Studies on the genotoxic and mutagenic potentials of mefloquine

PURPOSE: The detection of mefloquine mutagenicity has not been achieved by the use of Salmonella typhimurium his TA1535; TA1537 as tester strains. With the introduction of improved and more sensitive strains; it is of interest to evaluate the current mutagenic and genotoxic status of the drug. This study presents data on the in-vitro mutagenic and genotoxic potentials of mefloquine hydrochloride clinically used as an antimalarial agent. METHOD : The mutagenicity potentials was investigated in the Escherichia coli WP[2] trp and WP[2] uvrA trp tester strains containing the plasmids; pEB017 and pKM101; and the Salmonella typhimurium TA97 containing pKM101. The genotoxicity potential was determined using the microscreen phage-induction assay. RESULTS: The presence of plasmids pEBO17 and pKM101 enhanced the detection of mutagenicity of mefloquine. Microsomal-activated mefloquine unequivocally elicited base-pair substitution mutagenicity. The genotoxicity test indicated that mefloquine was generally not genotoxic but was of the same potential mutagenicity as chloroquine phosphate. CONCLUSION: Melfloquine hydrochloride exhibits base pair substitution mutagenesis; but not potentially genotoxic; even though it showed concentration dependent cytotoxicity. Its use as a last line antimalarial agent should still be encouraged

Antimutagenic activity of extracts of leaves of four common edible vegetable plants in Nigeria (west Africa).

Organic solvent extracts of leaves of 4 common edible vegetable plants--Bryophyllum pinnatum, Dialium guincense, Ocimum gratissimum and Vernonia amygdalina--had inhibitory activity for His- to His+ reverse-mutations induced by ethyl methanesulfonate acting on Salmonella typhimurium TA100. The concentrated ethyl acetate, methanol and petroleum ether extracts were heat-stable when dissolved in dimethyl sulfoxide. The Bryophyllum ethyl acetate extract was fractionated into alkaloidal/water-soluble, acids, polar lipid and non-polar lipid fractions. The polar and non-polar lipid fractions inhibited reversion mutations induced by ethyl methanesulfonate acting on TA100 or TA102, and were also active against reversions induced by 4-nitro-O-phenylenediamine and 2-aminofluorene in TA98. The alkaloidal/water-soluble and the acid fractions had no appreciable antimutagenic activities.

Properties of R-plasmid pEB017, which confers both enhanced UV-radiation resistance and mutability to wild-type, recA and umuC strains of Escherichia coli K12.

The R-plasmid, pEB017, restored recombination ability to recA56 and conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability to wild-type, recA56 and umuC36 strains of Escherichia coli K12. Comparatively, pEB017 enhanced UV-radiation mutability in a umuC strain, and also enhanced UV-radiation and nitrofuran mutability in a wild-type strain several-fold more than did another R-plasmid, pKM101. Plasmid pEB017 also mediated about a 3-fold enhancement of the SOS induction of beta-galactosidase synthesis in a recA strain, compared with the normal recA+ gene of E. coli. A BamHI fragment of pEB017 DNA was cloned into plasmid vector pBR322 to yield pEB021. The BamHI fragment in pEB021 (3.5 kb) is about 170 bp longer between the BamHI and PstI sites on the left end of the recA-like fragment, compared with published data on a similarly cloned recA gene from E. coli. Plasmid pEB021 conferred enhanced resistance to UV-radiation and enhanced UV-radiation mutability in wild-type and recA strains, and restored recombination ability in a recA strain. The introduction of pEB021 into a umuC strain made the cells slightly more resistant to killing by UV-irradiation, and promoted a small amount of UV-mutability in an otherwise nonmutable strain. These results suggest that R-plasmid pEB017 has a recA-like gene that mediates the enhanced resistance to UV-radiation and enhanced UV-radiation mutability, but which seems different in several important aspects from the normal recA gene in E. coli.

Trimethoprim/sulphamethoxazole resistance in Escherichia coli and Klebsiella spp. urinary isolates.

The susceptibility of 40 Escherichia coli and 35 Klebsiella spp. urinary isolates to trimethoprim/sulphamethoxazole (cotrimoxazole) was determined. The determination was based on the activity of the standard multodiscs-cotrimoxazole (25 micrograms), (Oxoid); sulphafurazole (500 micrograms), (Mastring-S); and the minimum inhibitory concentration of trimethoprim and sulphamethoxazole singly against the isolates. Thirty-two (80%) of 40 isolates of Escherichia coli, and 26 (74%) of 35 isolates of Klebsiella spp. were resistant to cotrimoxazole. All the isolates were resistant to more than 500 mg/l sulphamethoxazole. Twenty of the 32 (62%) cotrimoxazole-resistant E. coli strains and 18 of the 26 (69%) cotrimoxazole-resistant Klebsiella strains were resistant to more than 2000 mg/l trimethoprim. These high level trimethoprim-resistant strains invariably carried transferable resistance to at least the sulphonamides, ampicillin and tetracycline, which could be transferred en bloc to known sensitive recipients by the process of conjugation. The high incidence of the R-plasmid-mediated resistance to high levels of trimethoprim suggests the presence of a selective pressure from the increased, and probably the uncontrolled, use of trimethoprim/sulphonamide proprietary formulations in the society.

