Gentamicin Concentrations Research Articles

Intranasal and Serum Gentamicin Concentration: Comparison of Three Topical Administration Protocols in Dogs.

Antimicrobials' topical administration efficacy has not been assessed in dogs with upper respiratory tract disease. The aim was to compare the concentration of gentamicin in nasal lavage fluid (NALF) and in serum after three topical protocols. This was a prospective crossover study of ten healthy dogs. Gentamicin was nebulized for a duration of 1 week, twice a day, for 10 min in the first protocol (10-min protocol) and for 3 min in the second protocol (3-min protocol), while the third protocol consisted of the administration of 0.25 mL of gentamicin in each nostril (drop protocol). Median concentrations of gentamicin in NALF were 9.39 µg/mL (8.12-19.97 interquartile range), 4.96 µg/mL (4.60-6.43) and 137.00 µg/mL (110.5-162.00) in the 10-min protocol, 3-min protocol and drop protocol, respectively. The result for the drop protocol was significantly higher than those of both nebulization protocols in NALF (p = 0.039). In serum, the gentamicin concentration was 0.98 µg/mL (0.65-1.53) and 0.25 µg/mL (0.25-0.44) in the 10-min and 3-min protocols, respectively. Gentamicin was not detected in the serum of seven out of ten dogs in the drop protocol, and gentamicin was significantly higher in the 10-min protocol compared to the drop protocol (p = 0.001). This study found that the 10-min, 3-min and drop protocols achieved superior concentrations in NALF compared to the minimum inhibitory concentration for gentamicin-sensitive bacteria, while remaining below the toxic values in blood.

Study on the inhibitory effect of quercetin combined with gentamicin on the formation of Pseudomonas aeruginosa and its bioenvelope

ObjectiveThe potential effects of quercetin and gentamicin combination on the bacteriostatic activity and biofilm formation of Pseudomonas aeruginosa (PA) were examined, and the findings provided a theoretical basis for the development of quercetin as a new biofilm inhibitor. MethodsThe minimum inhibitory concentration (MIC) of eight PAs was determined by microdilution method and the partial inhibitory concentration index (FICI) of the combined drug was analyzed by micro-dilution method. Thereafter, the lowest film inhibitory concentration (MBIC) of quercetin and gentamicin alone and in combination was evaluated by crystal violet staining. Finally, scanning electron microscopy (SEM) and laser confocal microscopy (CLSM) were used to decipher the inhibitory effect of the combination on biofilm formation. OutcomeThe antibacterial activity of quercetin alone was relatively weak, but after combination with gentamicin, the antibacterial activity was significantly enhanced, as evident by FICI of 0.28 and 0.53 and manifested as synergistic or additive effect, which indicated that quercetin can enhance gentamicin antibacterial activity. The results of crystal violet staining revealed that quercetin and gentamicin alone exhibited a similar biofilm formation inhibitory effect, but the inhibitory effect was substantially weaker, and the antibiofilm activity was stronger and exhibited a dose-dependent response after the combination of the two with 1/2FICI. The results of scanning electron microscopy and laser confocal microscopy also showed that the treatment of PA biofilm after combining quercetin and gentamicin with 1/2FICI could completely destroy the spatial structure of the complete biofilm, significantly reduce the thickness of bacteria, and markedly reduce the proportion of viable bacteria in the membrane. ConclusionThe combination of quercetin and gentamicin can effectively inhibit the formation of PA as well as its biofilm, and exhibit synergistic and additive effects.

Antibimicrobial Susceptibility of Trimethoprim-Sulfamethoxazole and 3rd-Generation Cephalosporin-Resistant Escherichia coli Isolates Enumerated Longitudinally from Feedlot Arrival to Harvest in High-Risk Beef Cattle Administered Common Metaphylactic Antimicrobials.

