Comparison of the In Vitro Activity of Several Cephalosporin Antibiotics Against Gram-Negative and Gram-Positive Bacteria Resistant to Cephaloridine

The in vitro activity of each of two oral [cefatrizine (BL-S640), cephalexin] and three parenteral (cefamandole, cefazolin, cephapirin) cephalosporin antibiotics was compared with that of cephalothin against 168 clinical isolates of gram-negative and gram-positive bacteria selected as resistant to 20 μg of cephaloridine per ml on the basis of agar dilution susceptibility test data. Each of the five other cephalosporins inhibited a greater percentage of gram-negative bacillary isolates than did cephalothin or cephaloridine, with minimal inhibitory concentration values ranging 2- to 50-fold lower. Significant differences between minimal inhibitory concentrations of the compounds tested were also observed in tests against strains of Streptococcus faecalis and of methicillin-resistant Staphylococcus aureus. Potential advantages of including more than a single cephalosporin antibiotic in the panel of antibiotics used for routine susceptibility testing, suggested by these observations, are discussed.     

Lytic activity of a new cephalosporin, cefuroxime, against gram-negative bacteria.

Cefuroxime, a new cephalosporin antibiotic, was inhibitory at low concentrations to several types of gram-negative bacteria. It was considerably more stable than cephaloridine or cephalothin to the beta-lactamases produced by Escherichia coli (RP1), Klebsiella aerogenes K1 and Enterobacter cloacae P99. Concentrations of cefuroxime much greater than the minimum inhibitory concentration (MIC) were usually necessary to induce lysis of beta-lactamase and non-beta-lactamase producers. In contrast, cephaloridine, cephalothin and ampicillin were rapidly bacteriolytic at, or near, the respective MIC against non-beta-lactamase producers, whereas the activity of these three antibiotics was considerably reduced against beta-lactamase-producing strains.     

Cephamycins, a New Family of β-Lactam Antibiotics. III. In Vitro Studies

Cephamycins A, B, and C are naturally produced cephalosporin-type antibiotics. Although A and B were found to be more active than C against gram-positive organisms, they were not so active against such strains as are cephalosporin C or the semisynthetic antibiotics cephaloridine and cephalothin. Against gram-negative organisms, cephamycin C was more active than A or B and, in general, was as active as the cephalosporins. In addition, cephamycin C was active in vitro against clinically isolated strains resistant to the cephalosporins, such as Proteus, Providencia, and Escherichia coli. The in vitro antibacterial activity of cephamycin C, cephalothin, and cephaloridine is primarily bactericidal. A 10,000-fold increase in inoculum of a strain of Proteus mirabilis resulted in 200-fold or greater increases in minimal inhibitory and minimal bactericidal end points of cephalothin and cephaloridine, but only 10- and 16-fold increases, respectively, for cephamycin C. After 15 passages through antibiotic-containing broths, during which time a culture of E. coli showed an increase in minimal inhibitory concentrations of streptomycin of >1,000-fold, end points for cephamycin C increased 4-fold, for cephalothin, 1.5-to 6-fold, and for cephaloridine, 128-fold.     

Biliary Tract Excretion of Cefazolin, Cephalothin, and Cephaloridine in the Presence of Biliary Tract Disease

The biliary tract excretion of three cephalosporins, cefazolin, cephaloridine, and cephalothin, was compared in patients with biliary tract disease. In the absence of obstruction, mean antibiotic levels in bile from gall bladder and common duct in patients undergoing cholecystectomy were highest for cefazolin (17 and 31 mug/ml, respectively) than either cephaloridine (7 and 9 mug/ml) or cephalothin (1 and 4 mug/ml). Biliary tract levels generally paralleled serum levels. In no patient with cystic duct obstruction were any of the cephalosporins detectable in appreciable amounts in gall bladder bile. In patients with T-tube drainage given each of the three different cephalosporins on separate days, concentrations of cefazolin in bile were many-fold higher than either cephaloridine or cephalothin. Peak levels of cefazolin in T-tube bile averaged 51 mug/ml after intravenous and 26 mug/ml after intramuscular administration, whereas mean peak levels of cephalothin and cephaloridine were only 6 and 16 mug/ml, respectively. Here, too, T-tube levels reflected serum concentrations and obstruction to biliary flow impaired excretion of each of the drugs.     

