Simultaneous breakdown of multiple antibiotic resistance mechanisms in S. aureus.

In previous studies, the oligo-acyl-lysyl (OAK) C12(ω7)K-ß12 added to cultures of gram-positive bacteria exerted a bacteriostatic activity that was associated with membrane depolarization, even at high concentrations. Here, we report that multidrug-resistant Staphylococcus aureus strains, unlike other gram-positive species, have reverted to the sensitive phenotype when exposed to subminimal inhibitory concentrations (sub-MICs) of the OAK, thereby increasing antibiotics potency by up to 3 orders of magnitude. Such chemosensitization was achieved using either cytoplasm or cell-wall targeting antibiotics. Moreover, eventual emergence of resistance to antibiotics was significantly delayed. Using the mouse peritonitis-sepsis model, we show that on single-dose administration of oxacillin and OAK combinations, death induced by a lethal staphylococcal infection was prevented in a synergistic manner, thereby supporting the likelihood for synergism to persist under in vivo conditions. Toward illuminating the molecular basis for these observations, we present data arguing that sub-MIC OAK interactions with the plasma membrane can inhibit proton-dependent signal transduction responsible for expression and export of resistance factors, as demonstrated for ß-lactamase and PBP2a. Collectively, the data reveal a potentially useful approach for overcoming antibiotic resistance and for preventing resistance from emerging as readily as when bacteria are exposed to an antibiotic alone.

Investigating the impact of bisphosphonates and structurally related compounds on bacteria containing conjugative plasmids.

Bacterial plasmids propagate through microbial populations via the directed process of conjugative plasmid transfer (CPT). Because conjugative plasmids often encode antibiotic resistance genes and virulence factors, several approaches to inhibit CPT have been described. Bisphosphonates and structurally related compounds (BSRCs) were previously reported to disrupt conjugative transfer of the F (fertility) plasmid in Escherichia coli. We have further investigated the effect of these compounds on the transfer of two additional conjugative plasmids, pCU1 and R100, between E. coli cells. The impact of BSRCs on E. coli survival and plasmid transfer was found to be dependent on the plasmid type, the length of time the E. coli were exposed to the compounds, and the ratio of plasmid donor to plasmid recipient cells. Therefore, these data indicate that BSRCs produce a range of effects on the conjugative transfer of bacterial plasmids in E. coli. Since their impact appears to be plasmid type-dependent, BSRCs are unlikely to be applicable as broad inhibitors of antibiotic resistance propagation.

Antibacterial & antiplasmid activities of Helicteres isora L.

BACKGROUND & OBJECTIVES: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. METHODS: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. RESULTS: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 microg/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. INTERPRETATION & CONCLUSIONS: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

In vitro studies of plasmid-mediated penicillinase from Streptococcus faecalis suggest a staphylococcal origin.

A strain of Streptococcus faecalis with plasmid-mediated penicillinase production was studied further. Partially purified penicillinase from the S. faecalis strain hydrolyzed penicillin, ampicillin, and ureido-penicillins but not penicillinase-resistant semisynthetic penicillins, cephalosporins, or imipenem; hydrolysis was inhibited by clavulanic acid. Hydrolysis of a given antibiotic correlated with a marked increase in the minimal inhibitory concentration (MIC) of that drug when a high inoculum was used. As with most enterococci, the MICs of cephalosporins and penicillinase-resistant semisynthetic penicillins were too high for clinical usefulness, although these agents did not show an inoculum effect. Based upon hybridization under stringent conditions of plasmid DNA from the S. faecalis strain to cloned penicillinase genes from Staphylococcus aureus, it appears that these resistance determinants are highly homologous and suggests that this enzyme was introduced into streptococci from staphylococci.

Metal Resistance and Its Association With Antibiotic Resistance.

Antibiotic resistance is recognised as a major global threat to public health by the World Health Organization. Currently, several hundred thousand deaths yearly can be attributed to infections with antibiotic-resistant bacteria. The major driver for the development of antibiotic resistance is considered to be the use, misuse and overuse of antibiotics in humans and animals. Nonantibiotic compounds, such as antibacterial biocides and metals, may also contribute to the promotion of antibiotic resistance through co-selection. This may occur when resistance genes to both antibiotics and metals/biocides are co-located together in the same cell (co-resistance), or a single resistance mechanism (e.g. an efflux pump) confers resistance to both antibiotics and biocides/metals (cross-resistance), leading to co-selection of bacterial strains, or mobile genetic elements that they carry. Here, we review antimicrobial metal resistance in the context of the antibiotic resistance problem, discuss co-selection, and highlight critical knowledge gaps in our understanding.

