DIAGN MICROBIOLINFECT DIS 1992;15:549-552
549
The Activity of Metal Compounds Against Aerobic and Anaerobic Bacteria Nai-xun Chin and Harold C. N e u
We evaluated the antimicrobial activity of two metal compounds, JM-1397 (OsO2[xylyl]2) and JM-2469 (AuCl[S2CPEt3]). Both inhibited methicillin-susceptible and methicillin-resistant Staphylococcus aureus at concentrations of 0.5-2 i~g/ml, with a minimum inhibitory concentration (MIC9o) of I izg/ml for JM-1397 and 0.5 i~g/ml for ]M2469. Similar concentrations inhibited methicillin-susceptible and -resistant coagulase-negative staphylococci (S. epidermidis, S. haemolyticus, and S. saprophyticus). JM-2469 inhibited group A, B, C, F, and G ~8-hemolytic streptococci and
viridans group streptococci at 1-8 i~g/ml (MICgo4 izg/ml) but Enterococcus faecalis and E. faecium had MICs of 8-16 I~g/ml. JM-1397 had MICs for these organisms of >64 I~g/ml. Bacteroides fragilis, other Bacteroides, and Clostridium species were inhibited by 64 i~g/ml. These studies show that osmium and gold compounds have potential as topical agents against Gram-positive and anaerobic species.
There has been continued interest in developing compounds that, on topical application, will inhibit both the growth of bacteria involved in w o u n d sepsis and, in particular, the organisms causing nosocomial venous or arterial line sepsis (Maki, 1989). Currently, there has been use of agents, such as bacitracin, polymyxin B, and neomycin, as topical agents. Also, excellent success has been achieved with silver sulfadiazine in burn w o u n d sepsis because of its activity against Gram-positive and -negative bacteria. Chlorhexidine, povidone iodine, and alcohol also have been used (Maki et al., 1991). This study evaluates the in vitro activity of several novel metal compounds against Gram-positive and Gramnegative bacteria. Compounds JM-1397 (OsO2[xylyl]2) and JM-2469 (AuCI[S2CPEt3]) were provided by Johnson Matthey Technology Centre (Reading, England). Com-
pounds JM-1397 and JM-2469 were dissolved in acetone at 10 mg/ml and then diluted in medium. Microorganisms came from patients admitted to the Columbia-Presbyterian Medical Center in N e w York. Antimicrobial susceptibilities of aerobic species were measured by an agar dilution method with Mueller-Hinton agar according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS, 1990). Activity against streptococcal species was determined with Mueller-Hinton agar supplemented with 5% sheep blood. Activity against anaerobic species was determined using WilkinsChalgren agar (NCCLS, 1989). For aerobic species a replicating spot device was used to apply 104 colonyforming units (CFU) to agar plates, and 105 CFU were used per spot for anaerobes. Plates were incubated at 35°C for 18-24 hr; however, anaerobic organisms were incubated for 48 hr in GASPAKjars. Both JM-1397 and JM-2469 inhibited Staphylococcus aureus, including methicillin-resistant isolates, at concentrations of 0.5-2 ~,g/ml for JM-1397 and -64 2-8 8->64 1-4 16->64 4-32 >64 8-16 ~64 32
1
0.5 1 0.5 1 0.5 0.5 0.25 64 2 64 4 64 8 >64 8 64 4 >64 4 >64 4 >64 16 >64 16 0.25 0.5 0.5 4 0.25 8 0.25 0.25 0.5 0.5 >64 32 >64 32
Note
551
TABLE 1
Continued MIC (~g/ml) No.
Organism
Tested
Agent
Citrobacter freundii
11
Enterobacter cloacae
11
Serratia marcescens
20
Pseudomonas aeruginosa
20
Haemophilus influenzae
15
Moraxella catarrhalis
10
Neisseria gonorrhoeae
10
JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469
50%
Range
>64 32 >64 >64 >64 >64 >64 >64 32
>64 32 >64 64 >64 >64 >64 >64
>64 16-64 >64 32->64 >64 32->64 >64 >64 2->64 0.5-4 0.12-1 1~8 1-4 0.5-2
90%
8 1
2
0.5 2
1 4
1 1
2 1
"Includes Staphylococcusepidermidis, S. haemolyticus, and S. saprophyticus. blncludes Bacteroidesovatus, B. distasonis, and B. thetaiotaomicron.
