J. T. Smith

R factor-mediated resistance to ultraviolet light in strains of Escherichia coli deficient in known repair functions.

The expression of resistance to u.v. irradiation mediated by R factor R46 has been studied in strains deficient in excision repair and recombination repair. The R factor protected wild-type bacteria and also wild-type cells in which repair had been inhibited by the substitution of bromouracil for chromosomal thymine. It increased the survival of strains defective in the endonucleolytic (uvr), repolymerizing (pol) and joining (lig) stages of the excision repair process. Recombination deficient bacteria mutant at the recB or recC loci were protected by R46, but the R factor had little effect on the survival of a recA strain or a recA recB double mutant. R46 increased the survival of cells that had been treated with chloramphenicol before u.v. irradiation, but did not protect cultures treated with chloramphenciol after irradiation. It is concluded that R46 confers resistance to the lethal effects of u.v. irradiation by a mechanism that is independent of excision repair. Resistance appears to be mediated by an inducible gene product, which is possibly a nuclease and dependent on a functional host recA gene for expression.

Function of the SOS Process in Repair of DNA Damage Induced by Modern 4‐Quinolones

Abstract— The recA13 mutant of Escherichia coli strain K‐12, which lacks recombination and SOS error‐prone DNA repair is hypersensitive to nalidixic acid and to the newer 4‐quinolones ciprofloxacin, norfloxacin and ofloxacin. However, whereas recombination‐proficient but SOS repair‐deficient strains, such as those carrying the lexA3 or recA430 alleles are no more sensitive to nalidixic than the lexA+ recA+ parent, they are more sensitive to the newer quinolones, although not as sensitive as the recA13 derivative. Nalidixic acid possesses only bactericidal mechanism A (which requires RNA and protein synthesis and is only effective on actively dividing cells), whereas the newer 4‐quinolones exhibit additional mechanisms B (which does not require RNA and protein synthesis and is effective on bacteria unable to multiply) and C (which requires RNA and protein synthesis but does not depend on cell division). Results obtained with bacteria suspended in phosphate‐buffered saline, which inhibits mechanism A, and with bacteria suspended in nutrient broth plus rifampicin, which inhibits mechanisms A and C, showed that the lexA3 mutant was still more sensitive than the lexA+ parent under these conditions. The results suggest that, unlike bactericidal mechanism A, DNA damage that results from bactericidal mechanisms B and C of the newer 4‐quinolones is subject to SOS error‐prone (mutagenic) repair.

Post-antibiotic effects of ofloxacin on Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pyogenes

A viable counting technique was used to determine the post-antibiotic effect (PAE) of ofloxacin against four bacterial species, treated with either once of four times the minimum inhibitory concentration for 1 or 3 h. Similar to the results obtained previously with ciprofloxacin, ofloxacin gave PAE values with Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes. Cell division of Klebsiella pneumoniae was inhibited on removal of ofloxacin, but no clear PAE was demonstrated with this species because once replication recommenced, the mean generation times of drug-treated cultures were much shorter than those of untreated controls. Therefore, although the results obtained with ciprofloxacin and ofloxacin imply a consistency of PAE for 4-quinolones within a species, the response to DNA damage induced by 4-quinolones is multifaceted and species dependent. 4-quinolones inhibit both DNA replication and cell division, whilst at the same time stimulating DNA repair pathways. Thus, in some cases PAEs result from an increased post-treatment lag phase which may be followed by nearly normal multiplication, whereas in other cases a long lag may be followed by abnormally rapid cell division, with the generation times of treated cultures being shorter than those of corresponding drug-free controls. The PAE of a drug-induced lag may thus be masked by rapid cell division once growth resumes.

Contributions of post-antibiotic lag and repair-recovery to the post-antibiotic effects of ciprofloxacin on Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pyogenes.

A viable counting technique was used to determine the post-antibiotic effect (PAE) of ciprofloxacin against four bacterial species, treated with either one or four times the minimum inhibitory concentration for 1 or 3 h. PAE were demonstrated with Escherichia coli, Staphylococcus aureus or Streptococcus pyogenes after exposure to either concentration for both times. No clear PAE was demonstrated for Klebsiella pneumoniae after any treatment. In some cases, PAE was due to an increased post-treatment lag phase, which was followed by nearly normal multiplication, whereas in other cases a long lag was followed by abnormally rapid cell division, with the generation times of treated cultures being much less than those of the corresponding drug-free controls. This is evidence of recovery of cells that have completed DNA repair. S. aureus, E. coli and K. pneumoniae all exhibited evidence of this type of repair even though K. pneumoniae gave no significant PAE. However, the post-treatment generation times of S. pyogenes, which produced the greatest PAE, gave no evidence of such repair. It is concluded that PAEs may result from a variety of factors.

THE BACTERICIDAL ACTION OF NALIDIXIC ACID CAN BE POTENTIATED BY AMINOGLYCOSIDES

The bactericidal activity of nalidixic acid can only occur when bacteria are able to synthesize RNA and protein (Deitz et al, 1966) hence bacterial killing by the drug can be antagonized by the addition of a bacteriostatic concentration of rifampicin or chloramphenicol, respectively (Smith, 1984). The aminoglycoside antibiotics also inhibit bacterial protein synthesis and because of this it would seem they too should antagonize the action of nalidixic acid. However, aminoglycosides have recently been discovered to possess another mechanism that operates at concentrations less than those inhibiting protein synthesis. This extra mechanism that takes place at such low concentrations interferes with the initiation of DNA synthesis (Tanaka et al, 1984). As nalidixic acids target site is also associated with DNA metabolism this study was undertaken to test whether the two classes of drugs could interact.