Multidrug resistant (MDR) Escherichia coli threaten the preservation of antimicrobials to treat infections in humans and livestock. Thus, it is important to understand where antimicrobial-resistant E. coli persist and factors that contribute to its their development. Crossbred cattle (n = 249; body weight = 244 kg ±25 kg standard deviation) were blocked by arrival date and assigned metaphylactic antimicrobial treatments of sterile saline control, tulathromycin (TUL), ceftiofur, or florfenicol at random. Trimethoprim-sulfamethoxazole (COTR) and third-generation cephalosporin (CTXR)-resistant E. coli were isolated from fecal samples on days 0, 28, 56, 112, 182, and study END (day 252 for block 1 and day 242 for block 2). Then, susceptibility testing was conducted on all confirmed isolates. MDR was detected in both COTR and CTXR E. coli isolates. In COTR isolates, the number of antimicrobials each isolate was resistant to and the minimum inhibitory concentration (MIC) for amoxicillin-clavulanic acid, ceftriaxone, and gentamicin was greatest on day 28 compared with all other days (p ≤ 0.04). Similarly, chloramphenicol MIC was greater on day 28 than on day 0 (p < 0.01). Overall, sulfisoxazole MIC was less for TUL than all other treatments (p ≤ 0.02), and trimethoprim-sulfamethoxazole MIC was greater for TUL than all other treatments (p ≤ 0.03). Finally, there was no effect of treatment, day, or treatment × day for tetracycline or meropenem MIC (p ≥ 0.07). In CTXR isolates, there was an effect of day for all antimicrobials tested except ampicillin and meropenem (p ≤ 0.06). In conclusion, administering a metaphylactic antimicrobial at feedlot arrival did influence the susceptibility of COTR and CTXR E. coli. However, MDR E. coli are widely distributed, and the MIC for most antimicrobials was not different from the initial value upon completion of the feeding period.

The Nocardial aph(2″) Gene Confers Tobramycin and Gentamicin Resistance and Is an Effective Positive Selection Marker in Mycobacteria and Nocardia.

The current study aimed to evaluate the feasibility of using the aminoglycoside 2″-O-phosphotransferase aph(2″) gene as a positive selection marker in N. asteroides, M. smegmatis, M. abscessus and M. tuberculosis. The aph(2″) gene, known to confer resistance to tobramycin, was PCR amplified from N. farcinica and cloned into two plasmid vectors, pMSG383 and pDB151, harboring hygromycin and zeocin selection markers, respectively. The recombinant plasmids were transformed into the target microorganisms, and selectability was assessed against varying concentrations of tobramycin and using an E-test against gentamicin. The results indicated that the aph(2″) gene is a useful selection marker in Mycobacteria and Nocardia against tobramycin, with a good selectability at 2.5-10 µg/mL for M. smegmatis mc2-155 and N. asteroides ATCC 19,247, and 60-160 µg/mL for M. abscessus ATCC 19,977 and M. tuberculosis H37Ra. The minimum inhibitory concentration (MIC) of gentamicin for recombinant N. asteroides, M. smegmatis and M. abscessus was >256 µg/mL, whereas respective MIC in wild-type strains was 0.125 µg/mL, 0.38 µg/mL and 8 µg/mL, respectively. These findings demonstrate the potential of aph(2″) as a positive selection marker for genetic manipulation processes in Mycobacteria and Nocardia, thus facilitating their research and improving the efficiency of biotechnology applications. Conclusions: the aph(2″) gene is a useful, new selection marker for genetic manipulation of Nocardia and various Mycobacteria.

Concentrations of penicillin and gentamicin following intrauterine administration of two compounded formulations