Neither of the compounds had appreciable activity against Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, or Pseudomonas aeruginosa with MICs >64 wg/ml for JM-1397 and 16 to >64 wg/ml for JM-2469. In contrast, JM-2469 inhibited Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae at concentrations between 1 and 8 v,g/ml. We investigated the effect of the assay medium on the activity of JM-1397 and JM-2469 (Table 2). The presence of blood markedly reduced the activity of JM-1397--the osmium c o m p o u n d - - b u t the gold compound's activity was minimally affected. Similarly, the addition of 2% NaCI did not alter the activity of either compound against S. aureus or coagulase-negative staphylococci which included 10 ciprofloxacin, gentamicin, and rifampin-resistant isolates (data not shown). In addition to the compounds shown, we evaluated JMACF-199, which is a AgC1/TiO2 composite that is insoluble but can be made into a suspension. This agent proved unsatisfactory with MICs > 64 ~g/ml for all of the organisms tested and indicated that the silver titanium oxide was not a suitable form. Nosocomial infection due to cutaneous organisms is a serious problem in hospitals (Jarvis et al., 1991). Topical application of metal agents, such as silver sulfadiazine, has proved useful in preventing burn wound sepsis, and is widely used as an agent when large cutaneous wounds are a problem in bedridden patients (Babb et al., 1977). Iodophors have been used to a major degree to prevent sepsis and infection at the site of insertion of catheters that will remain in place for protracted periods (Maki, 1989).
TABLE 2
The Effect of Growth Medium on the MICs of JM-1397 and JM-2469 Against Two Strains of Staphylococcus aureus MIC (~g/ml)
Medium Blood agar Blood agar~ Mueller-Hinton agar Mueller-Hinton agar + 2% NaCI Wilkins-Chargen agar
Isolate
JM1397
JM2469
1 2 1 1 2 1 2 1 2
>64 >64 >64 1 0.5 0.5 0.5 0.25 0.12
1 1 2 0.25 0.25 0.25 0.25 0.25 0.25
~Anaerobicconditions. JM-1397 is an osmium salt and JM-2469 a gold salt. As this study shows, the agents are active at low concentrations inhibiting staphylococci (including methicillin- and fluoroquinolone-resistant organisms) and anaerobic species. Whereas the gold salt, JM-2469, will inhibit hemolytic streptococci, the osmium salt, JM-1397, was not active in the presence of blood, which explained its poor activity against streptococci tested on blood-containing media. Neither agent inhibited Enterobacteriaceae or Pseudomonas aeruginosa. Further modification of such metal compounds may yield more active agents. The activity against anaerobic species might be of particular use in some
552
l o w e r b o d y c u t a n e o u s infections or w h e n groin lines are necessary. W h e t h e r a formulation of JM-1397 or JM-2469 w o u l d be useful to control methicillin-re-
Nai-xun Chin a n d H.C. N e u
sistant S. aureus or S. epidermidis, as is possible with mupirocin, will n e e d to be d e t e r m i n e d in animal and h u m a n studies.
REFERENCES Babb JR, Bridges K. Jackson DM, Lowbury EJL, Ricketts CR (1977) Topical chemoprophylaxis: trials in silver phosphate chlorhexidine, silver sulfadiazine and povidone-iodine preparations. Burns 3:65-71. Jarvis WR, Edwards JR, Culver DH, Hughes JM, Horan T, Emori TG, Banerjee S, Tolson J, Henderson T, Gaynes RP, Martone NJ (1991) Nosocomial infection rates in adult and pediatric intensive care units in the United States. Am J Med 91(3b):185-196. Maki DG (1989) Pathogenesis, prevention, and management of infections due to intravascular devices used for infusion therapy. In Infections Associated with Indwelling Medical Devices. Eds, A Bisno and F. Waldvogel. Washington, DC: American Society for Microbiology, pp 161-177.
Maki DG, Ringer M. Alvarado CJ (1991) Prospective randomized trial of povidone-iodine, alcohol, and chlorhexidine for prevention of infection associated with central venous and arterial catheters. Lancet 338:339343. National Committee for Clinical Microbiology Standards (NCCLS) (1989) Tentative standard Mll-T2: methods for antimicrobial susceptibility testing of anaerobic bacteria. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1990) Approved standard, M7-A2: standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova, PA: NCCLS.
