Bactericidal activity of netilmicin compared with gentamicin and streptomycin, alone and in combination with penicillin, against penicillin tolerant viridans streptococci and enterococci

Netilmicin was compared with gentamicin and streptomycin for in-vitro activity against 30 strains of penicillin-tolerant streptococci including 16 strains of enterococci. Both netilmicin and gentamicin tested alone at 4 mg/l caused 99.9% kill of more than half of the 13 strains of viridans streptococci tested, whereas streptomycin, 4 mg/l, had no bactericidal effect against these strains. Netilmicin, gentamicin and streptomycin tested alone at 8.0 mg/l against 10 strains of Streptococcus faecalis resulted in 99.9% kill of six, one and zero strains respectively. Combinations of penicillin with 2 mg/l of either netilmicin or gentamicin resulted in bactericidal synergy against 12 of 13 strains of viridans streptococci and all 10 strains of S. faecalis after 18 to 24 h incubation. Parallel experiments showed that higher concentration of penicillin were required to obtain 99.9% kill of 10 streptococcal strains when 4 mg/l streptomycin was compared with 2 mg/l of the other aminoglycosides. Killing curves showed similar bactericidal synergy for netilmicin-penicillin and gentamicin-penicillin combinations against most streptococci tested after 24 h incubation but there was sometimes a greater bactericidal effect noted with netilmicin after only 6 h incubation of the broth or after 48 h incubation. The results of this in-vitro study suggest that netilmicin is at least as effective as gentamicin as a bactericidal synergic agent with penicillin against penicillin-tolerant viridans streptococci and S. faecalis strains isolated from patients with endocarditis. Neither gentamicin or netilmicin were effective as bactericidal synergic agents with penicillin against 4 of 6 strains of S. faecium tested.     

Synergism, killing kinetics, and antimicrobial susceptibility of group A and B streptococci.

The susceptibility of 110 group A and 179 group B streptococci to 25 antimicrobics was tested by broth microdilution and agar disk diffusion tests. Representative strains were used in killing kinetics, penicillin-gentamicin synergy, and minimal bactericidal concentration tests. Group A streptococci were more susceptible than group B streptococci to 17 of the 25 antimicrobics tested. Group A and B streptococci were killed at the same rate if the amount of penicillin used was equivalent to their respective penicillin minimal inhibitory concentrations. Synergism was demonstrated for both group A and B streptococci when penicillin was used at concentrations equal to each respective minimal inhibitory concentration and subinhibitory concentration of gentamicin. This synergy could be demonstrated best using minimal bactericidal concentrations obtained by culturing 3- and 6-h cultures from the microdilution checkerboard tests rather than from 24-h subcultures. A greater synergistic effect was achieved by adding penicillin first and then adding gentamicin rather than in the reverse order, or simultaneously.     

Interaction between penicillin, clindamycin or metronidazole and gentamicin against species of clostridia and anaerobic and facultatively anaerobic gram-positive cocci

Seven anaerobic and facultative Gram-positive cocci and 12 clostridial species were tested for in-vitro and in-vivo susceptibilities to penicillin, clindamycin, and metronidazole, used singly or in combination with gentamicin. The in-vitro tests consisted of determination of minimal inhibitory concentration (MIC), done without or with constant amounts of gentamicin. When used alone or in combination with penicillin or metronidazole, gentamicin had negligible effects on the bacteria. When used with clindamycin, gentamicin significantly reduced the MIC for one strain each of Peptococcus magnus and Clostridium difficile. The in-vivo tests were carried out in mice and consisted of studying the bacterial contents of abscesses induced by subcutaneous injection of bacterial suspensions. Synergy between gentamicin and penicillin, clindamycin or metronidazole was shown respectively in five, three and one strain. Consistency between in-vitro and in-vivo findings was present in the above mentioned strains only between gentamicin and clindamycin. The synergy between penicillin, clindamycin or metronidazole and gentamicin in Gram-positive anaerobic and facultative organisms may have clinical implications.     

