Impact of specific inhibitors on metallo-ß-carbapenemases detected in Escherichia coli and Klebsiella pneumoniae isolates.

Carbapenems are widely regarded as the drugs of choice for the treatment of severe infections caused by extended-spectrum beta lactamases producing Enterobacteriaceae. The emergence of carbapenem-resistant organisms is worrisome due to the limited treatment options. Detection of carbapenemase-producing bacteria is critical for the choice of appropriate therapy. However, Inhibition of carbapenemases is an alternative approach to combat resistance to carbapenms. In this study, Escherichia coli and Klebsiella pneumoniae carbapenem resistant isolates were recovered from 300 clinical isolates. They were subjected phenotypically for detection of class B metallo-carbapenemase (MBL) producers (by carbapenem disks with or without EDTA), and were subjected for confirmation genotypically by PCR. In addition, the synergistic activities of MBL-inhibitors in combination with carbapenems were elucidated. Two E. coli and 15 K. pneumoniae isolates were carbapenem resistant. The genes encoding blaNDM-1 carbapenemase were detected in 16/17 isolates solely, or collaboratively with either blaVIM, or blaIMP or both in all carbapenem resistant isolates, by PCR method. The VIM-carbapenemase was encoded by one isolate. In pre-clinical trials for development of MBL-specific inhibitors, Sub-inhibitory concentrations of citric acid, malic acid, ascorbic acid and ciprofloxacin in combination with imipenem or meropenem exerted synergistic activities against metallo-carbapenemases. Their activities are probably attributed to the chelation of zinc ions in the active site of carbapenemase. Conclusively, these promising combined therapies might represent a new strategy for combating such serious infections caused by metallo-B-carbapenemase producers of K. pneumoniae and E. coli isolates.

Ciprofloxacin-Mediated Mutagenesis Is Suppressed by Subinhibitory Concentrations of Amikacin in Pseudomonas aeruginosa.

Resistance to antibiotics is a global health problem. Activation of the SOS response, and the subsequent elevation in mutagenesis, contributes to the appearance of resistance mutations. Among currently used drugs, quinolones are the most potent inducers of the SOS response. In the present study, we show that amikacin inhibits ciprofloxacin-mediated SOS induction and mutagenesis in Pseudomonas aeruginosa.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella typhimurium and Salmonella typhi.

OBJECTIVES: Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. METHOD: We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RT-PCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. RESULTS: Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. CONCLUSIONS: The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon γ (IFNγ), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumin's ability to hinder the bactericidal action of ciprofloxacin and IFNγ might significantly augment Salmonella pathogenesis.

Ciprofloxacin-induced antibacterial activity is reversed by vitamin E and vitamin C.

In the present study, we investigated the possible involvement of oxidative stress in ciprofloxacin-induced cytotoxicity against several reference bacteria including Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, and clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). Oxidative stress was assessed by measurement of hydrogen peroxide generation using a FACScan flow cytometer. The antibacterial activity of ciprofloxacin was assessed using the disk diffusion method and by measuring the minimum inhibitory concentration (MIC). Ciprofloxacin induced a dose-dependent antibacterial activity against all bacteria where the highest tested concentration was 100 ug/ml. Results revealed that E. coli cells were highly sensitive to ciprofloxacin (MIC = 0.21 µg/mL ± 0.087), P. aeruginosa and S. aureus cells were intermediately sensitive (MIC = 5.40 µg/mL ± 0.14; MIC = 3.42 µg/mL ± 0.377, respectively), and MRSA cells were highly resistant (MIC = 16.76 µg/mL ± 2.1). Pretreatment of E. coli cells with either vitamin E or vitamin C has significantly protected cells against ciprofloxacin-induced cytotoxicity. These results indicate the possible antagonistic properties for vitamins C or E when they are used concurrently with ciprofloxacin.

Study on the susceptibility of Enterococcus faecalis from infectious processes to ciprofloxacin and vancomycin

Enterococcus faecalis is considered a pathogen responsible for hospital infections and, due to its frequent multi-resistant profile, has caused preoccupations among many medical authorities. The objective of this study was to determine the antimicrobial susceptibility of 74 strains isolated from blood cultures and purulent secretions to vancomycin and ciprofloxacin through the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) by using the Microdilution test. The results showed a greater efficacy of vancomycin compared to ciprofloxacin (98.6% of the strains were inhibited by vancomycin at lower concentrations: 0.06 - 1 mg/ml). However, in the MBC analysis 73% of the strains showed a MBC of vancomycin only at high concentrations (equal to or higher than 64 mg/ml). For ciprofloxacin, the strains showed a broad sensitivity with MICs and MBCs distributed along all the MIC classes. Results also revealed a probability that some strains are tolerant to vancomycin, which indicates the need of other tests to confirm this characteristic

Evolution of ciprofloxacin-resistant Staphylococcus aureus in in vitro pharmacokinetic environments.