[Plasmid elimination effect of usnic acid on antibiotic-resistant Staphylococcus aureus].

OBJECTIVE: To observe the influence of Usnic acid on the antibiotic-resistant plasmid in Staphylococcus aureus (Sa). METHODS: The antibiotic-resistant plasmid was abstracted from the clinical divided strain of Sa and plasmid elimination test was performed in vitro. RESULTS: Plasmid elimination test showed that Usnic acid could eliminate the resistant plasmid in Sa effectively. At 24th and 48th hour after the treatment of Usnic acid, the elimination rate of resistant plasmid was 5.2% and 16.4% respectively. CONCLUSION: Usnic acid can eliminate the antibiotic-resistant plasmid in Sa. It is possible to use Usnic acid to treat the infection of antibiotic-resistant Sa in clinic.

[Influence of increased partial argon and nitrogen pressure on the R-plasmid Escherichia coli transfer frequency].

Subject of the study was frequency of transfer of bacterial plasmids determining the antibiotic resistance (R-plasmid) in argon-containing gas mixtures. Investigated were reference strains of Escherichia coli K-12. Conjugation took place during incubation in pAr/pN2/pO2, ata (1 ata = 10(5) kPa): No. 1 - 0.0/0.8/0.2 (air control); No. 2 - 1.0/0.8/0/2; No. 3 - 0.0/1.8/0.2; No. 4 - 1.0/1.8/0.2. E.coli conjugation was found inhibited after 24 hrs. of incubation with pAr elevated to 1 ata, whereas elevated partial pressure of argon (1 ata) and nitrogen (1.8 ata) brought transfer frequency down to the minimum.

Changing patterns of hospital infections: implications for therapy. Changing mechanisms of bacterial resistance.

During the past decade there has been a marked increase in resistance of bacteria to antimicrobial agents. Microorganisms have developed the ability to make altered receptors for antimicrobial agents, have prevented agents from reaching their receptors within the bacterial cell, now have enzymes to destroy antibiotics, and have resistant metabolic pathways. Altered penicillin receptors, penicillin-binding proteins, have been found in Streptococcus pneumoniae, Streptococcus faecalis, Staphylococcus aureus, Neisseria gonorrhoeae, Clostridium, and Pseudomonas aeruginosa. Resistance based on decreased entry of drugs has been found for penicillins, cephalosporins, aminoglycosides, and tetracyclines in the Enterobacteriaceae and Pseudomonas aeruginosa. Beta-lactamase resistance has increased significantly being encountered in Neisseria, Haemophilus, Enterobacteriaceae, and Pseudomonas species. Chromosomal inducible beta-lactamases, which function as cephalosporinases, have been a particular problem in Enterobacter and Citrobacter species, and organisms resistant to the third-generation cephalosporins have been isolated from patients. It is clear that beta-lactamases and changes in cell wall permeability will play an extremely important role in the future of the new penicillins and cephalosporins.

Loss of plasmid linked antibiotic resistance in Escherichia coli on treatment with some phenolic compounds.

Treatment of E. coli 46R641 cells carrying multidrug resistant TP181 plasmid with phenolic compounds namely bharangin, gossypetin, gossypin and quercetin lead to the concurrent loss of all the six plasmid linked antibiotic resistance markers. Among these test compounds, bharangin exhibited higher efficiency in curing of plasmids belonging to IncF1, H1 and X groups. However, multicopy plasmids with ColE1 origin of replication were totally refractory to these curing agents under similar conditions. The curing activity of the test compounds was much higher as compared to some of the known curing agents.

Analysis of neomycin, kanamycin, tobramycin and amikacin resistance mechanisms in gentamicin-resistant isolates of Enterobacteriaceae.