Synergism between penicillin, clindamycin, or metronidazole and gentamicin against species of the Bacteroides melaninogenicus and Bacteroides fragilis groups

Clinical isolates of the Bacteroides melaninogenicus and Bacteroides fragilis groups were tested for in vitro and in vivo susceptibility to penicillin, clindamycin, and metronidazole, used singly or in combination with gentamicin. The in vitro tests consisted of determinations of minimal inhibitory concentrations (MICs) carried out with or without constant amounts of gentamicin. When used alone, gentamicin had negligible effects on the bacteria but significantly reduced the MICs of penicillin, clindamycin, and metronidazole against 11, 10, and 3, of the 15 strains of the B. melaninogenicus group, respectively. The 15 strains of the B. fragilis group were all beta-lactamase producers and were highly resistant to penicillin or the combination of penicillin and gentamicin. However, gentamicin reduced the MICs of clindamycin and metronidazole against 1 and 7 strains of this group, respectively. The in vivo tests were carried out in mice and consisted of measurements of the effects of the antimicrobial agents on the sizes and bacterial content of abscesses induced by subcutaneous injection of bacterial suspensions. The results of the in vivo tests were generally consistent with those obtained in vitro with strains of the B. melaninogenicus group. Synergism between gentamicin and penicillin, clindamycin, or metronidazole was shown in 13, 10, and 3 strains of this group, respectively. In vivo synergism was not clearly demonstrated with the strains of the B. fragilis group, possibly because clindamycin and metronidazole used alone were highly efficacious. We suggest that the synergistic effect of gentamicin is due to its increased transport into the bacterial cell in the presence of penicillin and, possibly, other antimicrobial agents. The newly recognized in vitro and in vivo synergism between penicillin and other antimicrobial agents and an aminoglycoside in B. melaninogenicus may have clinical implications that deserve to be investigated.     

Antibiotic resistance and penicillin tolerance in clinical isolates of group B streptococci.

The aim of this study was to determine the susceptibility patterns of 100 group B streptococcal strains isolated in our hospital and to ascertain tolerance to penicillin by determining quantitative killing curves. We found two strains with intermediate susceptibility to penicillin and eight strains to ampicillin. Seventeen isolates were tolerant to penicillin, with bacterial counts decreasing 2 to 3 log during the first 8 h but still above 10(2) CFU/ml after 24 h. The kinetic study shows that penicillin tolerance is not rare among group B streptococci isolated in our hospital.     

Influences of linezolid,penicillin, and clindamycin,alone and in combination,on streptococcal pyrogenic exotoxin a release

An in vitro model was used to compare the effects of linezolid, clindamycin, and penicillin, alone and in combination, on streptococcal pyrogenic exotoxin A (SPE A) release against virulent group A streptococci (GAS). All regimens exhibited lower (P < 0.05) SPE A release at 1 h than those with penicillin alone. Linezolid and clindamycin, alone or in combination with penicillin, may optimize the treatment of GAS infections by reducing bacterial burden and exotoxin release.     

Antibiotic synergism against Listeria monocytogenes

The effectiveness of ampicillin, penicillin, streptomycin, and gentamicin against 20 strains of Listeria monocytogenes was studied in vitro. For all strains, the minimal bactericidal concentration (MBC) of both ampicillin and penicillin was much higher than the minimal inhibitory concentration (MIC). The MBC of both streptomycin and gentamicin was close to the MIC, but relatively high concentrations of these antibiotics were necessary to inhibit the growth of most of the strains of Listeria. The combination of penicillin plus streptomycin was synergistic against 19 of 20 strains and in the remaining strain produced enhanced killing (but of less magnitude than our criterion for synergism). Combinations of penicillin plus gentamicin, ampicillin plus streptomycin, and ampicillin plus gentamicin produced enhanced killing against all strains tested. No antagonism was observed when ampicillin or penicillin was combined with streptomycin or gentamicin.     

Enhanced activity of the combination of penicillin G and gentamicin against penicillin-resistant viridans group streptococci.

We evaluated the effects of the combination of penicillin G and gentamicin against 10 penicillin-resistant bacteremic isolates of viridans group streptococci for which the MICs of penicillin were 4 to 64 micrograms/ml. In time-kill studies, the combination resulted in more killing of eight isolates for which the MICs of penicillin were from 8 to 64 micrograms/ml than any of the antimicrobial agents tested alone. In general, clearly enhanced antimicrobial activity was observed with the combination.     

High incidence of erythromycin-resistant streptococci in Taiwan.

The activities of nine antimicrobial agents against 247 isolates of group B, C, F, and G streptococci and viridans group streptococci were studied by the broth microdilution method. Erythromycin resistance was found in 29.7, 41.7, 81.8, 23.5, and 53.3% of the strains of group B, C, F, and G streptococci and viridans group streptococci tested, respectively. Macrolides are not considered an optimal alternative to penicillin in the treatment of streptococcal infections, at least empirically, in Taiwan.     

High-level penicillin resistance and penicillin-gentamicin synergy in Enterococcus faecium.