The development of novel antibacterial agents is decreasing despite increasing resistance to presently available agents among common pathogens. Insights into relationships between pharmacodynamics and resistance may provide ways to optimize the use of existing agents. The evolution of resistance was examined in two ciprofloxacin-susceptible Staphylococcus aureus strains exposed to in vitro-simulated clinical and experimental ciprofloxacin pharmacokinetic profiles for 96 h. As the average steady-state concentration (C(avg ss)) increased, the rate of killing approached a maximum, and the rate of regrowth decreased. The enrichment of subpopulations with mutations in grlA and low-level ciprofloxacin resistance also varied depending on the pharmacokinetic environment. A regimen producing values for C(avg ss) slightly above the MIC selected resistant variants with grlA mutations that did not evolve to higher levels of resistance. Clinical regimens which provided values for C(avg ss) intermediate to the MIC and mutant prevention concentration (MPC) resulted in the emergence of subpopulations with gyrA mutations and higher levels of resistance. A regimen producing values for C(avg ss) close to the MPC selected grlA mutants, but the appearance of subpopulations with higher levels of resistance was diminished. A regimen designed to maintain ciprofloxacin concentrations entirely above the MPC appeared to eradicate low-level resistant variants in the inoculum and prevent the emergence of higher levels of resistance. There was no relationship between the time that ciprofloxacin concentrations remained between the MIC and the MPC and the degree of resistance or the presence or type of ciprofloxacin-resistance mutations that appeared in grlA or gyrA. Regimens designed to eradicate low-level resistant variants in S. aureus populations may prevent the emergence of higher levels of fluoroquinolone resistance.

[Synthesis and antibacterial activity of pyridonecarboxylic acid derivatives containing 2-methyl-5-nitroimidazol].

AIM: To study the synthesis and antibacterial activity of pyridonecarboxylic acid derivatives containing 2-methyl-5-nitroimidazol. METHODS: Pyridonecarboxylic acid derivatives containing 2-methyl-5-nitroimidazol were synthesized primarily from 2-methyl-5-nitroimidazol, norfloxacin, ciprofloxacin, enoxacin via nucleophilic substitution and esterification. The antibacterial activity of the nine target compounds were tested. RESULTS: Nine new compounds were synthesized (IIa-c and IIIa-f). The structure of the title compounds were identified by 1HNMR, MS as well as elementary analysis. CONCLUSION: Compounds IIa, IIb and IIc showed antibacterial activity, and were worth further studying.

Two short peptides including segments of subunit A of Escherichia coli DNA gyrase as potential probes to evaluate the antibacterial activity of quinolones.

Quinolones constitute a family of compounds with a potent antibiotic activity. The enzyme DNA gyrase, responsible for the replication and transcription processes in DNA of bacteria, is involved in the mechanism of action of these drugs. In this sense, it is believed that quinolones stabilize the so-called 'cleavable complex' formed by DNA and gyrase, but the whole process is still far from being understood at the molecular level. This information is crucial in order to design new biological active products. As an approach to the problem, we have designed and synthesized low molecular weight peptide mimics of DNA gyrase. These peptides correspond to sequences of the subunit A of the enzyme from Escherichia coli, that include the quinolone resistance-determining region (positions 75-92) and a segment containing the catalytic Tyr-122 (positions 116-130). The peptide mimic of the non-mutated enzyme binds to ciprofloxin (CFX) only when DNA and Mg2+ were present (Kd = 1.6 x 10(-6) M), a result previously found with DNA gyrase. On the other hand, binding was reduced when mutations of Ser-83 to Leu-83 and Asp-87 to Asn-87 were introduced, a double change previously found in the subunit A of DNA gyrase from several CFX-resistant clinical isolates of E. coli. These results suggest that synthetic peptides designed in a similar way to that described here can be used as mimics of gyrases (topoisomerases) in order to study the binding of the quinolone to the enzyme-DNA complex as well as the mechanism of action of these antibiotics.

[The participation of the transport-barrier functions of the plasma membrane in the development of fluoroquinolone (ciprofloxacin) resistance in Acholeplasma laidlawii]. / Uchastie transportno-bar'ernykh funktsii plazmaticheskoi membrany v razvitii rezistentnosti k ftorkhinolonam (tsiprofloksatsinu) u Acholeplasma laidlawii.

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin was investigated. It was shown that ethidium bromide used as fluoroquinolone analogue in plasmatic membrane efflux pump was accumulated in ciprofloxacin-resistant cells in much less amount. It was estimated that ethidium bromide efflux depended on temperature, glucose and transmembrane electro-chemical proton potential. Inhibitors of efflux systems--reserpine and verapamil enhanced the ethidium bromide accumulation much more intensively in ciprofloxacin resistant cells. The results of investigation allowed to consider the existence of active efflux system for toxic agents in acholeplasma; in the case of ciprofloxacin-resistant strain these systems are inducible.

