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In vitro activity of MK0787 (N-formimidoyl thienamycin) and other beta-lactam compounds against Bacteroides spp.

The susceptibilities of 82 strains of the Bacteroides fragilis group to eight beta-lactam compounds, lincomycin, and metronidazole were determined by using an agar dilution technique. MK0787 (N-formimidoyl thienamycin) was the most active compound, inhibiting all strains at a concentration of 1 microgram/ml. Metronidazole was the only other drug of similar activity. Of the beta-lactam compounds, cefoxitin and MK0787 showed uniform activity against all species, whereas most other compounds were relatively less active against Bacteroides distasonis and Bacteroides thetaiotaomicron than against B. fragilis and Bacteroides vulgatus. Using a well diffusion technique, we determined the relative stability of each beta-lactam compound to sonicated cultures of selected resistant strains. Whereas MK0787 was completely stable to inactivation--and with one exception, cefoxitin was also--ceftriaxone, cefotaxime, cephaloridine, and cefoperazone always showed some inactivation, often quite substantial. Moxalactam and ceftazidime were completely stable to some of the enzyme preparations.

Anti-Bacterial Agents↗

Chemotherapy of an experimental Fusobacterium (Sphaerophorus) necrophorum infection in mice.

An experimental animal model for testing antibiotics in vivo against Fusobacterium (Sphaerophorus) necrophorum has been developed. It incorporates the subcutaneous injection of the bacteria into mice followed by intraperitoneal administration of the antibiotic at 24, 48, 72, and 96 h. Mean effective dose values are based on the number of survivors 21 days after challenge. Tetracycline was the most effective drug tested, with a mean effective dose of 5.0 mg/kg, compared with mean effective dose values of 11.1 for clindamycin, 11.8 for penicillin-G, and 52.9 for lincomycin.

Animals↗

Susceptibility of pneumococci and Haemophilus influenzae to antibacterial agents.

Strains of Diplococcus pneumoniae and Haemophilus influenzae were tested for susceptibility to numerous antibiotics by a twofold agar dilution method using an inocula replicator. Undiluted, fully grown broth cultures were used as inocula for both species, and cultures of pneumococci diluted 1:1,000 were also tested. The antibiotics included most of those in common use in the United States as well as some chemical modifications recently approved and others that are under investigation. The most striking aspect of the results was the marked susceptibility of the pneumococci to all the antibiotics tested except the polymyxins and most of the aminoglycoside antibiotics, although some new aminoglycosides were active in quite low concentrations. Some of the strains of pneumococci were of decreased susceptibility to penicillin G (minimal inhibitory concentrations, 0.2 to 0.4 mug/ml), but none were tetracycline resistant, although such strains had been reported previously from this laboratory. The strains of H. influenzae, which were all serologically nontypable, exhibited different patterns of susceptibility to the groups of antibiotics and to the individual chemically related ones. None of these strains (isolated early in 1972) were ampicillin resistant. The most active agents against H. influenzae were: carbenicillin and ampicillin, analogues related to each of them, rifampin, chloramphenicol, and the polymyxins. However, the tetracycline analogues other than tetracycline, some aminoglycosides, notably tobramycin, kanamycin, gentamicin, and verdamicin, erythromycin, and some new lincomycin analogues were also active in low concentrations. Trimethoprim alone was highly active, and in combination with sulfamethoxazole it was even more active and synergistic against strains of both D. pneumoniae and H. influenzae.

Aminoglycosides↗

Electrotransformation of Clostridium thermocellum.

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.

Anti-Bacterial Agents↗

Susceptibility of salmonellae to cephalosporins and to nine other antimicrobial agents.

Three cephalosporin-related antibiotics and nine other antimicrobial agents were studied for in vitro effectiveness against 54 recently isolated strains of Salmonella. Minimal inhibitory concentrations determined by the plate dilution method demonstrated the following percentages of resistance: ampicillin, 6%; tetracycline, 13%; streptomycin, 52%; sulfadiazine, 94%; cephaloglycin, 96%; and lincomycin, 100%. No strains were resistant to cephalothin, cephaloridine, chloramphenicol, colistimethate, kanamycin, and polymyxin B. The commonest serotype studied, S. typhimurium, showed the greatest antibiotic resistance, with 21% resistant to ampicillin, 36% resistant to tetracycline, and 71% resistant to streptomycin. Cephalothin and cephaloridine were highly effective in vitro but inhibitory concentrations of 20 to 40 mug of cephaloglycin per ml were required for the majority of Salmonella strains.

Ampicillin↗

Chemotherapeutic studies of mycobacterial infections in mice.