Thirty-seven Enterococcus faecium strains with different levels of penicillin susceptibility were studied in time-kill experiments with a fixed concentration (5 micrograms/ml) of gentamicin combined with different penicillin concentrations (6 to 600 micrograms/ml). Synergy was defined as a relative decrease in counts of greater than 2 log10 CFU per milliliter after 24 h of incubation when the combination of the antibiotics was compared with its most active component alone. The minimal synergistic penicillin concentrations found were 6 micrograms/ml for 16 of 16 strains for which penicillin MICs were < or = 25 micrograms/ml, 20 to 100 micrograms/ml for 14 of 17 strains for which penicillin MICs were 50 to 200 micrograms/ml, and 200 to 500 micrograms/ml for 4 of 4 strains for which MICs penicillin were > 200 micrograms/ml. Penicillin-gentamicin synergy was observed even in high-level penicillin-resistant E. faecium strains at penicillin concentrations close to one-half the penicillin MIC. The possibility of treating infections caused by high-level penicillin-resistant E. faecium strains with penicillin-gentamicin combinations in particular cases may depend on the penicillin levels attainable in vivo.     

Comparative Synergistic Activity of Nafcillin, Oxacillin, and Methicillin in Combination with Gentamicin Against Enterococci

The effectiveness of three semisynthetic, penicillinase-resistant penicillins alone and in combination with gentamicin was tested against 29 clinical isolates of enterococci. The minimal inhibitory concentrations of nafcillin were considerably lower than those of oxacillin and methicillin but were slightly higher than those of penicillin. At clinically achievable concentrations, the combination of nafcillin plus gentamicin produced enhanced killing against 13 of 14 strains of enterococci and was synergistic (by very rigid criteria) against 10 of 14 strains. In contrast, combinations of oxacillin plus gentamicin were synergistic against only 3 of 14 strains, and methicillin plus gentamicin produced synergistic killing against only 1 of 14 strains.     

Importance of Medium in Demonstrating Penicillin Tolerance by Group B Streptococci

A total of 30 clinical isolates of group B streptococci were studied for penicillin tolerance in vitro. Minimal inhibitory and bactericidal concentrations of penicillin were determined simultaneously in three test media which have been used for group B streptococci, tryptose phosphate, Mueller-Hinton, and Todd-Hewitt broths, using a logarithmic-phase inoculum of 10(5) colony-forming units per ml. Minimal inhibitory concentrations in the three media did not differ significantly. However, minimal bactericidal concentrations were significantly higher in tryptose phosphate broth (mean, 1.04 mug/ml) than in Mueller-Hinton broth (0.22 mug/ml) or Todd-Hewitt broth (0.15 mug/ml). Similarly, ratios of minimal bactericidal to minimal inhibitory concentrations were significantly greater in tryptose phosphate broth than in Mueller-Hinton or Todd-Hewitt broth. After incubation in tryptose phosphate broth for an additional 24 h, the minimal bactericidal concentration consistently fell to levels which were only twice or equal to the minimal inhibitory concentration. This study illustrates the importance of the medium in the demonstration of penicillin tolerance and of controlling laboratory variables in the susceptibility testing of group B streptococci with penicillin.     

Synergy of imipenem or penicillin G and aminoglycosides against enterococci isolated from patients with infective endocarditis.

We tested the synergistic activity of imipenem (formerly imipemide, N-formimidoyl thienamycin, or MK 0787) (20 micrograms/ml) or penicillin (20 micrograms/ml) in combination with increasing concentrations of either streptomycin (5, 10, and 20 micrograms/ml) against 13 strains of streptomycin-susceptible enterococci or gentamicin (1, 3, and 5 micrograms/ml) against 13 strains of streptomycin-susceptible enterococci and 7 strains of streptomycin-resistant enterococci. At 24 h, penicillin together with each increment in streptomycin concentration resulted in a significant increase (P less than 0.001) in killing of streptomycin-susceptible enterococci compared with imipenem and the corresponding concentration of streptomycin. Similarly, at 24 h, the magnitude of killing of streptomycin-susceptible enterococci by a combination of penicillin plus each increment of gentamicin concentration was significantly greater (P less than 0.001) than that of the combination of imipenem and the corresponding concentration of gentamicin. Against streptomycin-resistant enterococci, penicillin together with each increment of gentamicin concentration killed significantly more enterococci (P less than 0.02) than did the combination of imipenem and the corresponding concentration of gentamicin. When combined with an aminoglycoside, the synergistic activity in vitro against enterococci of imipenem was significantly less than that of penicillin.     