Elimination of fecal Enterobacteriaceae by intravenous ciprofloxacin is not inhibited by concomitant sucralfate--a microbiological and pharmacokinetic study in patients.

Intravenously administered ciprofloxacin is partially secreted into the intestinal lumen and thereby eliminates fecal Enterobacteriaceae. Sucralfate inhibits the antimicrobial activity of ciprofloxacin by chelate binding. In a prospective study, we investigated the impact of intravenous ciprofloxacin on the intestinal microflora during oral administration of sucralfate. A total of 45 stool specimens were analyzed in 20 hospitalized patients who were treated with 200 mg of ciprofloxacin i.v. bid. Ten patients concomitantly received 1 g sucralfate p.o. tid (group A). After more than 3 days of i.v. ciprofloxacin, the mean fecal ciprofloxacin concentration was 185.3 +/- 158.7 micrograms/g in patients of group A and 108.7 +/- 76.9 micrograms/g in patients without concurrent sucralfate (group B). There was no significant difference in mean fecal ciprofloxacin levels between both groups (Wilcoxon's test). Enterobacteriaceae were below the threshold of detection (10(2) cfu/g) in all patients of group B after 3 days of treatment whereas small numbers were found in only 2 samples of patients of group A (10(4) cfu/g). Intravenous ciprofloxacin eliminates or largely reduces intestinal Enterobacteriaceae irrespective of concurrent administration of sucralfate.

In vitro activities of ciprofloxacin and rifampin alone and in combination against growing and nongrowing strains of methicillin-susceptible and methicillin-resistant Staphylococcus aureus.

We characterized the effects of ciprofloxacin and rifampin alone and in combination on Staphylococcus aureus in vitro. The effects of drug combinations (e.g., indifferent, antagonistic, or additive interactions) on growth inhibition were compared by disk approximation studies and by determining the fractional inhibitory concentrations. Bactericidal effects in log-phase bacteria and in nongrowing isolates were characterized by time-kill methods. The effect of drug combinations was dependent upon whether or not cells were growing and whether killing or growth inhibition was the endpoint used to measure drug interaction. Despite bactericidal antagonism in time-kill experiments, our in vitro studies suggest several possible explanations for the observed benefits in patients treated with a combination of ciprofloxacin and rifampin for deep-seated staphylococcal infections. Notably, when growth inhibition rather than killing was used to characterize drug interaction, indifference rather than antagonism was observed. An additive bactericidal effect was observed in nongrowing bacteria suspended in phosphate-buffered saline. While rifampin antagonized the bactericidal effects of ciprofloxacin, ciprofloxacin did not antagonize the bactericidal effects of rifampin. Each antimicrobial prevented the emergence of subpopulations that were resistant to the other.

[The appearance of ciprofloxacin resistance in the microbial strains isolated from gynecologic patients]. / Za poiavata na rezistentnost kum tsiprofloksatsin u mikrobni shtamove, izolirani ot ginekologichno bolni zheni.

The sensitivity of 415 microbial strains isolated from clinically taken samples from gynecologically diseased women was determined. The urine strains showed sensitivity up to 93.3%, but 7 Gram/-/ strains isolated in the last 3 months of the study were resistant to Ciprofloxacin. The cervical secretion strains, those of CD aspirates, of IUP and wound secretions were found sensitive up to 81.2%. The 50 Enterococcus strains isolated in gynecological infections were found in 28% resistant to Ciprofloxacin, with 7 of the 19 resistant strains were isolated in the last 3 months of the study. The appearance of resistant strains in some microbial species, pointed out in the literature as markedly sensitivity to Ciprofloxacin, as well as the data of a fast developed resistance Ciprofloxacin in the course of treatment, allowed us to recommend their application in the infections caused by microorganisms resistant to the habitual antibiotics.

[Yersinia pseudotuberculosis sensitivity to fluoroquinolones and the efficacy of pefloxacin in experimental pseudotuberculosis]. / Izuchenie chuvstvitel'nosti Yersinia pseudotuberculosis k ftorkhinolonam i éffektivnosti pefloksatsina pri éksperimental'nom psevdotuberkuleze.

The sensitivity of Y. pseudotuberculosis of different origin and plasmid profile to Russian preparations of the ftoroquinolones group (cyprofloxacin, pefloxacin, norfloxacin) was studied. Of the 3 preparations under study, pefloxacin was found to be most active with respect to Y. pseudotuberculosis. The absence of relationship between antibiotic resistance in 4 Y. pseudotuberculosis strains to cyprofloxacin, pefloxacin and norfloxacin and their additional conjugative R-plasmid with molecular weights of 32 and 38 mD was noted. Pefloxacin was shown to give a good sanitation and protective effect in experimental pseudotuberculosis.