Of six antibiotics investigated, streptovaricin C had the most marked chemotherapeutic effect on Mycobacterium kansasii infections in mice. By the intraperitoneal route this antibiotic caused elimination of the pathogens from all organs. Kanamycin eliminated the pathogens from the lungs of all animals and from the spleens and livers of most of them. Bluensomycin also removed the pathogens from the lungs of all animals, and spectinomycin and lincomycin, from the lungs of the majority of the animals. The three latter antibiotics lowered the bacterial counts in liver and spleen. Streptovaricin C also decreased the bacterial counts in brain, spleen, and liver of mice inoculated intracerebrally with M. kansasii. In one experiment it completely eliminated this pathogen from the spleen and almost completely from the liver. The effect of streptovaricin C on the cerebral infection was more marked than that of streptovaricin complex. Respiratory and cerebral infections of mice with M. avium, serotypes I and II, were limited by streptovaricin C, and marked decreases of the bacterial counts in brain, lungs, spleen, and liver were observed.

Animals↗

Comparative inhibition of methicillin-resistant strains of Staphylococcus aureus by lysostaphin and other antibiotics.

Sixteen methicillin-resistant strains of Staphylococcus aureus obtained from Europe were found to be sensitive to the lytic activity of lysotaphin. With only minor exceptions, the strains were found to be sensitive to novobiocin, erythromycin, fusidic acid, and lincomycin, and slightly less sensitive to vancomycin and chloramphenicol. All strains were resistant to tetracycline, penicillinase-sensitive penicillins (benzylpenicillin, ampicillin, and propicillin), penicillinase-resistant penicillins (methicillin, nafcillin, ancillin, oxacillin, cloxacillin, and dicloxacillin), and two cephalosporin antibiotics (cephalothin and cephaloridine).

Anti-Bacterial Agents↗

Microbiological and pharmacological behavior of 7-chlorolincomycin.

Replacement of the 7-(R) hydroxyl group of lincomycin by a 7-chloro-substituent produced a compound with greater in vitro activity than the parent. Laboratory studies of this compound showed it to be highly active against all of the following strains of gram-positive organisms examined, including penicillinase- and nonpenicillinase-producing staphylococci, Diplococcus pneumoniae, Streptococcus viridans and Streptococcus pyogenes. The enterococci, as well as all the gram-negative organisms tested, with the exception of some strains of Haemophilus, were uniformly insensitive to this agent. The activity of 7-chlorolincomycin was not affected by serum or inoculum size. Resistance developed in a slow stepwise pattern. Peak levels of approximately 2 mug/ml were achieved in the serum of volunteers after ingestion of 150 mg either in the fasting state or after a meal. No untoward effects were noted. The antibiotic appears to be of potential value in the treatment of infections due to gram-positive organisms, with the exception of enterococcus.

Adult↗

Susceptibility of genital mycoplasmas to antimicrobial agents.

The susceptibility of 11 T-strains, 12 strains of Mycoplasma hominis, and a single strain of M. fermentans to 15 antimicrobial agents was determined by study of inhibition of metabolic activity in a broth dilution system. All three species were inhibited by tetracycline, chloramphenicol, streptomycin, gentamicin, and kanamycin, and were relatively resistant to cephalothin, cephaloridine, polymyxin, vancomycin, and ampicillin. Three antimicrobial agents had significant differential effects on these species. Erythromycin was more active against T-strains than against M. hominis or M. fermentans. Lincomycin, clindamycin, and nitrofurantoin had greater activity against M. hominis and M. fermentans than against T-strains. The activity of the drugs tested was generally uniform over a wide range of inocula. The effect of pH and the difference between minimal inhibiting and minimal mycoplasmacidal concentrations of the drugs tested were consistent with expectations based on the effects of these drugs on bacteria.

Ampicillin↗

Lateral gene transfer in vitro in the intracellular pathogen Chlamydia trachomatis.

Genetic recombinants that resulted from lateral gene transfer (LGT) have been detected in sexually transmitted disease isolates of Chlamydia trachomatis, but a mechanism for LGT in C. trachomatis has not been described. We describe here a system that readily detects C. trachomatis LGT in vitro and that may facilitate discovery of its mechanisms. Host cells were simultaneously infected in the absence of antibiotics with an ofloxacin-resistant mutant and a second mutant that was resistant to lincomycin, trimethoprim, or rifampin. Selection for doubly resistant C. trachomatis isolates in the progeny detected apparent recombinant frequencies of 10(-4) to 10(-3), approximately 10(4) times more frequent than doubly resistant spontaneous mutants in progeny from uniparental control infections. Polyclonal doubly resistant populations and clones isolated from them in the absence of antibiotics had the specific resistance-conferring mutations present in the parental mutants; absence of the corresponding normal nucleotides indicated that they had been replaced by homologous recombination. These results eliminate spontaneous mutation, between-strain complementation, and heterotypic resistance as general explanations of multiply resistant C. trachomatis that originated in mixed infections in our experiments and demonstrate genetic stability of the recombinants. The kind of LGT we observed might be useful for creating new strains for functional studies by creating new alleles or combinations of alleles of polymorphic loci and might also disseminate antibiotic resistance genes in vivo. The apparent absence of phages and conjugative plasmids in C. trachomatis suggests that the LGT may have occurred by means of natural DNA transformation. Therefore, the experimental system may have implications for genetically altering C. trachomatis by means of DNA transfer.