The use of broad-spectrum antimicrobial therapy either by intrauterine or systemic route is commonplace in equine practice, however, much remains unknown about antibiotic efficacy in this environment. We recently demonstrated the activity of penicillin and gentamicin against common endometrial pathogens ex vivo.1 The objective of this study was to measure the concentration of gentamicin and penicillin in the uterine fluid of mares following intrauterine infusion of either a standard (PPGent) or long-acting (LA-PPGent) compounded formulation of procaine penicillin and gentamicin. We hypothesized that both drug formulations would result in therapeutic concentrations following administration, with total concentrations sustained for longer using the long-acting formulation (LA-PPGent). Ten reproductively sound mares in early estrus were administered 2378mg of procaine penicillin and 200mg of gentamicin via a single intrauterine infusion in either a standard (n=5) or a lyophilized formulation suspended in a slow-release matrix (n=5). Intrauterine fluid was collected at 0.5, 2, 4, 8, 24, 32, 48 and 72 h following infusion. A pre-weighed section of umbilical tape was introduced into the uterus through a double-guarded pipette for 30 seconds and then weighed to determine the absorbed fluid volume. Penicillin and gentamicin analyses were performed using high-performance liquid chromatography(HPLC) and ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), respectively. Penicillin and gentamicin concentrations were assessed between groups and over time using a repeated measures analysis with treatment, time, and their interaction in the model statement with significance set at p<0.05. Mean intrauterine drug concentrations of PPGent peaked at 0.5 hours (Penicillin: 10,123.0 ± 4,298.0 µg/mL, Gentamicin: 3,397.3 ± 1,338.5 µg/mL), well above concentrations previously shown to inhibit bacterial growth ex vivo ([1] Von Dollen et al. J Equine Vet Sci 2019;79:121-26), and exceeded MIC for relevant organisms for 72 hours (Penicillin: 2.59 ± 6.34 µg/mL, Gentamicin: 2.14 ± 2.4 µg/mL). Mean concentrations of LA-PPG were lower at peak (Penicillin: 2,213.8 ± 967.8 µg/mL – p<0.05, Gentamicin: 1,859 ± 2,413 µg/mL) and exceeded MIC for 24h and 32h, respectively. These results support the use of procaine penicillin and gentamicin as intrauterine antimicrobial therapeutics. The long-acting formulation was not found to extend the time over MIC of antibiotics in the uterus of estrus mares. Acknowledgements: LA-PPGent was prepared by SR Veterinary Technologies LLC, Windsor CO using previously lyophilized PPGent. This research was supported by the Grayson-Jockey Club Research Foundation.

Is fat-free mass-based gentamicin dosing regimen preferable than whole-body weight in neonates?

Body fluid dynamics and renal maturation status vary during the neonatal period. We hypothesized that differences in peak and trough gentamicin concentrations could be expected. To predict the peak and trough gentamicin concentrations in critically ill neonates and to predict the changes in the predicted peak plasma concentrations of gentamicin following fat-free mass dosing. Critically ill neonates that received gentamicin and have gentamicin concentration measured were recruited. Fat mass was estimated using skinfold thicknesses. Changes in the peak plasma concentrations (Cmax) using whole-body weight (estimated using the current dosing regimen) and predicted concentrations following the fat-free mass-based dosing were the outcome measures. Eighty-nine critically ill neonates were recruited. Sub-therapeutic Cmax was estimated using the current dosing regimen in 32.6%, and 22.5% neonates following the first and second doses of gentamicin. Preterm neonates had significantly higher fat mass compared to term neonates. All except one had Cmax above 12 μg/ml after the first dose and all had after the second gentamicin dose following the predicted fat-free mass-based gentamicin dosing. The recommended doses are as follows: extreme preterm: 7.95 mg/kg every 48 h; very preterm: 7.30 mg/kg every 36-48 h; late preterm: 5.90 mg/kg every 36-48 h; and term neonates at 5.10 mg/kg every 24 h. Fat-free mass dosing may be considered for obtaining optimal therapeutic effects in the neonatal population.

Increase in Multidrug Resistant Neisseria gonorrhoeae FC428-Like Isolates Harboring the Mosaic penA 60.001 Gene, in Nanjing, China (2017-2020).