549
The Activity of Metal Compounds Against Aerobic and Anaerobic Bacteria Nai-xun Chin and Harold C. N e u
We evaluated the antimicrobial activity of two metal compounds, JM-1397 (OsO2[xylyl]2) and JM-2469 (AuCl[S2CPEt3]). Both inhibited methicillin-susceptible and methicillin-resistant Staphylococcus aureus at concentrations of 0.5-2 i~g/ml, with a minimum inhibitory concentration (MIC9o) of I izg/ml for JM-1397 and 0.5 i~g/ml for ]M2469. Similar concentrations inhibited methicillin-susceptible and -resistant coagulase-negative staphylococci (S. epidermidis, S. haemolyticus, and S. saprophyticus). JM-2469 inhibited group A, B, C, F, and G ~8-hemolytic streptococci and
viridans group streptococci at 1-8 i~g/ml (MICgo4 izg/ml) but Enterococcus faecalis and E. faecium had MICs of 8-16 I~g/ml. JM-1397 had MICs for these organisms of >64 I~g/ml. Bacteroides fragilis, other Bacteroides, and Clostridium species were inhibited by 64 i~g/ml. These studies show that osmium and gold compounds have potential as topical agents against Gram-positive and anaerobic species.
There has been continued interest in developing compounds that, on topical application, will inhibit both the growth of bacteria involved in w o u n d sepsis and, in particular, the organisms causing nosocomial venous or arterial line sepsis (Maki, 1989). Currently, there has been use of agents, such as bacitracin, polymyxin B, and neomycin, as topical agents. Also, excellent success has been achieved with silver sulfadiazine in burn w o u n d sepsis because of its activity against Gram-positive and -negative bacteria. Chlorhexidine, povidone iodine, and alcohol also have been used (Maki et al., 1991). This study evaluates the in vitro activity of several novel metal compounds against Gram-positive and Gramnegative bacteria. Compounds JM-1397 (OsO2[xylyl]2) and JM-2469 (AuCI[S2CPEt3]) were provided by Johnson Matthey Technology Centre (Reading, England). Com-
pounds JM-1397 and JM-2469 were dissolved in acetone at 10 mg/ml and then diluted in medium. Microorganisms came from patients admitted to the Columbia-Presbyterian Medical Center in N e w York. Antimicrobial susceptibilities of aerobic species were measured by an agar dilution method with Mueller-Hinton agar according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS, 1990). Activity against streptococcal species was determined with Mueller-Hinton agar supplemented with 5% sheep blood. Activity against anaerobic species was determined using WilkinsChalgren agar (NCCLS, 1989). For aerobic species a replicating spot device was used to apply 104 colonyforming units (CFU) to agar plates, and 105 CFU were used per spot for anaerobes. Plates were incubated at 35°C for 18-24 hr; however, anaerobic organisms were incubated for 48 hr in GASPAKjars. Both JM-1397 and JM-2469 inhibited Staphylococcus aureus, including methicillin-resistant isolates, at concentrations of 0.5-2 ~,g/ml for JM-1397 and -64 2-8 8->64 1-4 16->64 4-32 >64 8-16 ~64 32
1
0.5 1 0.5 1 0.5 0.5 0.25 64 2 64 4 64 8 >64 8 64 4 >64 4 >64 4 >64 16 >64 16 0.25 0.5 0.5 4 0.25 8 0.25 0.25 0.5 0.5 >64 32 >64 32
Note
551
TABLE 1
Continued MIC (~g/ml) No.
Organism
Tested
Agent
Citrobacter freundii
11
Enterobacter cloacae
11
Serratia marcescens
20
Pseudomonas aeruginosa
20
Haemophilus influenzae
15
Moraxella catarrhalis
10
Neisseria gonorrhoeae
10
JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469 JM-1397 JM-2469
50%
Range
>64 32 >64 >64 >64 >64 >64 >64 32
>64 32 >64 64 >64 >64 >64 >64
>64 16-64 >64 32->64 >64 32->64 >64 >64 2->64 0.5-4 0.12-1 1~8 1-4 0.5-2
90%
8 1
2
0.5 2
1 4
1 1
2 1
"Includes Staphylococcusepidermidis, S. haemolyticus, and S. saprophyticus. blncludes Bacteroidesovatus, B. distasonis, and B. thetaiotaomicron.
