Macrophages (Mphi) play a pivotal role in the protection system by recognizing and eliminating invading pathogenic bacteria. Phagocytosis and the killing of invading bacteria are major effector functions of Mphi. Although the phagocytic and bactericidal activities of Mphi have been analyzed via several methods using a light microscope, a fluorescence microscope, or a fluorescence-activated cell sorter, expensive materials and equipment are usually required, and the methods are rather complicated. Moreover, it is impossible to determine both the phagocytic and bactericidal activities of Mphi simultaneously using these methods. In this review, we describe a simple, reproducible, inexpensive, yet old-fashioned method (antibiotic protection assay) for determining the phagocytic and bactericidal activities of Mphi.
Anti-Bacterial Agents/*pharmacology ; Gentamicins/*pharmacology ; Macrophages/*drug effects ; Phagocytosis/*drug effects

OBJECTIVETo investigate the prevalence of aminoglycoside resistance and genotyping of acetyltransferase in Escherichia coli.

METHODSResistance phenotypes to 12 antibiotics of 44 Escherichia coli isolates were analyzed using agar dilution method and 3 aminoglycoside resistance genes aac(3)-I, II and aac(6')-I were determined by PCR method.

RESULTSIn 44 clinical isolates, the occurrence of ESBLs was 45.45%, resistance rates were discrepant for amikacin (18.18%), gentamicin (56.82%) and tobramycin (61.36%), the prevalence of phenotype TG (tobramycin and gentamicin) indicative of aac(3)-II production and TGA (tobramycin, gentamicin and amikacin) indicative of aac(6')-I production were 36.36% and 18.18%, respectively. The most common aminoglycoside resistance genotype of acetyltransferase was aac(3)-II (52.27%) and aac(6')-I was lower (29.55%), but no aac(3)-I was detected.

CONCLUSIONAt least 2 acetyltransferase genes exist in this area i.e. aac(3)-II and aac(6')-I.

Acyltransferases ; genetics ; Amikacin ; pharmacology ; Aminoglycosides ; pharmacology ; Drug Resistance, Bacterial ; genetics ; Escherichia coli ; enzymology ; genetics ; Genotype ; Gentamicins ; pharmacology ; Phenotype ; Tobramycin ; pharmacology

OBJECTIVETo investigate the antimicrobial resistance of clinical isolates of Stenotrophomonas matophilia (SMA) and the mechanisms of their drug resistance.

METHODSDisc diffusion method (NCCLS) was used to detect the resistant patterns of 88 initial SMA isolates resistant to 12 antibiotics isolated from a local hospital in the past 4 years. PCR was used to detect the 7 aminoglycosides modifying enzymes genes (AME) against amikacin and gentamicin. Metal-beta-lactamases (MBLs) were screened by synergic method, and extended-spectrum beta-lactamases (ESBLs) were detected by double-disk synergy test.

RESULTSThe resistance rates of the SMA isolates were 0%-9.7% to minocycline, 12.5%-22.6% to ticarcillin-clavulanic acid, 12.5%-28.6% to levofloxacin, 18.8%-33.3% to doxycycline, 18.8%-40% to sulfamethoxazole compound, 50%-65.7% to ciprofloxacin, 50%-66.7% to cehazindme, 54.8%-66.7% to amikacin, 75%-100% to gentamicin, 81.3%-100% to piperacillin, 87.5%-100% to aztreonam and 93.5%-100% to imipenem. Aac(3)-I and ant(4')-II were not detected in these strains. The positive rates of the other 5 AME genes of aac(3)-II, ant(2'')-I, aac(6')-I, aac(3)-III, aac(3)-IV were 2.3%, 5.7%, 8%, 10%, and 10%, respectively. SMA strains producing ESBLs were found at the rate of 38.6%; 25% of the strains were MBL-producing, and 13.6% produced both ESBLs and MBLs.

CONCLUSIONMost of the SMAs we isolated are multidrug-resistant through various mechanisms. The choice of antibiotics should be made according to the susceptibility results.

Amikacin ; pharmacology ; Drug Resistance, Multiple, Bacterial ; Gentamicins ; pharmacology ; Humans ; Imipenem ; pharmacology ; Microbial Sensitivity Tests ; Stenotrophomonas maltophilia ; drug effects ; isolation & purification