Since the first Chinese report of the ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone in 2016, additional FC428-like, penA 60.001 isolates have been identified in China. To document the rise in penA 60.001 isolates in Nanjing, China, and characterize their molecular and epidemiological features. N. gonorrhoeae minimum inhibitory concentrations (MICs, mg/L) for ceftriaxone, cefixime, penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, gentamicin and zoliflodacin were determined by agar dilution. MICs for ertapenem were measured by E-test. N. gonorrhoeae antimicrobial sequence typing (NG-STAR) of seven loci (penA, mtrR, porB, ponA, gyrA, parC and 23S rRNA) was analyzed together with N. gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST). Phylogenetic analysis was also performed using whole genomic sequencing (WGS). Fourteen FC428-related penA 60.001 N. gonorrhoeae infections were identified out of 677 infections from 2017 to 2020, in Nanjing, representing an incremental yearly rise in the percentage of the city's N. gonorrhoeae isolates that were FC428-related. Seven FC428-related N. gonorrhoeae infections were acquired in Nanjing, proper; four others in eastern Chinese cities and three from unknown locations. All FC428-related isolates were resistant to ceftriaxone, cefixime, ciprofloxacin, tetracycline and penicillin but susceptible to spectinomycin, gentamicin, ertapenem and zoliflodacin; three strains were resistant to azithromycin. penA 60.001 isolates displayed closely related MLST types and NG-STAR types but relatively distant NG-MAST types. WGS showed a phylogenetic analysis that intermingled with other international isolates. penA 60.001 N. gonorrhoeae isolates emerged in Nanjing, China, beginning in 2017, and have continued to rise.

P43 | Minimal inhibitory and bactericidal concentrations of gentamicin, enrofloxacin, colistin and Melaleuca armillaris essential oil against Pseudomonas aeruginosa isolated from canine otitis externa

Journal of Veterinary Pharmacology and TherapeuticsVolume 46, Issue S1 p. 106-106 POSTER PRESENTATIONS P43 | Minimal inhibitory and bactericidal concentrations of gentamicin, enrofloxacin, colistin and Melaleuca armillaris essential oil against Pseudomonas aeruginosa isolated from canine otitis externa First published: 11 June 2023 https://doi.org/10.1111/jvp.13322Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. Volume46, IssueS1Special Issue: 15th International Congress of the European Association for Veterinary Pharmacology and Toxicology held Bruges, Belgium, July 2–5, 2023June 2023Pages 106-106 RelatedInformation

Sub-inhibitory gentamicin pollution induces gentamicin resistance gene integration in class 1 integrons in the environment

Antibiotics at sub-inhibitory concentrations are often found in the environment. Here they could impose selective pressure on bacteria, leading to the selection and dissemination of antibiotic resistance, despite being under the inhibitory threshold. The goal of this study was to evaluate the effects of sub-inhibitory concentrations of gentamicin on environmental class 1 integron cassettes in natural river microbial communities. Gentamicin at sub-inhibitory concentrations promoted the integration and selection of gentamicin resistance genes (GmRG) in class 1 integrons after only a one-day exposure. Therefore, sub-inhibitory concentrations of gentamicin induced integron rearrangements, increasing the mobilization potential of gentamicin resistance genes and potentially increasing their dissemination in the environment. This study demonstrates the effects of antibiotics at sub-inhibitory concentrations in the environment and supports concerns about antibiotics as emerging pollutants.

Epigenetic-Based Regulation of Transcriptome in Escherichia coli Adaptive Antibiotic Resistance.

Adaptive antibiotic resistance is a transient metabolic adaptation of bacteria limiting their sensitivity to low, progressively increased, concentrations of antibiotics. Unlike innate and acquired resistance, adaptive resistance is dependent on the presence of antibiotics, and it disappears when the triggering factor is removed. Low concentrations of antibiotics are largely diffused in natural environments, in the food industry or in certain body compartments of humans when used therapeutically, or in animals when used for growth promotion. However, molecular mechanisms underlying this phenomenon are still poorly characterized. Here, we present experiments suggesting that epigenetic modifications, triggered by low concentrations of ampicillin, gentamicin, and ciprofloxacin, may modulate the sensitivity of bacteria to antibiotics. The epigenetic modifications we observed were paralleled by modifications of the expression pattern of many genes, including some of those that have been found mutated in strains with permanent antibiotic resistance. As the use of low concentrations of antibiotics is spreading in different contexts, our findings may suggest new targets and strategies to avoid adaptive antibiotic resistance. This might be very important as, in the long run, this transient adaptation may increase the chance, allowing the survival and the flourishing of bacteria populations, of the onset of mutations leading to stable resistance. IMPORTANCE In this study, we characterized the modifications of epigenetic marks and of the whole transcriptome in the adaptive response of Escherichia coli cells to low concentrations of ampicillin, gentamicin, and ciprofloxacin. As the transient adaptation does increase the chance of permanent resistance, possibly allowing the survival and flourishing of bacteria populations where casual mutations providing resistance may give an immediate advantage, the importance of this study is not only in the identification of possible molecular mechanisms underlying adaptive resistance to antibiotics, but also in suggesting new strategies to avoid adaptation.

