Low Concentrations Of Gentamicin Research Articles

The Antimicrobial Activity of Combination of Vitamin D3 and Omega-3 against Pathogenic Microorganisms.

Food supplements such as vitamin D3 and omega-3 have a significant role in activating the immune system and impacting the diversity of gut microbiota; thus, controlling the growth of invading pathogens indirectly. This study aims to evaluate the direct antimicrobial activity of vitamin D3 and omega- 3 individually, combined together, and combined with low concentrations of gentamicin or amphotericin B against selected pathogenic microorganisms. In addition, this study hypothesizes the potential antimicrobial mechanism and recommends suitable studies to be conducted. Minimum inhibitory concentration of different serial dilutions of vitamin D3 [0.7μg/mL-83.3μg/mL] or omega-3 [0.7mg/mL-100mg/mL] or combined [vitamin D3:1.3μg/mL-83.3μg/mL and omega-3:1.56mg/mL-100mg/mL] with/without antibiotic have been investigated on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans using check board technique. The highest concentration of vitamin D3 [83.3 μg/mL] demonstrated a complete eradication of the tested microorganisms. Conversely, omega-3 had a lower effect on them. The highest concentration of combining vitamin D3 and omega-3 with/without gentamicin resulted in a complete eradication of the S. aureus, E. coli and P. aeruginosa with a 6.8 to 7 log reduction. On the other hand, C. albicans was inhibited when using vitamin D3 [83.3 μg/mL] or when this concentration is combined with 100mg/mL of omega-3. However, when these two concentrations were added to amphotericin B the log reduction dropped to 0.45 suggesting antagonistic effect. These findings suggested that, unlike omega 3, vitamin D3 possesses good antimicrobial effects against pathogenic microorganisms. The combination of the studied food supplement showed enhanced microbial inhibition at high concentration, while they had antagonistic effect when combined with amphotericin B and applied on C. albicans combined. Further studies on the exact antimicrobial mechanism are still required to understand the measured data here.

Plantaricin NC8 \u03b1\u03b2 prevents Staphylococcus aureus-mediated cytotoxicity and inflammatory responses of human keratinocytes

Multidrug resistance bacteria constitue an increasing global health problem and the development of novel therapeutic strategies to face this challenge is urgent. Antimicrobial peptides have been proven as potent agents against pathogenic bacteria shown by promising in vitro results. The aim of this study was to characterize the antimicrobial effects of PLNC8 αβ on cell signaling pathways and inflammatory responses of human keratinocytes infected with S. aureus. PLNC8 αβ did not affect the viability of human keratinocytes but upregulated several cytokines (IL-1β, IL-6, CXCL8), MMPs (MMP1, MMP2, MMP9, MMP10) and growth factors (VEGF and PDGF-AA), which are essential in cell regeneration. S. aureus induced the expression of several inflammatory mediators at the gene and protein level and PLNC8 αβ was able to significantly suppress these effects. Intracellular signaling events involved primarily c-Jun via JNK, c-Fos and NFκB, suggesting their essential role in the initiation of inflammatory responses in human keratinocytes. PLNC8 αβ was shown to modulate early keratinocyte responses, without affecting their viability. The peptides have high selectivity towards S. aureus and were efficient at eliminating the bacteria and counteracting their inflammatory and cytotoxic effects, alone and in combination with low concentrations of gentamicin. We propose that PLNC8 αβ may be developed to combat infections caused by Staphylococcus spp.

Impact of Gentamicin Concentration and Exposure Time on Intracellular Yersinia pestis.