OBJECTIVES: Staphylococcus epidermidis (S. epidermidis) is a Gram-positive opportunistic pathogen that often causes hospital infections. With the abuse of antibiotics, the resistance of S. epidermidis gradually increases, and drug repurposing has become a research hotspot in the treating of refractory drug-resistant bacterial infections. This study aims to study the antimicrobial and antibiofilm effects of simeprevir, an antiviral hepatitis drug, on S. epidermidis in vitro. METHODS: The micro-dilution assay was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of simeprevir against S. epidermidis. Crystal violet staining assay was used to detect the biofilm inhibitory effect of simeprevir. The antimicrobial activity of simeprevir against S. epidermidis and its biofilm were explored by SYTO9/PI fluorescent staining. The combined effect between simeprevir and gentamycin was assessed by checkerboard assay and was confirmed by time-inhibition assay. RESULTS: Simeprevir showed significant antimicrobial effects against S. epidermidis type strains and clinical isolates with the MIC and MBC at 2-16 μg/mL and 4-32 μg/mL, respectively. The antimicrobial effects of simeprevir were confirmed by SYTO9/PI staining. Simeprevir at MIC could significantly inhibit and break the biofilm on cover slides. Similarly, simeprevir also significantly inhibit the biofilm formation on the surface of urine catheters either in TSB [from (0.700±0.020) to (0.050±0.004)] (t=54.03, P<0.001), or horse serum [from (1.00±0.02) to (0.13±0.01)] (t=82.78, P<0.001). Synergistic antimicrobial effect was found between simeprevir and gentamycin against S. epidermidis with the fractional inhibitory concentration index of 0.5. CONCLUSIONS Simeprevir shows antimicrobial effect and anti-biofilm activities against S. epidermidis.
Humans ; Simeprevir ; Antiviral Agents ; Anti-Bacterial Agents/pharmacology* ; Cross Infection ; Gentamicins

Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.
Animals ; Gentamicins/pharmacology* ; Humans ; Phenotype ; Reproducibility of Results ; Salmonella Infections, Animal ; Salmonella enteritidis

OBJECTIVETo develop antibacterial coatings for orthopedic implants with a sustained release of drugs.

METHODSWollastonite coatings were deposited on the titanium substrates by an atmospheric plasma spray system. After soaking in weight percent of 5% AgNO(3) solution for 24 h, the wollastonite coatings loading silver were obtained. Gentamicin were loaded on the wollastonite coatings by collagen grafting process. The release rates of drugs from wollastonite coatings were investigated by the in vitro solution soaking test. One strain of S. aureus was used in zone of inhibition test to evaluate the antibacterial properties of drug loaded wollastonite coatings, and the cell culture test was used to evaluate their cytotoxicity.

RESULTSSilver and gentamicin loaded wollastonite coatings were successfully prepared. The release of silver ions from the silver loaded wollastonite coatings lasted 50 d in deionized water, effectively inhibiting the growth of S. aureus for 40 d. While an initial burst release of gentamicin was found during the in vitro solution soaking test. The gentamicin released from gentamicin loaded wollastonite coatings can inhibit the growth of S. aureus for 18 d. Both the two kinds of antibacterial wollastonite coatings showed no adverse effect on cellular adhesion, proliferation and alkaline phosphatase expression.

CONCLUSIONSCompared with gentamicin loaded wollastonite coatings, silver loaded wollastonite coatings may have more promising clinical applications due to the even and long-time antibacterial agent release.

Anti-Bacterial Agents ; pharmacology ; Calcium Compounds ; Cells, Cultured ; Coated Materials, Biocompatible ; pharmacology ; Gentamicins ; pharmacology ; Materials Testing ; Microbial Sensitivity Tests ; Osteoblasts ; cytology ; drug effects ; Silicates ; Silver ; pharmacology

To make a universal gene targeting vector fitting for most gene and delete positive selection gene after targeting successfully, a vector named pA2T was constructed by inserting one neomycin gene (neo) for positive selection and two same herpes simplex virus thymidine kinase gene HSV-tk1 and HSV-tk2 for negative selection into the vector of pGEM-3Z, and two locus of crossing-over (x) in P1 (LoxP) and two different multiple cloning sites (MCS) were inserted into two flanks of neo separately. There were eight rare cloning sites between neo and HSV-tk1 and five rare cloning sites between neo and HSV-tk2, and neo, HSV-tk1 and HSV-tk2 could be translated respectively in the pA2T. Transfection of the pA2T into goat fetus fibroblast cells with Lipofectamine 2000 conferred resistance to geneticin (G418) and resistance to ganciclovir (GAC) in the cells, which suggested the positive and negative selectable markers could express in the cells and thus the vector pA2T could be used as a universal gene targeting vector. Transformation of the pA2T into the BM25.8 expressing Cre recombinase conferred neo was deleted in the pA2T, which suggested the LoxP was active. Thus, this vector can be inserted by most gene sequences as homologous sequences and positive selection gene can be deleted after targeting successfully, which is very convenience for the production of transgenic animals using gene targeting method.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Molecular ; Ganciclovir ; pharmacology ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; Gentamicins ; pharmacology ; Goats ; Integrases ; genetics ; Neomycin ; pharmacology ; Phosphotransferases ; genetics ; metabolism