A sensitive surface-enhanced Raman spectroscopy detection for gentamicin and tobramycin using γ-Al2O3-modified silver nanoparticles coated with bovine serum albumin as substrate

Two aminoglycoside antibiotics (AGs), gentamicin (GEN) and tobramycin (TOB), have good antibacterial activity against most pseudomonas aeruginosa and staphylococcus. The molecular structure of these drugs lack chromogenic groups, which brings challenges to their detection. In this project, the detecting methods for GEN and TOB utilizing surface-enhanced Raman spectroscopy (SERS) based on γ-Al2O3-modified silver nanoparticles (AgNPs) coated with bovine serum albumin (BSA) were established. The enhancement factors (EFs) of GEN and TOB were 2.44 × 105 and 2.67 × 106, respectively. The transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and spectrophotometric techniques were used to characterize the substrate and the combination of the substance with drugs. The pH, the addition amounts for the substrate and coagulant, as well as the mixing time were optimized. On the basis of single factor experiments, a more scientific response surface model was established. The concentrations of GEN and TOB showed good linear relationships with their Raman signals in the ranges of 6.67 × 10−8 – 2.00 × 10−6 and 6.67 × 10−9 – 3.00 × 10−7 mol L−1 respectively. The limits of detection (LODs) were 11.88 and 1.26 nmol L−1 for GEN and TOB, respectively. The methods were used successfully for the samples determination of the two AGs in commercial drugs and meat products.

Effect of gentamicin on CD3+ T-lymphocyte proliferation for treatment of equine recurrent uveitis: An in vitro study.

The objective of the study was to determine the effect of gentamicin on CD3+ T-lymphocyte proliferation and cell viability using an in vitro cell culture model as a means of investigating the mechanism of action of low-dose intravitreal gentamicin injection. Three adult horses with no evidence of ophthalmic or systemic disease. Peripheral blood lymphocytes were treated with gentamicin at concentrations 37.5 μg/mL, 112.5 μg/mL, 187 μg/mL, 375 μg/mL, or 750 μg/mL then stimulated to proliferate with concanavalin A (ConA). 4',6-diamidino-2-phenylindole (DAPI) and carboxyfluoroscein succinimidyl ester (CSFE) were used as markers of cell viability and cell proliferation, respectively. Following 5-day culture, live cell counts and CSFE fluorescent intensity data were collected via automated cell count and flow cytometry. The experimental design was duplicated using preservative-free gentamicin and a proprietary brand formulation. Statistical analysis was performed using two-way ANOVA with Tukey's multiple comparison test. No statistically significant comparisons in CD3+ T-lymphocyte live cell counts and geometric mean fluorescent intensity of CSFE were identified between gentamicin concentrations or formulations. Gentamicin had no effect on equine peripheral blood CD3+ T-lymphocyte cell viability and proliferation in concentrations ranging from "safe" to "retinotoxic" in relation to intravitreal injection volumes. Low-dose intravitreal gentamicin may not suppress the Th1- and Th17-mediated immune response.

Evaluation of Dosing Guidelines for Gentamicin in Neonates and Children.