The mutagenic activity of chlorpromazine.

The mutagenic activity of chlorpromazine hydrochloride based on the Ames plate incorporation test and the modified fluctuation test in the presence and absence of liver microsomal enzyme (S9 fraction) and NADPH was determined. The results indicated that chlorpromazine required activation for mutagenic activity for the reversion of some of the tester bacterial strains from tryptophan and histidine dependence to independence respectively. The positive response of Escherichia coli WP2 trp, uvrA, E. coli WP2 trp (pKM101 and pAQ1) and Salmonella typhimurium his TA102 indicated that chlorpromazine mediates base-pair substitution and frame-shift mutagenesis.

Expression of a novel R-plasmid pEB017 compared to pKM101 in Escherichia coli wild-type, recA and uvrA strains.

The expression of bacterial resistance to UV irradiation and nitrofurantoin by a novel R-plasmid pEB017 in DNA-repair-proficient (wild-type) and -deficient (recA; uvrA) host strains was compared to the effects of plasmid pKM101 in the isogenic strains. pEB017 partially protected the uvrA strain, and completely protected the wild-type and recA strains from the killing effect of UV irradiation; pKM101 had no effect on the recA strain and only enhanced the survival of the wild-type and the uvrA strains after UV irradiation. pEB017 conferred nitrofurantoin resistance 10-fold on the wild-type and the recA strains and 4-fold on the uvrA strain; pKM101 did not confer nitrofurantoin resistance on the wild-type and recA strains but gave 4-fold resistance in the uvrA strain.

A survey of antibiotic outpatient prescribing and antibiotic self-medication.

In order to assess patterns of antibiotic prescribing and self-medication, a survey was carried out of patients from Government and private hospitals (500 each) and of 1000 apparently healthy adults in Benin City, Nigeria. Ampicillin and tetracycline were the antibiotics commonly used for self-medication; the commonest reasons given for the self-medication were the treatment of sexually transmitted diseases, cough, stomach upsets and diarrhoea. Ampicillin was the commonest prescribed antibiotic; the commonest indications for prescription were soft-tissue, sexually transmitted, upper respiratory and gastro-intestinal infections. According to an assessment by four clinicians from a panel of eight in Government and private practice, 52% of the total prescriptions were judged to be appropriate whereas 30% were judged to be inappropriate by a majority of the physicians. The implications of this study for the control of bacterial resistance to antibiotics are discussed.

Faecal Escherichia coli mediating transferable multi-antibiotic resistance and undesirable extra-chromosomal genes.

A conjugative R-plasmid PE004, Inc F11, conferring resistance to ampicillin, tetracycline, streptomycin, kanamycin and trimethoprim was obtained from an E. coli serotype 026 isolate from the stool of a child with acute diarrhoea. The R-plasmid PE004 also co-transfers an enteropathogenicity antigen without the production of enterotoxins or manifestation of invasiveness. It is not yet known whether this transferable antigen mediates enterocyte damage with consequent diarrhoea. The R-plasmid was of molecular weight 2.4 megadaltons (3.7 kilobase) with a transfer frequency of 6 x 10(-4) cfu/ml E. coli J53-1. The uncontrolled mediation with antibiotics in cases of acute diarrhoea could select gut bacteria not only possessing R-plasmids conferring resistance to several antibiotics but with associated undesirable extrachromosomal genes.

Aspects of chloroquine mutagenicity.

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP2, WP2hcr, WP6 and WP67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP2, WP2hcr; WP6 and WP67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 micrograms/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.

Antimicrobial properties of amniotic fluid from some Nigerian women.

Fifty-one amniotic fluids were aspirated via the vaginal route from 51 pregnant Nigerian mothers. Their antimicrobial activity was tested against Staphylococcus aureus, Escherichia coli and Candida albicans. Inhibition rates were 39.2% for Staph. aureus 19.6% for E. coli and 41.2% for C. albicans. The overall inhibitory capacity was 64.7%. Sixteen fluids (31.4%) were active against one organism, three fluids (5.9%) were active against two organisms and one fluid (2%) was active against the three organisms. Age, parity and meconium-staining of liquor had no correlation with antimicrobial properties. The possible explanations for these are given.

Activity of fosfomycin and R-plasmid conferring fosfomycin resistance among some clinical bacteria isolates in Nigeria.

The in vitro activity of fosfomycin (an antibiotic that has clinically not been widely used in Nigeria) against 516 clinical bacteria isolates and the screening for the presence of R plasmid conferring resistance to fosfomycin among the test bacteria isolates were determined. In the presence of added glucose-6-phosphate (25 micrograms/ml) to the growth medium, all the isolates were inhibited at fosfomycin minimum inhibitory concentrations (MIC) of less than or equal to 32-64 micrograms/ml. Without the glucose-6-phosphate, fosfomycin had MIC75, MIC70, MIC48, and MIC20 at 64 micrograms/ml against Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., and Serratia spp., respectively, while the rest of the strains maintained about the same susceptibility as in the presence of glucose-6-phosphate. An R plasmid of about 59 megadaltons in size, conferring resistance to streptomycin, ampicillin, carbenicillin, and fosfomycin, was obtained from a Serratia liquifacens isolated in an area where fosfomycin had not been clinically used.