Antagonism between bactericidal activities of 4-quinolones and coumarins gives insight into 4-quinolone killing mechanisms.

At concentrations exceeding their MICs, novobiocin and coumermycin antagonised the bactericidal activities of nalidixic acid, ciprofloxacin, ofloxacin and norfloxacin against Escherichia coli KL16. The sensitivities to killing by ciprofloxacin of four mutant derivatives of KL16 carrying gyrA, nalB, nal24 or nal31 alleles were also antagonised by novobiocin. The activities of drug combinations were tested in nutrient broth, which allowed expression of 4-quinolone killing mechanisms A, B and C. They were also tested in nutrient broth plus rifampicin to inhibit mechanisms A and C of the 4-quinolones, and in phosphate-buffered saline, which inhibited mechanism A. Results showed that novobiocin antagonised mechanism C, but not B, of both ciprofloxacin and ofloxacin, but did not antagonise mechanism C of norfloxacin. A review of these and other data indicates that mechanism B may result from the activities of SOS error-prone DNA repair on an irreversibly-bound drug-gyrase-DNA complex, and that mechanism C is mediated via drug interaction with the B subunit of DNA gyrase.

Antibacterial antagonism between fusidic acid and ciprofloxacin.

A routine laboratory disk susceptibility testing of a resistant Staphylococcus aureus strain showed that around the ciprofloxacin disk, placed by chance in proximity to a fusidic acid disk, the inhibition zone was truncated. Follow-up of this observation by a planned disk approximation method showed that there is a real antagonism between these two antibacterial agents. The antagonism was observed while testing S. aureus isolates including the standard ATCC 25923 strain, with Bacillus subtilis ATCC 6633 spores and also with a mutant Escherichia coli made fusidic acid susceptible. The antagonistic property was found structure-specific, only associated with those fluoroquinolones containing the cyclopropyl substituent at the N1-position: ciprofloxacin, enrofloxacin, sparfloxacin and WIN 57273. Fluoroquinolones without this substituent such as enoxacin, norfloxacin, pefloxacin and ofloxacin were not antagonized by fusidic acid, the steroidal Gram-positive active antibiotic.

Protection of Pseudomonas aeruginosa against ciprofloxacin and beta-lactams by homologous alginate.

Pseudomonas aeruginosa-derived alginate but no other neutral and negatively charged polysaccharides protected mucoid and nonmucoid strains of that organism against ciprofloxacin, gentamicin, ticarcillin, and ceftazidime. Data indicate that alginate has an intrinsic protective effect which is independent of diffusion, charge, or biofilm phenomena.

In vitro synergy studies with ciprofloxacin and selected beta-lactam agents and aminoglycosides against multidrug-resistant Pseudomonas aeruginosa.

In order to determine the effect of combining ciprofloxacin with beta-lactam and aminoglycoside agents against multidrug-resistant P. aeruginosa, ciprofloxacin was tested in vitro with the checkerboard broth microdilution technique with various current beta-lactam and aminoglycoside agents. In both inhibitory and bactericidal testing, the combination of ciprofloxacin with a beta-lactam agent (ceftazidime, piperacillin, mezlocillin, azlocillin, or imipenem) was more likely to show a synergistic effect or additive effect and lack of antagonism (96.5% of all inhibitory and cidal tests) than was a combination of ciprofloxacin with an aminoglycoside [(amikacin, tobramycin, or gentamicin), 90% of all inhibitory and cidal tests]. Finding of antagonism in some testing, especially with aminoglycosides, indicates that results are unpredictable. These results may influence selection of agents for selection in clinical situations.

Activity and mechanism of action of DuP 105 and DuP 721, new oxazolidinone compounds.

The in-vitro activities of DuP 721 and DuP 105, new oxazolidinone antibacterials, were compared with those of cefazolin, cephalexin, ciprofloxacin, clindamycin, oxacillin, penicillin, and vancomycin against Gram-positive cocci. DuP 721 was approximately four-fold more active than DuP 105 with an MIC of 2.0 mg/l for 90% of the Staphylococcus aureus, beta-haemolytic streptococcus and Streptococcus faecalis strains tested, and an MIC of 4.0 mg/l for 90% of the Str. faecium, penicillin-resistant Str. pneumoniae and viridans streptococcus strains tested. DuP 105 was most active against strains of Staph. epidermidis with an MIC of 4.0 mg/l for 90% of the strains tested. There was no cross resistance between these and the other antibacterial agents that were tested. Both oxazolidinones had bacteriostatic activity in broth against susceptible organisms. Both DuP 721 and DuP 105 inhibited ribosomal protein synthesis in a cell-free system. These synthetic, orally absorbable compounds represent a new series of antibacterial agents unrelated by chemical structure to any other currently available antimicrobial agents.