Although aminoglycosides are frequently prescribed to neonates and children, the ability to reach effective and safe target concentrations with the currently used dosing regimens remains unclear. This study aims to evaluate the target attainment of the currently used dosing regimens for gentamicin in neonates and children. We conducted a retrospective single-center cohort study in neonates and children receiving gentamicin between January 2019 and July 2022, in the Beatrix Children's Hospital. The first gentamicin concentration used for therapeutic drug monitoring was collected for each patient, in conjunction with information on dosing and clinical status. Target trough concentrations were ≤1 mg/L for neonates and ≤0.5 mg/L for children. Target peak concentrations were 8-12 mg/L for neonates and 15-20 mg/L for children. In total, 658 patients were included (335 neonates and 323 children). Trough concentrations were outside the target range in 46.2% and 9.9% of neonates and children, respectively. Peak concentrations were outside the target range in 46.0% and 68.7% of neonates and children, respectively. In children, higher creatinine concentrations were associated with higher gentamicin trough concentrations. This study corroborates earlier observational studies showing that, with a standard dose, drug concentration targets were met in only approximately 50% of the cases. Our findings show that additional parameters are needed to improve target attainment.

Zebrafish (Danio rerio) larvae as a predictive model to study gentamicin-induced structural alterations of the kidney.

Nephrotoxicity is an important drug safety aspect to be assessed during drug discovery and development. To study renal toxicity, in vitro cell-based assays are often used. Unfortunately, translating the results of such cell assays to vertebrates including human remains challenging. Therefore, we aim to evaluate whether zebrafish larvae (ZFL) could serve as a vertebrate screening model to detect gentamicin-induced changes of kidney glomeruli and proximal tubules. To validate the model, we compared the results of ZFL with those obtained from kidney biopsies of gentamicin-treated mice. We used transgenic zebrafish lines expressing enhanced green fluorescent proteins in the glomerulus to visualize glomerular damage. Synchrotron radiation-based computed tomography (SRμCT) is a label-free approach providing three-dimensional representations of renal structures with micrometre resolution. Clinically used gentamicin concentrations induce nephrotoxicity and affect glomerular and proximal tubular morphology. Findings were confirmed in mice and ZFL. There was a strong correlation between fluorescent signals in ZFL, SRμCT- derived descriptors of glomerular and proximal tubular morphology and the histological analysis of mouse kidney biopsies. A combination of SRμCT and confocal microscopy provides unprecedented insights into anatomical structures of the zebrafish kidney. Based on our findings, we suggest to use ZFL as a predictive vertebrate screening model to study drug-induced nephrotoxicity and to bridge the gap between cell culture-based test systems and experiments in mammals.

Stimulation of Aquatic Bacteria from Mammoth Cave, Kentucky, by Sublethal Concentrations of Antibiotics

Many microorganisms secrete secondary metabolites with antibiotic properties; however, there is debate whether the secretions evolved as a means to gain a competitive edge or as a chemical signal to coordinate community growth. The objective of this research was to investigate if select antibiotics acted as a weapon or as a chemical signal by exposing communities of aquatic cave bacteria to increasing concentrations of antibiotics. Water samples were collected from six cave locations where actinobacterial mats appeared to be plentiful. Bacterial growth was measured using colony counts on 10 % tryptic soy agar augmented with increasing concentrations of erythromycin, tetracycline, kanamycin, gentamicin, or quaternary ammonia compounds (QAC). Colony counts generally decreased as the gentamicin, kanamycin and QAC dose increased. In contrast, the colony numbers increased on agar plates supplemented with 0.01 mg L−1, 0.10 mg L−1 and 1.00 mg L−1 erythromycin or tetracycline. A 10.00 mg L−1 dose of each antibiotic treatment reduced bacteria colonies by 98 % or more. Community-level physiological capabilities were evaluated using Ecolog plates inoculated with cave water dosed with either 0.00 mg L−1 or 0.10 mg L−1 of erythromycin. Incubation with the antibiotic almost doubled the number of food substrates used in the first 24 hours. There was a significant increase in the use of acetyl glucosamine, arginine, and putrescine when bacteria were exposed to 0.10 mg L−1 erythromycin triggered by the antibiotic acting as a chemical messenger. Principal component analysis confirmed a shift in substrate preferences when erythromycin was added. A conceptual ecological model is proposed based on the response of aquatic cave bacteria to sublethal antibiotics.