Twenty-four gentamicin-resistant isolates of Enterobacteriaceae, obtained from the clinical laboratories of three health centres in Nablus, Palestine, were tested for susceptibility to neomycin, kanamycin, tobramycin and amikacin. Resistance rates were 29.2% for neomycin, 58.3% for kanamycin, 45.8% for tobramycin and 8.3% for amikacin. Fourteen (58.3%) isolates were noted to be multiresistant, i.e., resistant to gentamicin and two or more other aminoglycosides; resistance to gentamicin, kanamycin and tobramycin was the most common pattern of multiple resistance. This pattern implies the involvement of adenyltransferase ANT(")-I activity. Plasmid profiles and curing experiments suggested a plasmid localisation of gentamicin, neomycin, kanamycin and tobramycin resistance genes. However, a chromosomal location is proposed for plasmid-deficient strains. Cross-resistance in two isolates to all aminoglycosides tested suggested membrane impermeability to aminoglycosides as the mechanism of resistance.

Emergence of tetracycline resistance due to a multiple drug resistance plasmid in Vibrio cholerae O139.

Of the 173 clinical strains of Vibrio cholerae O139 isolated from India, Bangladesh, and Thailand tested, six strains from India were resistant to tetracycline, ampicillin, chloramphenicol, kanamycin, and gentamicin. These six strains harbored a self-transmissible plasmid that mediated resistance to tetracycline, ampicillin, chloramphenicol, kanamycin, gentamicin, sulfamethoxazole, trimethoprim, and O/129. The multiple drug resistance plasmids were 200 kb in size and belonged to the incompatibility group C. Although a majority of the O139 strains (94.8%) were highly resistant to streptomycin, sulfamethoxazole, trimethoprim, and O/129, the tetracycline-susceptible strains so far tested were plasmid-negative. The data suggest the existence of two distinct multiple antimicrobial agent resistance (MAR) patterns in V. cholerae O139.

Pyrimethamine-sulfadoxine still effective against Plasmodium falciparum in Jayapura, Irian Jaya: RI-type resistance in 2 of 18 patients.

The sensitivity of Plasmodium falciparum infections to pyrimethamine-sulfadoxine was studied in 18 Indonesian patients in Jayapura, Irian Jaya. In 16 of the 18 patients parasitaemia was cleared by day 6 and the patients remained without parasitaemia through day 28. Two of the 18 patients had late recrudescences consistent with RI-type resistance; one each on days 14 and 21. Pyrimethamine-sulfadoxine is still an effective antimalarial for most patients with falciparum malaria in Jayapura.

Population shift in drug resistant and sensitive Escherichia coli during antibiotic treatment of children with diarrhea.

Changes in composition of Escherichia coli were followed during antibiotic therapy of nin children with acute diarrhea. In five cases enteropathogenic serotypes of E. coli were multiple-drug resistant, and in four other this feature characterized nonpathogenic E. coli strains. The results point to the importance of choice of the drugs effective not only against pathogenic serotypes, but also against nonpathogenic ones, and uselessness and even harmfulness of chemotherapy "in blindly".

Effect of EDTA-tris on an Escherichia coli isolate containing R plasmids.

Solutions of ethylenediaminetetraacetate (EDTA)-tris combined with antibiotics have been shown to be effective in treating selected cases of persistent bacterial infections. Basic techniques in microbial genetics, including mating frequencies, chemical elimination of R plasmids, isolation of plasmid DNA and agarose gel electrophoresis, were used to determine if EDTA-tris has a curing effect on an R plasmid as part of its clinical action. Results of this study indicated that EDTA-tris by itself eliminated an antibiotic resistance marker from a clinical isolate of Escherichia coli and when combined with another chemical curing agent altered the isolate's mating frequency.

Inhibition by paromomycin of R-factor transfer of tetracycline resistance between Escherichia coli and Salmonella pullorum.

In vitro assays showed that low concentrations (5--10 and 20 microgram/ml) of the antimicrobial paromomycin sulfate are able to block or diminish significantly the transfer of the tetracycline resistance R-factor between Escherichia coli and Salmonella pullorum. This observation is important because it offers the possibility of preventing the formation of tetracycline-resistant pathogens, a limiting factor of tetracycline use in both human and veterinary medicine.

Loss of penicillinase plasmids of Staphylococcus aureus after treatment with L-ascorbic acid.

When 2 clinical strains of plasmid-mediated penicillin-resistant Staphylococcus aureus were treated with 1 mM sodium ascorbate for 6 h, 12-35% colony-forming units (CFU) irreversibly lost their ability to produce beta-lactamase. Agarose gel electrophoresis showed the disappearance of the plasmid bands from the lysates of colonies in which susceptibility to penicillin was induced by ascorbate treatment. Since ascorbic acid is a compound that is completely non-toxic, the possibility of its therapeutic use in the treatment of multiresistant bacterial diseases is proposed.