Neither of the compounds had appreciable activity against Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, or Pseudomonas aeruginosa with MICs >64 wg/ml for JM-1397 and 16 to >64 wg/ml for JM-2469. In contrast, JM-2469 inhibited Haemophilus influenzae, Moraxella catarrhalis, and Neisseria gonorrhoeae at concentrations between 1 and 8 v,g/ml. We investigated the effect of the assay medium on the activity of JM-1397 and JM-2469 (Table 2). The presence of blood markedly reduced the activity of JM-1397--the osmium c o m p o u n d - - b u t the gold compound's activity was minimally affected. Similarly, the addition of 2% NaCI did not alter the activity of either compound against S. aureus or coagulase-negative staphylococci which included 10 ciprofloxacin, gentamicin, and rifampin-resistant isolates (data not shown). In addition to the compounds shown, we evaluated JMACF-199, which is a AgC1/TiO2 composite that is insoluble but can be made into a suspension. This agent proved unsatisfactory with MICs > 64 ~g/ml for all of the organisms tested and indicated that the silver titanium oxide was not a suitable form. Nosocomial infection due to cutaneous organisms is a serious problem in hospitals (Jarvis et al., 1991). Topical application of metal agents, such as silver sulfadiazine, has proved useful in preventing burn wound sepsis, and is widely used as an agent when large cutaneous wounds are a problem in bedridden patients (Babb et al., 1977). Iodophors have been used to a major degree to prevent sepsis and infection at the site of insertion of catheters that will remain in place for protracted periods (Maki, 1989).
TABLE 2
The Effect of Growth Medium on the MICs of JM-1397 and JM-2469 Against Two Strains of Staphylococcus aureus MIC (~g/ml)
Medium Blood agar Blood agar~ Mueller-Hinton agar Mueller-Hinton agar + 2% NaCI Wilkins-Chargen agar
Isolate
JM1397
JM2469
1 2 1 1 2 1 2 1 2
>64 >64 >64 1 0.5 0.5 0.5 0.25 0.12
1 1 2 0.25 0.25 0.25 0.25 0.25 0.25
~Anaerobicconditions. JM-1397 is an osmium salt and JM-2469 a gold salt. As this study shows, the agents are active at low concentrations inhibiting staphylococci (including methicillin- and fluoroquinolone-resistant organisms) and anaerobic species. Whereas the gold salt, JM-2469, will inhibit hemolytic streptococci, the osmium salt, JM-1397, was not active in the presence of blood, which explained its poor activity against streptococci tested on blood-containing media. Neither agent inhibited Enterobacteriaceae or Pseudomonas aeruginosa. Further modification of such metal compounds may yield more active agents. The activity against anaerobic species might be of particular use in some
552
l o w e r b o d y c u t a n e o u s infections or w h e n groin lines are necessary. W h e t h e r a formulation of JM-1397 or JM-2469 w o u l d be useful to control methicillin-re-
Nai-xun Chin a n d H.C. N e u
sistant S. aureus or S. epidermidis, as is possible with mupirocin, will n e e d to be d e t e r m i n e d in animal and h u m a n studies.
REFERENCES Babb JR, Bridges K. Jackson DM, Lowbury EJL, Ricketts CR (1977) Topical chemoprophylaxis: trials in silver phosphate chlorhexidine, silver sulfadiazine and povidone-iodine preparations. Burns 3:65-71. Jarvis WR, Edwards JR, Culver DH, Hughes JM, Horan T, Emori TG, Banerjee S, Tolson J, Henderson T, Gaynes RP, Martone NJ (1991) Nosocomial infection rates in adult and pediatric intensive care units in the United States. Am J Med 91(3b):185-196. Maki DG (1989) Pathogenesis, prevention, and management of infections due to intravascular devices used for infusion therapy. In Infections Associated with Indwelling Medical Devices. Eds, A Bisno and F. Waldvogel. Washington, DC: American Society for Microbiology, pp 161-177.
Maki DG, Ringer M. Alvarado CJ (1991) Prospective randomized trial of povidone-iodine, alcohol, and chlorhexidine for prevention of infection associated with central venous and arterial catheters. Lancet 338:339343. National Committee for Clinical Microbiology Standards (NCCLS) (1989) Tentative standard Mll-T2: methods for antimicrobial susceptibility testing of anaerobic bacteria. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (NCCLS) (1990) Approved standard, M7-A2: standard methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Villanova, PA: NCCLS.