Antimicrobial therapy of experimental endocarditis caused by nutritionally variant viridans group streptococci.

Rabbits with nutritionally variant viridans group streptococcal experimental endocarditis were treated three times daily for 3 days with procaine penicillin (1.2 X 10(6) U) alone or together with low-dose streptomycin (2 mg/kg), high-dose streptomycin (8 mg/kg), low-dose gentamicin (0.32 mg/kg), or high-dose gentamicin (1.05 mg/kg). The mean 0.5-h serum concentrations of streptomycin were 5.3 and 22.5 micrograms/ml in the low- and high-dose group, respectively, and the concentrations of gentamicin were 0.7 and 2.5 micrograms in the low- and high-dose groups, respectively. The combination of procaine penicillin with each dose of aminoglycoside was significantly more effective (P less than 0.001) than was procaine penicillin alone. In combination with procaine penicillin, the higher dose of streptomycin was significantly more effective (P less than 0.02) than the lower dose of streptomycin. The higher dose of streptomycin was not significantly more effective than either dose of gentamicin. The results of treatment with the high or low dose of gentamicin were virtually identical.     

Lack of in vivo and in vitro bactericidal activity of N-formimidoyl thienamycin against enterococci.

The minimal bactericidal concentrations of N-formimidoyl thienamycin (N-f-thienamycin) against 21 strains of enterococci isolated from patients with infective endocarditis were determined by macro- and microdilution methods. By a macrodilution technique with the minimal bactericidal concentration defined as greater than or equal to 99.9% killing of an initial inoculum, all 21 strains of enterococci were found to have minimal bactericidal concentration/minimal inhibitory concentration ratios of greater than or equal to 32. The mean minimal inhibitory concentration was 1.5 micrograms/ml (range, 0.5 to 4 micrograms/ml), and the minimal bactericidal concentration was greater than or equal to 128 micrograms/ml. The disparity between the results of our study and those published elsewhere, which reported that N-f-thienamycin is bactericidal in vitro against enterococci, may represent the relative insensitivity of the microdilution method in determining greater than or equal to 99.9% killing. The lack of in vitro bactericidal activity of N-f-thienamycin against enterococci was confirmed in vivo in the rabbit model of experimental endocarditis. N-f-Thienamycin was no more effective than penicillin alone in the treatment of experimental enterococcal endocarditis and was less effective than the combination of penicillin and gentamicin. The results indicate that N-f-thienamycin should not be used alone in the treatment of enterococcal endocarditis.     

Penicillin-induced effects on streptomycin uptake and early bactericidal activity differ in viridans group and enterococcal streptococci.

In vitro studies with penicillin and [3H]streptomycin in four strains of streptococci (S. faecalis, S. sanguis, and S. mitis) were performed by simultaneously measuring the rates of bacterial killing and uptake of streptomycin. In S. faecalis, penicillin stimulated streptomycin uptake, as has been shown by Moellering and Weinberg (R. C. Moellering, Jr., and A. N. Weinberg, J. Clin. Invest. 50:2580-2584, 1971). Moreover, the antibiotic combination was associated with an enhanced bactericidal rate which temporally correlated with beta-lactam-induced aminoglycoside uptake. In contrast, in viridans group streptococci (S. sanguis and S. mitis) penicillin had no effect on streptomycin uptake and a minimal effect on bactericidal rate when compared with either drug alone. These data suggested that the stimulation of streptomycin uptake in streptococci by penicillin is strain or species specific and that important differences exist between enterococci and viridans group streptococci regarding the mechanisms of beta-lactam-aminoglycoside potentiation.     

Synergistic Activity of Gentamicin with Trimethoprim or Sulfamethoxazole-Trimethoprim Against Escherichia coli and Klebsiella pneumoniae

The effect of combinations of gentamicin with trimethoprim or sulfamethoxazole-trimethoprim against clinical isolates of Escherichia coli (11 strains) and Klebsiella pneumoniae (12 strains) was examined by using a microdilution checkerboard technique. All isolates were susceptible to each antimicrobial agent. Synergism, defined as at least a 2-log(2)-dilution lowering of the minimal inhibitory concentration of either antibiotic in the combination compared with the minimal inhibitory concentration of the antibiotic alone, was observed with 15 of 23 (65%) isolates tested against trimethoprim and gentamicin and 14 of 23 (61%) isolates tested against sulfamethoxazole-trimethoprim and gentamicin. A 3-log(2)-dilution lowering of the minimal inhibitory concentration of either antibiotic was observed in 7 of 23 (30%) trimethoprim and gentamicin trials and 3 of 23 (13%) sulfamethoxazole-trimethoprim and gentamicin trials. Antagonism was observed in 3 of 46 combination trials and only with strains of K. pneumoniae.     

