ml. Blood inhibits the activity of vermisporin against streptococcal and staphylococcal species, and there is a 16- to 32-fold difference in MICs and MBCs for staphylococci. Further research may reveal whether a c o m p o u n d of this type could b e useful against anaerobic organisms and m a y suggest structural aspects which could be explored in other anti-anaerobic agents. It is conceivable that vermisporin could be used as an oral agent against Clostridiurn difficile and thereby decrease the use of oral vancomycin, which may be contributing to a rise in resistant enterococci.
References 1. National Committee for Clinical Laboratory Standards:Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 2nd edition. Approved Standard MT-A2. NCCLS, Villanova, PA, 1990. 2. Jorgenson JH, Reflding JS, Maher IA, Howell AW: Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. Journal of Clinical Microbiology 1987, 25: 2105-2113.
4 1 2 0.5
8 1
3. National Committee for Clinical Laboratory Standards: Methods for antimicrobial susceptibility testing of anaerobic bacteria, 2nd edition. Approved Standard Mll-A2. NCCLS, Villanova, PA, 1991. 4. PearsonRD, Steigbigel RT, Davis HT, Chapman SW: Method of reliable determination of minimal lethal antibiotic concentrations. Antimierobial Agents and Chemotherapy 1980, 18: 688--708.
m
,lllllllll
In Vitro Activity of L-627 against Anaerobic Bacteria C.E. N o r d 1'2., A . L i n d m a r k 2, I. P e r s s o n 2
The in vitro activity of L-627 against 370 anaerobic bacterial strains including anaerobic cocci, Propionibacterium aches, Clostridlura
perfringens, Clostridium difficile, Bacteroldes fragilis, o t h e r Bacteroides spp. and fusobacteria was determined by the agar dilution method. This activity was compared with that of plperaeillin, cefoxitin, imipenem, meropenem, clindamycin, metronidazole and chloramphenicol. L-627, im. ipenem, meropenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested. L-627 had in vitro activity similar to that of the other carbapenems tested. 1Department of Microbiology, Huddinge University Hospital, Karolinska Institute, 141 86 Huddinge, Sweden. 2National Bacteriological Laboratory, 105 21 Stockholm.