The study of intracellular bacterial pathogens in cell culture hinges on inhibiting extracellular growth of the bacteria in cell culture media. Aminoglycosides, like gentamicin, were originally thought to poorly penetrate eukaryotic cells, and thus, while inhibiting extracellular bacteria, these antibiotics had limited effect on inhibiting the growth of intracellular bacteria. This property led to the development of the antibiotic protection assay to study intracellular pathogens in vitro. More recent studies have demonstrated that aminoglycosides slowly penetrate eukaryotic cells and can even reach intracellular concentrations that inhibit intracellular bacteria. Therefore, important considerations, such as antibiotic concentration, incubation time, and cell type need to be made when designing the antibiotic protection assay to avoid potential false positive/negative observations. Yersinia pestis, which causes the human disease known as the plague, is a facultative intracellular pathogen that can infect and replicate in macrophages. Y. pestis is sensitive to gentamicin and this antibiotic is often employed in the antibiotic protection assay to study the Y. pestis intracellular life cycle. However, a large variety of gentamicin concentrations and incubation periods have been reported in the Y. pestis literature without a clear characterization of the potential influences that variations in the gentamicin protection assay could have on intracellular growth of this pathogen. This raised concerns that variations in the gentamicin protection assay could influence phenotypes and reproducibility of data. To provide a better understanding of the potential consequences that variations in the gentamicin protection assay could have on Y. pestis, we systematically examined the impact of multiple variables of the gentamicin protection assay on Y. pestis intracellular survival in macrophages. We found that prolonged incubation periods with low concentrations of gentamicin, or short incubation periods with higher concentrations of the antibiotic, have a dramatic impact on intracellular growth. Furthermore, the degree of sensitivity of intracellular Y. pestis to gentamicin was also cell type dependent. These data highlight the importance to empirically establish cell type specific gentamicin protection assays to avoid potential artificial data in Y. pestis intracellular studies.

Staphylococcus aureus develops increased resistance to antibiotics by forming dynamic small colony variants during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis often develops to chronicity despite antimicrobial treatments that have been found to be susceptible in in vitro tests. The complex infection strategies of S. aureus, including host cell invasion and intracellular persistence via the formation of dynamic small colony variant (SCV) phenotypes, could be responsible for therapy-refractory infection courses. To analyse the efficacy of antibiotics in the acute and chronic stage of bone infections, we established long-term in vitro and in vivo osteomyelitis models. Antibiotics that were tested include β-lactams, fluoroquinolones, vancomycin, linezolid, daptomycin, fosfomycin, gentamicin, rifampicin and clindamycin. Cell culture infection experiments revealed that all tested antibiotics reduced bacterial numbers within infected osteoblasts when treatment was started immediately, whereas some antibiotics lost their activity against intracellular persisting bacteria. Only rifampicin almost cleared infected osteoblasts in the acute and chronic stages. Furthermore, we detected that low concentrations of gentamicin, moxifloxacin and clindamycin enhanced the formation of SCVs, and these could promote chronic infections. Next, we treated a murine osteomyelitis model in the acute and chronic stages. Only rifampicin significantly reduced the bacterial load of bones in the acute phase, whereas cefuroxime and gentamicin were less effective and gentamicin strongly induced SCV formation. During chronicity none of the antimicrobial compounds tested showed a beneficial effect on bone deformation or reduced the numbers of persisting bacteria. In all infection models rifampicin was most effective at reducing bacterial loads. In the chronic stage, particularly in the in vivo model, many tested compounds lost activity against persisting bacteria and some antibiotics even induced SCV formation.

Suitability of Becton Dickinson Vacutainer rapid serum tube for collecting and storing blood samples for antibiotic and anticonvulsant drug monitoring

AimsTo investigate the suitability of newly developed Becton Dickinson Vacutainer rapid serum tube (RST) for therapeutic drug monitoring of antibiotics and anticonvulsants.MethodsTwo pools of citrated whole blood were created by...

In vitro activity (MICs and rate of kill) of AFN-1252, a novel FabI inhibitor, in the presence of serum and in combination with other antibiotics

AFN-1252 is a novel inhibitor of FabI, an essential enzyme in fatty acid biosynthesis in Staphylococcus spp. AFN-1252 exhibits typical MIC90 values of ⩽0·015 μg/ml against diverse clinical isolates of S. aureus, oral absorption, long elimination half-live and efficacy in animal models. We now report high binding (∼95%) to serum proteins of mouse, rat, dog and humans, associated with an eight-fold increase in minimal inhibitory concentration (MIC) and which may be responsible for the long elimination half-lives on pharmacokinetic studies. Unlike daptomycin, AFN-1252 activity is not reduced in the presence of lung surfactant. AFN-1252 exhibits a short post-antibiotic effect of 1·1 hours against methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) following a 4-hour exposure period. The AFN-1252 unique spectrum of activity is not compromised by interactions with major antibiotic classes, but demonstrates synergy with low concentrations of gentamicin against MSSA and MRSA. These studies support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.