Bactericidal Activity of Cephalosporins in an In Vitro Model Simulating Serum Levels

The bactericidal activity of cefazolin, cephaloridine, and cephalothin in a simulated intramuscular study (500 mg) and a simulated intravenous drip infusion study (2 g/2 h) is reported. In both model systems, the bactericidal activity of cefazolin surpassed that of cephalothin, and there were certain differences between cefazolin and cephaloridine in the simulated intramuscular study when human serum was used as a medium. In a routine reference static system, the drug levels were constant at the simulated peak level of each cephalosporin by both routes. In this system the three cephalosporins were equal in activity. In a third experiment, the effect of drug concentrations and exposure time on bactericidal activity of the cephalosporins was studied. The bactericidal activity of cephaloridine was the strongest of the three drugs when exposure time was 2 h and drug concentration was less than four times the minimal inhibitory concentration. At concentrations above four times the minimum inhibitory concentration, all three cephalosporins were equal in activity when the exposure time was 2 h.     

Antibacterial Activity of Cefamandole, a New Cephalosporin Antibiotic, Compared with that of Cephaloridine, Cephalothin, and Cephalexin

The in vitro antibacterial activity of cefamandole, a new cephalosporin antibiotic, was compared with that of cephaloridine, cephalothin, and cephalexin against 1,213 strains of gram-positive and gram-negative bacteria recently isolated from clinical sources. The decreasing order of activity of the four agents against gram-positive cocci was cephaloridine, cephalothin, cefamandole, and cephalexin. However, cefamandole was the most active of the four against Haemophilus species and gram-negative bacilli susceptible to cephalosporins. It was also active against many strains resistant to the other cephalosporins, such as Enterobacter species and indole-positive Proteus species, but there was a marked inoculum effect with all of these organisms, and minimal bactericidal concentrations were usually considerably higher than minimal inhibitory concentrations. Cefamandole, like other cephalosporins, had no useful activity against Pseudomonas species.     

In vitro and in vivo comparative evaluations of injectable cephalosporin derivatives and ampicillin.

The in vitro and in vivo activity of the 5 injectable cephalosporins, cefazolin, cephaloridine, cephalothin, cephapirin, cephacetrile, and ampicillin was compared. No gram-positive organisms resistant to any of the cephalosporin derivatives were encountered. The MIC of ampicillin for gram-positive organisms was distributed over a wide range. The antibacterial activity of cefazolin, cephaloridine and ampicillin was high against gram-negative organisms. Ampicillin was potently effective especially against P. mirabilis and cefazolin especially against K. pneumoniae. The protecting effect of the 5 cephalosporins on experimental infections in mice was compared. The effect of cefazolin and cephaloridine was more marked than that of the others. These results may be explained by the fact that the other 3 cephalospoirins are rapidly metabolized in the living body.     

Comparative serum levels and protective activity of parenterally administered cephalosporins in experimental animals

Six cephalosporin antibiotics were administered subcutaneously to mice at a level of 20 mg/kg. The serum levels of each were determined at five time intervals ranging from 5 to 120 min after dosing. Urinary recovery and the presence of active metabolites in mouse urine were determined. The peak serum levels and serum half-lives in mice were found to be positively correlated with the mean effective dose values obtained after lethal challenge with Escherichia coli. The administration of cefazolin and cephanone resulted in the highest serum level and the best protection. Good protection was obtained with cephaloridine despite somewhat lower serum levels. The cephalosporins with the acetoxy side chain (cephalothin, cephapirin, and cephacetrile) showed lower serum levels and the poorest protection. Cefazolin, cephaloridine, and cephalothin serum levels were also determined in dogs, squirrel monkeys, and rabbits. A mixed response was obtained in these species, with cefazolin peak serum levels being highest in rabbits and cephaloridine peak highest in dogs.     

Laboratory Evaluation of BL-S786, a Cephalosporin with Broad-Spectrum Antibacterial Activity

Biological and physicochemical properties of BL-S786 were compared with those of cephalothin, cephaloridine, and cefazolin. With few exceptions, BL-S786 was more active than the reference compounds against major gram-negative pathogenic species and its antibacterial spectrum was broader than that of cephalosporins currently available for clinical use. Although BL-S786 was generally less active than the control cephalosporins against gram-positive pathogens, it inhibited their growth at concentrations that should readily be achieved in humans after standard parenteral dosage. Streptococcus faecalis, a species relatively unsusceptible to cephalosporins in general, was an exception. BL-S786 was an effective bactericidal agent for strains of various gram-negative organisms. After intramuscular administration to mice, BL-S786 achieved high concentrations in blood, and its biological half-life was longer than that of the other three cephalosporins.     