The quality of some earlier developed antibiotics is usually ensured by the combination of HPLC purity and microbiological potency measurement in the pharmacopoeias of various countries because the relationship between their purity and potency is not clearly quantified. Due to potency is assessed using certain units of measurement, it can not be directly traced to the international system of units (SI unit). This has become a hotspot in the study of the quantitative relationship between purity and potency of antibiotics. It would be quite an achievement to simultaneously determine both purity and potency using HPLC methods during quality control. This study evaluated a multicomponent antibiotic product, gentamycin, as a test sample. First, pure samples of the C components of gentamycin: C1a, C2, C2a and C1 were prepared, separately. Second, quantitative relationship (theoretical potency) between the purity and potency of each C component of gentamycin were determined using 1H NMR, HPLC-ELSD and microbiological assay method. One milligram of gentamycin C1a, C2, C2a and C1 was equal to 1 286.98, 1 095.74, 1 079.52 and 739.61 gentamycin units, respectively. Finally, a method for the determination of gentamycin potency was established based on the proportion and content of C components of gentamycin. The unification of purity and potency for gentamycin was achieved using only HPLC-ELSD. It is also demonstrated that C components of gentamycin and micronomicin produce the same responses under ELSD, which means that it is not necessary to prepare separate reference standards for each C component of gentamycin and that quantitative testing can be performed accurately using only one micronomicin reference standard. This study simplified the previous method for the determination of the content of C components of gentamycin using HPLC-ELSD. The developed method is suitable for regular use as a part of quality control and can simplify the rigmarole quality control procedures provided in current pharmacopeias.
Chromatography, High Pressure Liquid ; Gentamicins ; chemistry ; pharmacology ; Magnetic Resonance Spectroscopy ; Microbial Sensitivity Tests ; methods ; Molecular Structure ; Quality Control ; Reference Standards

PURPOSE: Community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) infections are increasing. Although gentamicin (GEN) is usually susceptible against CA-MRSA, GEN is rarely considered for treatment as monotherapy. We employed an in vitro pharmacodynamic model (IVPDM) to compare efficacies of GEN against CA-MRSA with two dosing regimens [thrice-daily (TD), once-daily (OD)]. MATERIALS AND METHODS: Using two strains of CA-MRSA, we adopted IVPDM comprised of two-compartments with a surface-to-volume ratio of 5.34 cm-1. GEN regimens were simulated with human pharmacokinetic data of TD and OD. Experiments were performed over 48 hours in triplicate for each strain and dosing regimen. RESULTS: MICs of GEN for YSSA1 and YSSA15 were 1 and 2 mg/L, respectively. In OD, indices of peak/MIC were > 8.6 at least, in contrast to < 6.4 in TD. A > or = 3-log10 reduction in CFU/mL was demonstrated prior to 4 hours in TD and OD, and continued until 8 hours for both strains. However, reductions in the colony counts at 24 and 48 hours were significantly larger for OD compared to TD in both strains (p < 0.001). During TD, resistance developed in YSSA1 and small colony variants (SCVs) were documented in YSSA15. No resistance or SCVs were observed during OD in both strains. CONCLUSION: TD and OD showed the same killing slopes until 8 hours. After the 24 hours of experiments, OD of GEN would be advantageous not only in having more reductions in colony counts, but also suppressing the development of resistance or SCVs for 48 hours.
Anti-Bacterial Agents/*administration & dosage/pharmacokinetics/*pharmacology ; Drug Administration Schedule ; Gentamicins/*administration & dosage/pharmacokinetics/*pharmacology ; Humans ; Methicillin-Resistant Staphylococcus aureus/*drug effects ; Microbial Sensitivity Tests

AIMTo investigate the antagonism of tetrandrine in acute renal injury induced by gentamicin in guinea pig.

METHODSThe experimental guinea pigs are divided into 4 groups by randomized blocks, they were group 1 (control), group 2 (tetrandrine), group 3 (gentamicin) and group 4 (gentamicin with tetrandrine). After 10 days, set apart urinary specimen for determination the activity of NAG, reserve renal specimen for observing the renal histology and measuring expression of actin and TGF-beta1.

RESULTSThe renal histology showed that the injury of group 3 was the most grievous, group 4 was lighter than group 3. In addition, the apoptosis was obviously less than group 3. The activity of urine of NAG in group 4 was lower than group 3. The levels of expression of actin and TGF-beta1 in group 4 were more than group 3.

CONCLUSIONWhen kidney is damaged by gentamicin, tetrandrine is able to reduce the activity of urinary NAG and inhibit the renal tubular epithelial cell apoptosis. Besides, tetrandrine can prevent breaking actin and evoke the endogenous transmission growth factor beta1 tune-up, the above results can indicate that tetrandrine has antagonism to acute renal injury induced by gentamicin in guinea pig.

Acetylglucosaminidase ; urine ; Actins ; metabolism ; Acute Kidney Injury ; chemically induced ; Animals ; Benzylisoquinolines ; pharmacology ; Drug Antagonism ; Drugs, Chinese Herbal ; pharmacology ; Gentamicins ; adverse effects ; Guinea Pigs ; Transforming Growth Factor beta1 ; metabolism