Construction of Aminoglycoside-Sensitive Burkholderia cenocepacia Strains for Use in Studies of Intracellular Bacteria with the Gentamicin Protection Assay

Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

Augmentation of the Antimicrobial Efficacy of Antibiotics by Filamentous Phage

A significant increase in sensitivity to several antibiotics was observed in vitro after infection of the two Pseudomonas aeruginosa strains O1 and K with the filamentous phage Pf3 and Pf1, respectively. Moreover, upon infection with phage Pf1 a P. aeruginosa K strain harboring a plasmid-borne gentamicin resistance gene could be resensitized to the antibiotic. We further show that BALB/c mice were rescued from lethal infections with P. aeruginosa K by concomitant treatment with phage Pf1 and low concentrations of gentamicin, neither of which was able to cure the infection when administered alone.

Gentamicin release from old cement during revision hip arthroplasty

Bone cement containing gentamicin may release antibiotic when fractured during revision operations. Tissue samples taken during surgery may be contaminated by gentamicin and give inaccurate microbiological assessment. We studied five patients in whom cement containing gentamicin had been used in the primary procedure. During revision hip replacement, samples of joint fluid, tissues and cement were taken both before and after disruption of the cement. With the exception of one sample of joint fluid, low concentrations of gentamicin were recorded in the samples taken before the cement was disrupted, but after disruption the specimens contained gentamicin at concentrations high enough to inhibit or prevent growth of sensitive organisms. The cement contained very high levels up to ten years after insertion. Our findings suggest that no reliance can be placed on the microbiological assessment of specimens taken once cement splitting has started and that specimens should therefore be taken as early as possible.

Gentamicin release from old cement during revision hip arthroplasty

Bone cement containing gentamicin may release antibiotic when fractured during revision operations. Tissue samples taken during surgery may be contaminated by gentamicin and give inaccurate microbiological assessment. We studied five patients in whom cement containing gentamicin had been used in the primary procedure. During revision hip replacement, samples of joint fluid, tissues and cement were taken both before and after disruption of the cement. With the exception of one sample of joint fluid, low concentrations of gentamicin were recorded in the samples taken before the cement was disrupted, but after disruption the specimens contained gentamicin at concentrations high enough to inhibit or prevent growth of sensitive organisms. The cement contained very high levels up to ten years after insertion. Our findings suggest that no reliance can be placed on the microbiological assessment of specimens taken once cement splitting has started and that specimens should therefore be taken as early as possible.

Efficacy of iontophoresis in the rat cornea.

Iontophoresis can enhance penetration of drugs into tissues. We examined the extent of penetration of gentamicin into the cornea of rats during iontophoresis and the effect of varying the concentrations of gentamicin, the duration of iontophoresis and the current densities during iontophoresis. Eight groups of rats underwent corneal iontophoresis using gentamicin dissolved in agar. Low and high concentrations of gentamicin were used, as well as low and high current densities and long and short durations of iontophoresis. Control groups received topical or subconjunctival gentamicin, topical saline solution and mock iontophoresis with the agar-gentamicin mixture. The Mann-Whitney test was used for statistical evaluation. Highly bactericidal concentrations of gentamicin were obtained in all the iontophoresis-treated corneas. The high concentration compared to the low concentration of gentamicin in agar significantly increased the concentration of gentamicin in the corneas, as did the longer duration of iontophoresis. However, higher current intensity did not significantly enhance the drug concentration in the cornea. Iontophoresis with a concentrated gentamicin-agar mixture may provide a rapid increase of gentamicin levels in the cornea.