Cephamycins, a New Family of β-Lactam Antibiotics: Antibacterial Activity and Resistance to β-Lactamase Degradation

The susceptibility to some cephalosporin antibiotics and to cephamycin C, a member of a new family of beta-lactam antibiotics, was evaluated for 466 cultures representing 11 different genera or species of gram-negative clinical isolates. The susceptibility of 39 gram-negative cultures known to produce beta-lactamase was also determined. The beta-lactamase activity of a representative group of the clinical isolates and the 39 enzyme producers was studied with the cephalosporins (cephalothin and cephaloridine) and cephamycin C as substrates and was related to the in vitro disc susceptibility to these same antibiotics. The significant resistance to beta-lactamase displayed by the cephamycins is reflected in the kinetics of enzyme activity (K(m) and V(max)) that are reported for the cephalosporins and the cephamycins. Resistance to beta-lactamase is probably one of the reasons that many cephalosporin-resistant cultures are susceptible to cephamycin C.     

Ascitic Fluid Cephalosporin Concentrations: Influence of Protein Binding and Serum Pharmacokinetics

Mongrel dogs with ascites created by inferior vena cava ligation were given cephalothin, cephaloridine, cefazolin, and cefamandole to evaluate the effect of protein binding and serum pharmacokinetics on the distribution of cephalosporins into ascitic fluid. Antibiotics were given intramuscularly (15 mg/kg) every 4 h for a total of eight doses. Antibiotic binding to dog serum and ascitic fluid was measured by ultracentrifugation. Binding of the cephalosporins to dog serum ranged from 31% for cephaloridine to 46% for cephalothin, considerably lower than human serum binding for cefazolin, cephalothin, and cefamandole. Antibiotic binding to ascitic fluid was only slightly lower than that to serum. Ascitic fluid antibiotic concentrations, which approached equilibrium at 16 to 28 h, were significantly higher for cefazolin and cephaloridine than for cephalothin and cefamandole. However, serum concentrations were also higher for cefazolin and cephaloridine, and percent penetration (ratio of serum peak to ascites peak × 100) was not statistically different among the four drugs. Binding of these cephalosporins to extravascular fluid protein was an important factor that determined the total ascitic fluid antibiotic level achieved. A formula utilizing the log mean serum level and binding to serum and extravascular fluid protein was used to accurately predict ascitic fluid drug levels at equilibrium.     

Use of a Heavy Inoculum in the In Vitro Evaluation of the Anti-Staphylococcal Activity of 19 Cephalosporins

The in vitro activity of 19 cephalosporins against 105 clinical isolates of Staphylococcus aureus and S. epidermidis was determined by using a heavy inoculum, i.e., 10(8) to 10(9) organisms per ml, to maximally challenge the antibiotics. The anti-staphylococcal activities of cephaloridine and 87/312 were consistently decreased by the use of a heavy inoculum when compared with the activity obtained with two less-concentrated inocula. The activity of most of the other compounds was also decreased with the use of a heavy inoculum, but this was observed only with selected isolates. Cephapirin, cephalothin, and cefazaflur were the most active drugs against the methicillin-susceptible isolates. Cephaloridine, cefamandole, cefazaflur, and 87/312 had substantial activity against methicillin-resistant staphylococci even with heavy inocula. With the exception of cefaclor against S. aureus, the orally absorbed cephalosporins were generally one-half to one-sixteenth as active as the parenterally administered cephalosporins. The median minimal inhibitory concentrations of five of the 12 parenteral cephalosporins were lower with the methicillin-susceptible S. aureus than with the methicillin-susceptible S. epidermidis strains.     