Combination of gentamicin and clindamycin as a promising therapy for multidrug-resistant (MDR) methicillin-resistant Staphylococcus aureus isolated in Enugu State

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is prevalent in Nigeria. Due to its increasing resistance to treatments with single antibiotics, such as vancomycin and others, it has become necessary to identify appropriate combination of antibiotics for treatment. This research tested the proneness of MRSA isolated in southeastern Nigeria to a combined treatment of selected antibiotics (vancomycin, clindamycin, gentamicin, and ceftriaxone), to identify the best combination for the treatment of MRSA isolated in the region. Methods: Various samples from patients were obtained in three laboratories in Enugu, Nigeria and cultured for isolation and purification. Minimum inhibitory concentration of vancomycin, clindamycin, gentamicin, and ceftriaxone antibiotics against the purified isolates was determined and the combined activity of the drugs on the isolates was also evaluated using Checkerboard-technique. Results: MRSA isolates were found to be highly prevalent in the clinical samples. They also showed multidrug-resistant traits. The different combinations of antibiotics against different species of MRSA indicated synergistic, indifferent, additive, and antagonistic effects. The best combination that could treat muti-drug resistant MRSA was found to be gentamycin and clindamycin. MRSA has developed resistance to many drugs, making its infections difficult to treat. The combination of gentamicin and clindamycin is a promising strategy for treating MRSA in the region.

Assessment of antibiotic resistance and biofilm formation of Enterococcus species isolated from different pig farm environments in Poland

BackgroundEnteroccocus spp. are human opportunistic pathogens causing a variety of serious and life-threating infections in humans, including urinary tract infection, endocarditis, skin infection and bacteraemia. Farm animals and direct contact with them are important sources of Enterococcus faecalis (EFA) and Enterococcus faecium (EFM) infections among farmers, veterinarians and individuals working in breeding farms and abattoirs. The spread of antibiotic-resistant strains is one of the most serious public health concerns, as clinicians will be left without therapeutic options for the management of enterococcal infections. The aim of the study was to evaluate the occurrence and antimicrobial susceptibility of EFA and EFM strains isolated from a pig farm environment and to determine the biofilm formation ability of identified Enterococcus spp. strains.ResultsA total numer of 160 enterococcal isolates were obtained from 475 samples collected in total (33.7%). Among them, 110 of genetically different strains were identified and classified into EFA (82; 74.5%) and EFM (28; 25.5%). Genetic similarity analysis revealed the presence of 7 and 1 clusters among the EFA and EFM strains, respectively. The highest percentage of EFA strains (16; 19.5%) was resistant to high concentrations of gentamicin. Among the EFM strains, the most frequent strains were resistant to ampicillin and high concentrations of gentamicin (5 each; 17.9%). Six (7.3%) EFA and 4 (14.3%) EFM strains showed vancomycin resistance (VRE - Vancomycin-Resistant Enterococcus). Linezolid resistance was found in 2 strains of each species. The multiplex PCR analysis was performed to identify the vancomycin resistant enterococci. vanB, vanA and vanD genotypes were detected in 4, 1 and 1 EFA strains, respectively. Four EFA VRE-strains in total, 2 with the vanA and 2 with the vanB genotypes, were identified. The biofilm analysis revealed that all vancomycin-resistant E. faecalis and E. faecium strains demonstrated a higher biofilm-forming capacity, as compared to the susceptible strains. The lowest cell count (5.31 log CFU / cm2) was reisolated from the biofilm produced by the vancomycin-sensitive strain EFM 2. The highest level of re-isolated cells was observed for VRE EFA 25 and VRE EFM 7 strains, for which the number was 7 log CFU / cm2 and 6.75 log CFU / cm2, respectively.ConclusionsThe irrational use of antibiotics in agriculture and veterinary practice is considered to be one of the key reasons for the rapid spread of antibiotic resistance among microorganisms. Owing to the fact that piggery environment can be a reservoir of antimicrobial resistance and transmission route of antimicrobial resistance genes from commensal zoonotic bacteria to clinical strains, it is of a great importance to public health to monitor trends in this biological phenomenon.