Control of Bacteria and Alteration of Plant Growth in Tissue Cultures of Tansy (Tanacetum vulgare L.) Treated with Antibiotics

Ten bacteria were isolated from the in vitro shoot cultures of different tansy (Tanacetum vulgare) genotypes. All isolates were Gram-negative. Five isolates belonged to Enterobacteriaceae, three isolates were strains of fluorescent Pseudomonas, and two isolates were strains of other aerobic bacteria. The combined treatment with gentamicin (50 mg·L–1) and rifampicin (25 mg·L–1) prevented the growth of all bacteria, whereas none of the antibiotics (ampicillin, cefotaxime, rifampicin, gentamicin, or streptomycin) controlled all bacteria when used alone. The antibiotics ranged based on their increasing adverse effects on shoot cultures as follows: rifampicin, cefotaxime, and gentamicin. Increased antibiotic concentration reduced the initiation and growth rates of shoots, roots and calli with some exceptions. Low concentrations of gentamicin and cefotaxime both increased the growth rate and shoot number per plant. Rifampicin stimulated the root growth of some of the biotypes tested. The growth rate of calli increased significantly in the presence of both gentamicin (25 mg·L–1) and rifampicin (25 mg·L–1) in the growth media, whereas the use of either antibiotic alone reduced the calli growth relative to the control. Viability of tansy protoplasts was enhanced by rifampicin and to a lesser extend by gentamicin and cefotaxime. The growth of bacteria isolated from tansy tissue culture can be prevented with a combined gentamicin and rifampicin treatment. The growth rate of plants decreased slightly when grown in the media supplemented with the same antibiotic combination. However, the growth retardation was not permanent, and the plants recovered and grew vigorously when transferred to antibiotic free medium.

Primary culture of proximal tubular cells from normal rat kidney as an in vitro model to study mechanisms of nephrotoxicity: Toxicity of nephrotoxicants at low concentrations during prolonged exposure

The aim of this study was to set up an in vitro system to study nephrotoxicity of xenobiotics which allows exposure at low concentrations for long periods (1–5 days). A very pure preparation of isolated proximal tubular cells (PTC) from rat kidney (Boogaard et al., Toxicol Appl Pharmacol 101: 135–143, 1989) was brought into primary culture. Cells grew to confluence in 3 days and could be maintained up to 8 days in a modification of Dulbecco's modified Eagle's medium Ham F 12 nutrient mixture supplemented with fetal calf serum. Fibroblast growth was completely suppressed by replacement of l-valine by d-valine and of l-arginine by l-ornithine. Polarity was retained: in cells grown on filters organic anions were transported at the basolateral membrane while d-glucose transport was located at the apical membrane. Inhibition of the latter was used to assess the functional integrity of the cells after exposure to nephrotoxins. The newly grown cells expressed γ-glutamyltranspeptidase activity since incubation with the glutathione-conjugate of 1,1-dichloro-2,2-difluoroethylene (DCDFE) induced cytotoxicity. Both β-lyase and acylase activities were expressed because the cysteine- S-conjugate and the corresponding mercapturate of DCDFE showed cytotoxicity. Cultured cells showed toxicity on prolonged exposure to very low concentrations of gentamicin, cephaloridine, cisplatin and the cysteine- S-conjugate of chlorotrifluoroethylene. The lowest concentrations at which toxicity can be observed are 1–3 orders of magnitude lower in primary cultures than in freshly isolated PTC in suspension. This indicates that this cell model is suitable to investigate mechanisms of nephrotoxicity in vitro, at prolonged exposure to the low concentrations that are relevant in vivo levels.

Early synergistic killing activity at concentrations attainable in CSF of amoxicillin or cefotaxime and aminoglycosides against Haemophilus influenzae.

Rapid eradication of bacteria from the CSF is critical for the outcome of Haemophilus influenzae meningitis in children. In 15 patients studied, the mean H. influenzae colony counts in CSF were 10(5) CFU/ml (range: 10(2) to 10(9) CFU/ml). Time-kill curves were determined for amoxicillin and cefotaxime alone and in combination with gentamicin or amikacin, against 60 clinical isolates of H. influenzae at concentrations equivalent to those found in CSF following systemic administration. Against beta-lactamase-negative strains (n = 44) a bactericidal effect was observed at 18 h for amoxicillin alone, at 5 h for amoxicillin plus aminoglycosides and at 2.5 h for cefotaxime with or without aminoglycosides. Against beta-lactamase-positive strains a bactericidal effect was observed at 18 h for cefotaxime, at 5 h for amoxicillin plus aminoglycosides and at 2.5 h for cefotaxime plus aminoglycosides. It appears that despite low concentrations of gentamicin or amikacin in the CSF, the accelerated killing of H. influenzae provides a rationale for the initial use of the combination of cefotaxime and aminoglycosides in the initial treatment of H. influenzae meningitis.