Anti-Bacterial Agents↗

Properties of ribosomes from Streptomyces erythreus and Streptomyces griseus.

Ribosomes from an erythromycin-producing strain, Streptomyces erythreus, lacked affinity for erythromycin and were also resistant to other macrolide antibiotics (leucomycin, spiramycin, and tylosin) and to lincomycin, whereas Streptomyces griseus B(3) ribosomes were susceptible to all of these antibiotics.

Ammonium Chloride↗

Transfer of plasmid-mediated antibiotic resistance from streptococci to lactobacilli.

The transmissible plasmid pAMbeta1, which codes for erythromycin and lincomycin resistance, was conjugally transferred from a Lancefield group F Streptococcus to a strain of Streptococcus avium. Both organisms served as pAMbeta1 donors for three strains of Lactobacillus casei. Introduction of pAMbeta1 into one of the L. casei strains caused the organism to lose its native 6.7 X 10(6)-dalton plasmid. Loss of the native plasmid produced no alterations in the organism's growth characteristics or fermentation pattern.

Conjugation, Genetic↗

Coresistance to neomycin and kanamycin by mutations in an Escherichia coli locus that affects ribosomes.

Mutant strains resistant to neomycin or to kanamycin sulfate were isolated from Escherichia coli K-12. Nine mutants were analyzed; all were resistant to both antibiotics (about 150 and 100 mug/ml, respectively), and were designated nek. In the mutant strains, the ribosomes are changed from those of the parental strain; for when they were used in assays for polypeptide formation directed by polyadenylic acid or polycytidylic acid, coding fidelity in presence of the drugs was increased and inhibition of synthesis by the drugs was lessened. Mating experiments and transduction tests showed that all of the nine nek mutants are either closely linked or allelic, and the nek locus is closely linked to two genes-str (streptomycin) and spc (spectinomycin)-known to affect the 30S ribosome. The two nek mutants tested were recessive to the sensitive, wild-type allele. When the nek mutants were compared to the parental strain, pleiotropic effects of the nek mutations were observed. Resistance to low levels of streptomycin and spectinomycin was increased, whereas resistance to chloramphenicol was decreased. Also, the mutants were less able to adapt to high concentrations of lincomycin, and could no longer show phenotypic suppression of an arginine requirement by neomycin or kanamycin. Such pleiotropic effects are suggested to be the rule for mutations in genes that participate in the biosynthesis of a cellular organelle.

Alleles↗

Improved GM1-enzyme-linked immunosorbent assay for detection of Escherichia coli heat-labile enterotoxin.

Previously described GM1 ganglioside enzyme-linked immunosorbent assays (GM1-ELISA) for the detection of Escherichia coli heat-labile enterotoxin (LT) showed sensitivity equal to the Y-1 adrenal cell assay when anti-LT (a reagent not commercially available) was used. However, when antitoxin to immunologically related (commercially available) cholera toxin was substituted, a marked loss in sensitivity occurred. We modified the GM1-ELISA that employed anti-cholera toxin to make it comparable in sensitivity to the Y-1 adrenal cell assay. When five media commonly used for LT production were compared, Mundell's Casamino Acids medium was shown to be significantly superior. Lincomycin (45 micrograms/ml) added to E. coli cultures significantly increased net optical densities in the GM1-ELISA, a direct measure of the amount of LT. Treatment of broth cultures or bacterial cell pellets with polymyxin B or extension of culture time to 48 h also significantly increased net optical density by allowing enhanced release of periplasmic LT. A major innovation involved the direct culture of E. coli strains in GM1-coated wells of microtiter plates followed by ELISA. This direct culture method GM1-ELISA (DCM-GM1-ELISA) saved not only assay time, but also materials and reagents. The net optical densities that result from this assay allow the test to be read visually without a spectrophotometer. Three independent observers read plates with E. coli tested by DCM-GM1-ELISA. Thirty-four of 35 adrenal cell-positive strains (97% sensitivity) and 30 of 30 LT-negative control E. coli strains (100% specificity) were identified by all three observers reading coded plates. The DCM-GM1-ELISA provides a simple, practical and efficient assay for LT for less sophisticated laboratories.

Animals↗

Clinical and bacteriological studies with clindamycin.

Fifty patients have been treated with clindamycin, a chemical analogue of lincomycin. Forty-four responded satisfactorily to treatment. Gastrointestinal side-effects were rare though five patients developed rashes. Most recently isolated staphylococci are clindamycin-sensitive.

Adolescent↗