The permeability barrier of Haemophilus influenzae type b against beta-lactam antibiotics

An evaluation was made of the role of the outer membrane of Haemophilus influenzae type b as a permeability barrier against beta-lactam antibiotics. Sonic extracts of H. influenzae containing beta-lactamase were assayed for the rates of hydrolysis of benzylpenicillin, ampicillin, cloxacillin, cephacetrile, cefazolin, cefamandole, cephalothin, cephaloridine, cephaloglycin, and cefaclor. Benzylpenicillin was hydrolyzed most rapidly, whereas cephacetrile, cephaloridine, and cephaloglycin were the poorest substrates for the beta-lactamase. The hydrolysis of these ten beta-lactams by intact cells was also determined; it was necessary to stabilize the cells with MgCl2 to prevent lysis and thereby to maintain the beta-lactamase in the periplasm. Calculations were made of the concentration of the antibiotics which had accumulated in the periplasm. The transmembrane permeability coefficient, C, was determined for the ten beta-lactam antibiotics. All of the compounds tested were able to diffuse across the outer membrane of H. influenzae type b very efficiently. The values of the permeability coefficient were compared with the partition coefficients of the antibiotics in a two-phase isobutanol/water mixture. For a ten-fold increase in hydrophobicity, there was a ten-fold decrease in the permeability coefficient. The outer membrane of haemophilus was not an effective barrier against the penetration of penicillins or cephalosporins. The activity of these compounds could be attributed either to their low hydrolysis by beta-lactamase or to the high affinity of binding to their sensitive targets.     

Cephalosporinase Activity in Bacteroides fragilis

Cephalosporinase activity was demonstrated in all of 10 strains of Bacteroides fragilis investigated. Low rates of hydrolysis of cephalosporins (0.25 to 3.5 mumol of cephaloridine per h per 10(9) cells) were found, but no activity against penicillin substrates was detected. In two strains the cephalosporinase activity was increased 40-and 80-fold by growing cells in the presence of penicillin. No permeability barrier for these antibiotics was demonstrated. In most cases the substrate profile showed decreasing activity in the following order: cephaloridine > cephalothin > cephaloglycin > cephalexin. The cephalosporinase from these organisms differed from that found in facultative and aerobic gram-negative bacilli in that it was inhibited by both cloxacillin and p-chloromercuribenzoate. Among nine strains of Bacteroides fragilis subspecies fragilis, correlation was found between in vitro resistance to cephaloridine and amount of beta-lactamase activity in sonically disrupted cells.     

Correlation of in vitro susceptibility with in vivo efficacy in mice for cefoxitin in comparison with cephalosporins.

Agar minimal inhibitory concentrations and mouse protection test effective doses were determined for each of four beta-lactam antibiotics against each of 12 Gram-negative and 3 Gram-positive bacterial cultures. The beta-lactamase activity of these cultures also was studied. The data were examined to determine whether relative in vivo efficacies could be predicted from relative in vitro activities. Although such predictions were quite accurate for cefoxitin and cefazolin, this was not true for cefamandole or for cephalothin. Such poor predictability was not necessarily associated with the susceptibility of these cephalosporins to hydrolysis by bacterial beta-lactamases. Although the clinical significance of these observations is not known, these data emphasize that relative in vitro activities should be used only with caution to estimate in vivo efficacies, since not all compounds show the excellent predictability observed here for cefazolin and cefoxitin.     

Quantitative analysis of beta-lactamase production of multiple resistance to beta-lactam antibiotics in clinical isolates of Escherichia coli

A simple test of procedure is described for the rapid evaluation of beta-lactamase substrate profiles against all the clinically important beta-lactam antibiotics. By use of this method 100 clinical isolates of Escherichia coli were classified into four beta-lactamase types: TEM-like, 35; chromosomal-like, 56; others, 3; beta-lactamase-less, 6 strains. They were tested for their susceptibilities to cefuroxime, cefoxitin, cefamandole, cephalexin, cefazolin, cephaloridine, cephalothin, ampicillin, carbenicillin and kanamycin. The isolates of each of TEM-like and chromosomal-like beta-lactamase types showed a good correlation between the beta-lactamase activity and the minimum inhibitory concentration (MIC) determined by the agar dilution method and the least square line analysis; MIC = ax + b, where x is enzyme activity, and a and b are constants. TEM-like beta-lactamase increased the resistance to ampicillin and carbenicillin markedly and that to cephalothin, cephaloridine and cefamandole moderately. In contrast, chromosomal-like beta-lactamase increased the resistance to ampicillin, cephalexin and cephalothin only slightly.     