Synergy between penicillins and low concentrations of gentamicin in the killing of group B streptococci.

Antimicrobial sensitivity, synergy, and timed-killing assays were determined for 20 strains of group B streptococci isolated from cultures of blood and cerebrospinal fluid (CSF) of infected neonates. The mean minimal inhibitory concentrations by the tube-dilution method were as follows: penicillin, 0.02 microgram/ml; ampicillin, 0.05 microgram/ml; and gentamicin, 4.5 micrograms/ml. No synergy was detected with any combination of penicillin or ampicillin and gentamicin by the checkerboard titration method. Killing kinetics were determined for combinations of penicillin or ampicillin and gentamicin at low concentrations of these antibiotics comparable to those attained in the CSF following systemic administration of these antibiotics. Addition of 0.1 microgram and 0.5 microgram of gentamicin/ml to penicillin or ampicillin significantly accelerated the killing of group B streptococci. Despite the "poor" permeation of gentamicin into the CSF, the accelerated killing of streptococci at low concentrations of this antibiotic provides a rationale for the initial use of a combination of penicillin or ampicillin and gentamicin in the treatment of group B streptococcal meningitis.

Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa.

The mode of interaction of the polycationic aminoglycoside antibiotics with the surface of Pseudomonas aeruginosa cells was studied with the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN). The addition of the aminoglycoside gentamicin to intact cells in the presence of NPN led to a shift in the fluorescence emission maximum from 460 to 420 nm. At the same time the NPN fluorescence intensity increased fourfold. Gentamicin caused no such effects when added to outer membrane vesicles, suggesting that the increased fluorescence resulted from the interaction of gentamicin with intact cells. Gentamicin-promoted NPN uptake was inhibited by the divalent cations Mg2+ and Ca2+, but occurred in the absence of gentamicin transport across the inner membrane. Low concentrations of gentamicin (2 micrograms/ml) caused NPN fluorescence to increase over a period of 4 min in a sigmoidal fashion. At higher concentrations (50 micrograms/ml) the increase occurred within a few seconds. The final fluorescence intensity was almost independent of the gentamicin concentration. A centrifugation technique was used to demonstrate that gentamicin caused actual uptake of NPN from the supernatant. The initial rate of NPN uptake varied according to the gentamicin concentration in a sigmoidal fashion. Similar data were obtained for seven other aminoglycoside antibiotics. The data, when reanalyzed as a Hill plot, gave a series of lines with a mean slope (the Hill number) of 2.26 +/- 0.26, suggesting that the interaction of aminoglycosides with the cell surface to permeabilize it to NPN involved at least three sites and demonstrated positive cooperativity. There was a statistically significant relationship between the pseudoassociation constant K, from the Hill plots and the minimal inhibitory concentrations for the eight antibiotics. These results are consistent with the concept that aminoglycosides interact as a divalent cation binding site on the P. aeruginosa outer membrane and permeabilize it to the hydrophobic prove NPN.

Synergy of penicillin and gentamicin against Enterococci.

Because combinations of penicillin plus streptomycin and penicillin plus kanamycin do not produce synergism against all strains of enterococci, the effect of penicillin plus gentamicin against enterococci was examined. A very high level of resistance to gentamicin was absent in 172 strains of enterococci isolated from clinical specimens. Penicillin plus gentamicin was synergistic against all of 30 strains tested. The size of the inoculum had a significant effect when low concentrations of gentamicin were tested against certain of the strains. Nonetheless, the combination of penicillin plus gentamicin produced enhanced killing of all enterococci tested when used in clinically achievable concentrations.