Incidence of penicillinase producing Neisseria gonorrhoeae (PPNG) strains and susceptibility of gonococcal isolates to antibiotics in Benin City, Nigeria.

Of 53 strains of Neisseria gonorrhoeae isolated in Benin City, Nigeria, in February 1983 to October 1984, 46 (87%) produced penicillinase. The minimum inhibitory concentrations (MICs) of penicillin G and ampicillin for these isolates were between 1 mg/l and and 50 mg/l. About 48% (22/46) of the penicillinase producing strains were also resistant to streptomycin, cotrimoxazole, and ampicillin and cloxacillin. All 53 isolates were sensitive to tetracycline, erythromycin, amoxycillin and clavulanic acid, nalidixic acid, spectinomycin, and the penicillinase stable cephalosporins. The high incidence of resistance may have been the result of indiscriminate and unsupervised use of antibiotics before patients presented for proper treatment in clinics and hospitals.

Preliminary report on the in vitro antibacterial activity of Bryophyllum pinnatum leaf juice.

The juice from the leaves of Bryophyllum pinnatum S. Kurtz (Crassulaceae) was tested for antibacterial activity. The extract at 5% v/v was bactericidal to a wide spectrum of Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Staphyllococcus aureus, Streptococcus pyogenes; Streptococcus faecalis; Escherichia coli; Proteus spp; Klebsiella spp; Shigella spp; Salmonella spp; Serratia marcescens; and Pseudomonas aeruginosa including the clinical isolates of these organisms possessing multiple antibiotic resistance.

Comparative in vitro activity of ceftriaxone against clinical bacterial isolates in Nigeria.

The in vitro activity of ceftriaxone, a cephalosporin derivative, recently introduced in Nigeria was compared with the activities of other related beta-lactam antibiotics such as cefotaxime, cefuroxime, cephaloridine, cloxacillin/ampicillin and ampicillin against 530 local clinical isolates of gram-negative and gram-positive bacteria. The spectra of activity and potency of ceftriaxone and cefotaxime were generally similar against the Enterobacteriaceae, Pseudomonas aeruginosa, Serratia spp., Streptococcus spp. and Neisseria gonorrhoeae, but less active against Staphylococcus aureus and Staphylococcus epidermidis when compared to cefotaxime. All the other beta-lactam antibiotics tested were less active than ceftriaxone. The action of ceftriaxone was bactericidal and the minimum inhibitory concentration values observed with R-plasmid beta-lactamase mediated resistant Escherichia coli, Klebsiella spp., Proteus spp. and N. gonorrhoeae strains did not exceed the maximum value obtained with the beta-lactamase-negative strains.

In vitro effects of the combination of nitrofurantoin and trimethoprim against sensitive and nitrofurantoin and trimethoprim-resistant Escherichia coli K12.

The combination of subinhibitory concentrations of trimethoprim (Tp) and nitrofurantoin (NT) seemed additively inhibitory against sensitive Escherichia coli K12, chromosomally mediated NT, R-plasmid mediated Tp and NT-resistant E. coli strains, respectively. The minimal inhibitory concentrations of 6 micrograms/NT/ml and 2 micrograms Tp/ml against the sensitive strain were respectively reduced 5-fold by a minimum inhibitory combination of Tp + NT. The 30-fold chromosomally mediated and 15-fold R-plasmid NT resistance were 3- and 5-fold respectively reduced by a minimum inhibitory combination of Tp + NT. The minimum inhibitory combination of Tp + NT also reduced more than 20-fold the at least 1,000-fold resistance level of the R-plasmid mediated Tp resistance. This in vitro activity of NT and Tp could be of clinical relevance in the therapy of urinary tract infections due to multiple antibiotic-resistant strains.

The effects of chlorate- and streptomycin-resistance mutations on nitrofurantoin resistance in Escherichia coli K-12.

Chlorate-resistant mutants with none of the usual pleiotropic effects such as defective nitrate reductase activity were isolated from Escherichia coli K-12. These chlorate-resistant mutants (designated chlHW) did not yield strains with a high level of nitrofurantoin resistance following selection with nitrofurantoin. The chlorate-resistance mutation reduced the nitrofurantoin resistance of high-level mutants to an intermediate level. Further mutation to resistance to streptomycin and other aminoglycoside antibiotics suppressed the effect of chlHW on the level of nitrofurantoin resistance. Other chlorate-resistance genes examined did not have the same effect on nitrofurantoin resistance as chlHW. The gene was cotransducible (Pl) with intermediate-level nitrofurantoin resistance and proC. It is suggested that the chlHW mutation may enhance the accumulation of nitrofurantoin inside the cell since it is known that a multiple aminoglycoside-resistance mutation with pleiotropic effects on the cell membrane can also confer high-level resistance to nitrofurantoin.