Antibiotic Susceptibility of Streptococcus bovis and Other Group D Streptococci Causing Endocarditis

Seventy-four strains of Streptococcus bovis and 35 strains of enterococci (Streptococcus faecalis and its varieties, Streptococcus faecium and Streptococcus durans), most of which were isolated from patients with endocarditis, were tested for their susceptibility to penicillin, ampicillin, erythromycin, cephalothin, vancomycin, methicillin, tetracycline, chloramphenicol, kanamycin, streptomycin, and gentamicin. Minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) were determined by a microtiter broth dilution technique. All of these organisms are group D streptococci, but the S. bovis strains are not enterococci. On the basis of both MIC and MBC, the S. bovis strains were much more susceptibile in general to antibiotics then were the enterococcal strains. For the S. bovis strains, the lowest MICs were obtained with penicillin, ampicillin, and erythromycin, and the lowest MBCs with penicillin and ampicillin. Although these antibiotics were also the most active against the enterococci, the MICs and MBCs were much higher than obtained with the S. bovis strains. Gentamicin was the most active aminoglycoside. On the basis of in vitro susceptibility results, the S. bovis strains resemble the viridans streptococci rather than enterococci.     

Comparative antimicrobial activity of O-demethylfortimicin A, a derivative of fortimicin A.

The in vitro antimicrobial activity of O-demethylfortimicin A (ODMF), a derivative of fortimicin A, was compared with those of fortimicin A and gentamicin against a spectrum of 256 organisms. All three antibiotics were active in low concentrations against all strains of Enterobacteriaceae, Acinetobacter sp., and Staphylococcus aureus, with ODMF most active against Proteus mirabilis, indole-positive Proteus, and Providencia and gentamicin most active against other species. Activity of each of the antibiotics against group D streptococci was poor. The overall activity of ODMF was superior to that of fortimicin A for all groups of organisms examined and was most pronounced, approximately three-fold, against strains of Pseudomonas aeruginosa. Both ODMF and fortimicin A were resistant to the action of several aminoglycoside-inactivating enzymes, with the exception of 3-N-acetyltransferase-I. ODMF and fortimicin A showed similar rapid bactericidal effects at multiples of the minimum inhibitory concentration and equivalent synergistic activity against enterococci when combined with penicillin G. The combination of carbenicillin with ODMF, fortimicin A, or gentamicin was synergistic for approximately 80% of the P. aeruginosa strains tested. Inactivation of ODMF and fortimicin A when combined with carbenicillin in vitro was minimal or absent, whereas gentamicin was substantially inactivated under similar conditions. ODMF, fortimicin A, and gentamicin exhibited protective activity in mice infected with Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, S. aureus, or P. aeruginosa. Gentamicin was the most active, followed by ODMF and fortimicin A. The superior in vitro activity of ODMF compared with fortimicin A against P. aeruginosa was confirmed in vivo.     

In vitro and in vivo activity of ciprofloxacin against enterococci isolated from patients with infective endocarditis.

In vitro activity of ciprofloxacin against 27 strains of enterococci was inoculum dependent. Using inocula of 10(5) to 10(6) or 10(7) to 10(8) CFU of enterococci per ml, the MICs for 50 and 90% of strains tested increased from 1 to greater than or equal to 128 micrograms of ciprofloxacin per ml with the higher inoculum compared with the lower inoculum. The MBC for 50% of strains tested increased from 2 to greater than 128 micrograms/ml and the MBC for 90% of strains tested increased from 8 to greater than 128 micrograms of ciprofloxacin per ml with the lower and higher inocula, respectively. The combination of penicillin-gentamicin was more effective in vitro than the combination of ciprofloxacin-gentamicin against the low or high inoculum of enterococci. Using two strains of enterococci, we studied the efficacy of ciprofloxacin in the treatment of enterococcal experimental endocarditis in rabbits. Ciprofloxacin used alone or combined with gentamicin was significantly less effective (P less than 0.01) than procaine penicillin alone or procaine penicillin combined with gentamicin for the treatment of enterococcal experimental endocarditis. The combination of ciprofloxacin-procaine penicillin was not a more effective therapy than procaine penicillin alone.