DO R‐FACTORS AFFECT THE ABILITY OF HOST BACTERIA TO COMPETE FOR NUTRIENTS?

Possession of R-factors by bacterid,i.e. the R+ state, confers an advant.agcl over bacterid lacking them (R-), when dntibiotlcs to wt11ci-i t h c RL bacteria are resistant are in use. It is often believea that should the use of antibiotics cease, the occurrence of R-factors would diminish. There is however little evidence to support this view and on the contrary most studies reveal little difference in the growth of R+ and Rbacteria. A major problem with this work is that in mixed cultures the R’ bd riel can t r , l n s f i ’ r , by rlcitiric], the Kfactor to Rbacteria, thus making results of mixed culture studies difficult to interpret. Consequently most workers have studied the growth of each type of organism individually with the attendent drawback that there is no direct competition between the R+ and Rbacteria.

R‐factor mediated resistance to ultraviolet light

organism, as judged by increase in generation time caused by concentrations of 2 and 4 mg ml-l whereas P. niirabilis F67 (TEM) showed a marked sensitivity to this agent, cell lysis occurring at a concentration of 2 mg ml-l. Evidence that R-factor DNA is associated with a cellular component, presumed to be the cytoplasmic membrane has been presented (Hershfield, LeBlanc & Falkow, 1973). In an attempt to elucidate the nature of the R-factor induced effect, spheroplasts were produced by treating exponential cells in DM medium containing 0.4 M sucrose with benzylpenicillin (1000 units ml-l). Spheroplast formation, as judged by phase contrast microscopy was virtually complete after 4 h. The sensitivity of the spheroplasts from each strain to lysis by sodium desoxycholate was similar, suggesting that the R-factors have not caused a gross change in the cytoplasmic membrane and furthermore that the different response of whole cells to sodium desoxycholate is associated with modification to a cell envelope component external to the cytoplasmic membrane resulting in an increased permeability.

CALORIMETRIC INVESTIGATIONS OF ESCHERICHIA COLI TREATED WITH CIPROFLOXACIN AND NALIDIXIC ACID

Calorimetry has been extensively used to study the mechanisms of action of antimicrobials (Beezer, 1980). Changes of heat output caused by antimicrobial agents acting on bacterial metabolism can provide information on the rapidity of the onset of action of such drugs. Furthermore the actual shape of the observed heat output-time curve helps to elucidate the mechanism of drug action. Nalidixic acid (NAL) and ciprofloxacin (CIP) were studied at their most bactericidal concentrations, which are 90 and 1.5pg/ml, respectively (Smith, 1984). In addition the effect of RNA synthesis inhibition was tested by adding rifampicin (RIF) to a concentration of 160 &ml. The heat output of about lo7 E.coli per ml in nutrient broth with and without the 4-quinolones and rifampicin was monitored in an adiabatic batch titration calorimeter. Each heat output-time curve was normalised according to the number of organisms present at the start of each experiment then characterised by the initial rate of heat output, the total heat output and the time at which heat output ceased. From replicate determinations the limits of error on the data in Table 1 are flOX; arising from errors involved in viable count determinations.

EFFECT OF TEMPERATURE ON BACTERIAL MUTATIONAL CIPROFLOXACIN RESISTANCE

The commonest mechanism causing clinical resistance of bacteria to antibiotics and chemotherapeutic agents is the possession of transferrable drug resistance plasmids. However, four exceptional antibacterials which do not suffer from plasmid-mediated resistance are polymyxin, nitrofurantoin, metronidazole and the g-quinolones. for bacteria is chromosomal mutation. mutational resistance in vitro with such drugs have more clinical relevance than such studies with other antibacterials. With E. coli mutational resistance to ciprofloxacin has been shown to occur more frequently at 30°C than at 25 or 37°C (Smith 1986). It was not known whether this effect was species-specific, so other bacteria were investigated as follows. Minimum inhibitory concentrations (MIC‘s) of ciprofloxacin were determined using 2 x 105-106 colony-forming units of Staph. aureus, Staph. epidermidis, Pseudomonas aerusinosa and E . coli inoculated on nutrient agar incubated at 25, 30 and 37°C. After 1 to 3 days (depending on the temperature) the lowest concentration of ciprofloxacin inhibiting colony formation was recorded. Bacterial cultures concentrated 20-fold by centrifugation were spread on nutrient agar containing 5 times the MIC of ciprofloxacin for each organism at each temperature and the plates incubated for up to 6 days. Colonies were counted and their frequency of occurrence used to calculate mutation rates. With these four the only possible mechanism of clinical resistance Consequently studies of the development of

ACTIVITY OF TETROXOPRIM AGAINST R-FACTOR MEDIATED TRIMETHOPRIM RESISTANT BACTERIA

The enzyme Dihydrofolate reductase (DHFR) catalyzes the production of tetrahydrofolate (THF) from dihydro-folate (DHF) in both prokaryotes and eukaryotes. THF and its derivatives play a vital role in one carbon unit metabolism and are the sole source of reduction potential in thymidine biosynthesis. Inhibitors of DHFR hence have pronounced effects on DNA and other syntheses. exist although to be of use in antibacterial chemotherapy they must be considerably more active against bacterial than mammalian DHFR. Until recently only one such inhibitor, trimethoprim (Tm, R = -CH3), which is 80,000 times more active against bacterial than mammalian DHFR (Burchall However, Heumann and Co. have now developed a new bacterial DHFR inhibitor, tetroxoprim (Tx, R = -(CH~)Z-O-CH~), which is at least 50,000 times more active against bacterial than mammalian DHFR (Aschhoff and Vergin 1979). Several such inhibitors