Decreased resistance to antibiotics and plasmid loss in plasmid-carrying strains of Staphylococcus aureus treated with ascorbic acid.

The effect of ascorbic acid on plasmid-coded antibiotic resistance in Staphylococcus aureus was investigated. Several strains of S. aureus were cultured in the presence of 1 mM ascorbate for 6 h. This treatment induced an increased loss of resistance markers in 4 of 6 strains tested, and agarose gel electrophoresis showed this disappearance of plasmid DNA in ascorbate-induced susceptible colonies. The presence of ascorbate induced a 50-75% decrease in minimal inhibitory concentrations of different antibiotics for resistant strains. When ascorbate is added, formerly subinhibitory concentrations of penicillin or tetracycline have an increased inhibitory effect on resistant strains and even induced the death of 25-93% of the initial population. These results suggest that ascorbate can induce the loss of several plasmids of S. aureus, and that the levels of antibiotic resistance are also affected by the presence of this compound.

Effects of the antimutagen cinnamaldehyde on reversion and survival of selected Salmonella tester strains.

The antimutagenic activity of trans-cinnamaldehyde (C6H5CH = CHCHO) on chemically induced mutagenesis has been shown in E. coli. Using the Ames Salmonella typhimurium tester strains TA1535 (hisG46 uvrB rfa) and TA100 (TA1535/pKM101), the effects of cinnamaldehyde on spontaneous reversions and reversions induced by 4-nitroquinoline-N-oxide(4NQO) and ethyl methanesulfonate (EMS) have been examined. To observe the effect of cinnamaldehyde in the absence of functional muc genes, a third strain, TA1535/pGW201 (pKM101 muc140: :Tn5) was included in the testing. Modifications of the standard Ames test procedures and direct-plating techniques were employed to study the "antimutagenic" response exerted by cinnamaldehyde. In all strains tested, concentrations of cinnamaldehyde up to 25 micrograms/ml slightly decreased the number of spontaneous reversions and induced reversions were more markedly reduced. The decreases in the numbers of 4NQO-induced revertants were greater than those decreases which occurred for EMS-induced reversions. There was no effect on viability in 1% (v/v) nutrient broth supplemented minimal medium containing 5-25 micrograms/ml of cinnamaldehyde. Cinnamaldehyde did not display any mucAB dependent or independent specificity against the mutagens used. On minimal medium supplemented with histidine and biotin, concentrations of cinnamaldehyde above 10 micrograms/ml were lethal for the strains tested. When the test medium was supplemented with 1-5% (v/v) liquid nutrient broth, viability was not affected at concentrations up to 25 micrograms/ml. For both TA100 and TA1535 the presence of 20 micrograms/ml of cinnamaldehyde in 1% (v/v) liquid nutrient broth-supplemented minimal glucose broth extended the lag phase for 2-4 h with no effect on survival. Depending on the test procedure employed, decreases in numbers of revertants may reflect lethality rather than antimutagenesis. When used to test for antimutagenesis rather than mutagenesis, modifications of the standard Ames test procedure may mimic an antimutagenic response due to a decrease in the total number of revertants seen even though enough cells survive to produce a background lawn.

Mutagenicities of nitrofuran derivatives on a bacterial tester strain with an R factor plasmid.

Many nitrofuran derivatives are known to be mutagenic on Escherichia coli WP2 but not on Salmonella typhimurium TA1535, TA1536, TA1537 or TA1538. Ames and coworkers recently obtained a new tester strain of S. typhimurium, TA100, by putting an R factor plasmid, pKM101, into TA1535. We found that all mutagenic nitrofuran derivatives previously found to be mutagenic on E. coli WP2 were mutagenic on this new strain (TA100).

6-Acetylmethylenepenicillanic acid (Ro 15-1903), a potent beta-lactamase inhibitor. I. Inhibition of chromosomally and R-factor-mediated beta-lactamases.

6-Acetylmethylenepenicillanic acid (Ro 15-1903) was seen to be a powerful inhibitor of various beta-lactamases. Nearly all of the chromosomally and R-factor-mediated beta-lactamases which were studied were inhibited at much lower concentrations than required with clavulanic acid or sulbactam. Ro 15-1903 protected beta-lactamase-labile penicillins and cephalosporins from hydrolysis. The compound itself was stable against hydrolysis and inhibition of beta-lactamase was irreversible.