Effect of Inoculum and of Beta-Lactamase on the Anti-Staphylococcal Activity of Thirteen Penicillins and Cephalosporins

Because there are few persuasive data for selecting one semisynthetic penicillin or cephalosporin over another for treatment of serious staphylococcal infections, 118 recent clinical isolates of Staphylococcus aureus were studied to determine to what extent the presence of beta-lactamase affected the relative anti-staphylococcal activity of six penicillins and seven cephalosporins. In addition, the effect of inoculum was studied for its possible effect on the anti-staphylococcal activity of the 13 beta-lactam antibiotics. By all criteria, methicillin and nafcillin were clearly more resistant to both the inoculum effect and the production of staphylococcal beta-lactamase, whereas benzylpenicillin and cephaloridine (especially benzyl-penicillin) were the most susceptible to these effects. Cephazolin was clearly more susceptible to staphylococcal beta-lactamase and heavy inocula than the other cephalosporins (with the exception of cephaloridine), whereas cephalothin was the most resistant cephalosporin to these factors. The minimal inhibitory concentration for benzylpenicillin for tests with undiluted inoculum, compared to results with inoculum diluted 10(-4), differed by a factor up to 16,384, whereas with methicillin and nafcillin the differences were rarely more than twofold. Ratios for the other 10 antibiotics fell between these extremes. These results suggest that methicillin or nafcillin is most stable to staphylococcal beta-lactamase, and that benzylpenicillin and cephaloridine are the most susceptible.     

BL-S 640, a Cephalosporin with a Broad Spectrum of Antibacterial Activity: Properties In Vitro

BL-S 640 was evaluated in vitro by comparison with cephalothin, cephaloridine, cefazolin, and cephalexin. The new compound was more active than the control cephalosporins against most major gram-negative and some gram-positive species. Moreover, its antibacterial spectrum included strains of Enterobacter, Proteus morganii, P. rettgeri, and Providencia stuartii, species generally resistant to the other cephalosporins. BL-S 640 was an effective bactericidal agent for strains of various species of Enterobacteriaceae. In human plasma, the compound was 58% protein bound.     

Inhibition of Amino Acid Transport in Escherichia coli by Some Beta-Lactam Antibiotics

Among 10 antibiotics tested, cephaloridine and cephalothin showed the greatest inhibition of proline active transport. Ethylenediaminetetraacetate pretreatment of the cells did not enhance inhibition by any of these 10 antibiotics. The inhibition of active transport of 10 additional amino acids by cephaloridine and cephalothin was studied. Both antibiotics inhibited transport of three amino acids which, like proline, have transport systems resistant to osmotic shock; neither antibiotic inhibited transport of the remaining amino acids, including three with shock-resistant and four with shock-sensitive systems.     

Activity of ten cephalosporins on biomass of methicillin-susceptible and -resistant Staphylococcus aureus

The growth curves automatically recorded and printed during the action of 10 cephalosporins on methicillin-susceptible and methicillin-resistant Staphylococcus aureus showed the following. (i) The biomass of methicillin-susceptible S. aureus exposed to the cephalosporins increased before lysis occurred (inoculum, 10⁶ colony-forming units per ml). Lysis was more rapid with cephalothin and cephaloridine, whose minimal inhibitory concentrations were lowest. (ii) The same biomass increase followed by lysis occurred with methicillin-resistant S. aureus, and the speed of lysis was not different from those of cephalothin (without any regrowth), cefoxitin (with regrowth of a few strains), and cephaloridine (regrowth of all strains), with methicillin-susceptible strains. A 2-log increase of inoculum (10⁸ colony-forming units per ml) did not modify significantly the speed of lysis with cephalothin, cephaloridine, and cefoxitin, but regrowth sometimes occurred. The early transitory lysis caused by cephaloridine, cephalothin, cefamandole, and cefoxitin was not suppressed by preincubation with 32 μg of methicillin per ml, but regrowth occurred more frequently. No lysis could be observed with cefazolin, cefotaxime, cephalexin, cephradine, cefuroxime, and cefaclor unless high concentrations were achieved. (iii) From a practical point of view, the early response of the growth curve (4 h) could not determine in every case whether a strain of S. aureus was resistant or susceptible to cephalosporin. A further study of the growth curve (18 of 24 h) was necessary for this purpose. Results obtained after a few hours with automated systems should be interpreted with great caution.