Untargeted metabolomics analysis of gentamicin-induced tolerant colonies of Klebsiella pneumoniae

PurposeAntibiotic resistance development in pathogenic bacteria like Klebsiella pneumoniae seriously threatens humankind. Therefore, it is important to understand the interaction of bacteria with antibiotic agents and how it acquires resistance at the molecular level. The current study describes metabolomics analysis of K. pneumoniae sensitive strains and its gentamicin-tolerant (resistant) strains. MethodsK. pneumoniae strains were treated at five different concentrations of gentamicin, increasing from a low dose (16.2 µg/mL) to the highest dose (250 µg/mL) at three incubation time periods (24 h, 48 h, and 72 h). Colonies obtained at various concentrations and time intervals were subjected to metabolomic analysis using GC–MS. ResultsA drastic change was observed in the morphology of K. pneumoniae colonies with the increasing gentamicin concentration. Moreover, K. pneumoniae strains grown at the highest concentration (250 µg/mL) were found tolerant to 1 mg/mL gentamicin (4-folds) and considered resistant strains. A total of 459 metabolites were identified. A sequential down/up-regulation in 4, 3, and 4 metabolites were observed in association with the increasing gentamicin concentration at 24 h, 48 h, and 72 h, respectively. While with the comparative analysis of resistant and sensitive strains, a total of seven down- and sixteen up-regulated metabolites were observed. The concentration of some fatty acids and sugars have been found to increase while, a few metabolites like inosine, tyrosine, 1-propionylproline, and 2-hydroxyacetic acid have been found down-regulated in resistant samples. ConclusionThese regulator metabolites might be associated with resistance development in K. pneumoniae against gentamicin and might be helpful in the rapid detection of gentamicin-resistant clinical strains.

Discovery of Novel Resistance Mechanisms of Vibrio parahaemolyticus Biofilm against Aminoglycoside Antibiotics.

Inappropriate use of antibiotics eventually leads to the emergence of antibiotic-resistant strains and invalidates the treatment of infectious diseases. Aminoglycoside antibiotics (AGAs) are a class of broad-spectrum cationic antibiotics widely used for the treatment of Gram-negative bacterial infections. Understanding the AGA resistance mechanism of bacteria would increase the efficacy of treating these infections. This study demonstrates a significant correlation between AGA resistance and the adaptation of biofilms by Vibrio parahaemolyticus (VP). These adaptations were the result of challenges against the aminoglycosides (amikacin and gentamicin). Confocal laser scanning microscope (CLSM) analysis revealed an enclosure type mechanism where the biological volume (BV) and average thickness (AT) of V. parahaemolyticus biofilm were significantly positively correlated with amikacin resistance (BIC) (p < 0.01). A neutralization type mechanism was mediated by anionic extracellular polymeric substances (EPSs). The biofilm minimum inhibitory concentrations of amikacin and gentamicin were reduced from 32 µg/mL to 16 µg/mL and from 16 µg/mL to 4 µg/mL, respectively, after anionic EPS treatment with DNase I and proteinase K. Here, anionic EPSs bind cationic AGAs to develop antibiotic resistance. Transcriptomic sequencing revealed a regulatory type mechanism, where antibiotic resistance associated genes were significantly upregulated in biofilm producing V. parahaemolyticus when compared with planktonic cells. The three mechanistic strategies of developing resistance demonstrate that selective and judicious use of new antibiotics are needed to win the battle against infectious disease.

Gentamicin Upregulates the Gene Expression of hla and nuc in Staphylococcus aureus

The current study aimed to detect the effect of gentamicin stress on the expression of hla (encodes hemolysin) and nuc (encodes nuclease) genes of Staphylococcus aureus. Fifty-eight isolates identified as S. aureus were isolated locally from different clinical specimens. Disk diffusion method was used to detect the resistance to S. aureus. The minimum inhibitory concentration (MIC) of gentamicin was estimated by broth microdilution method. hla and nuc genes were determined by polymerase chain reaction technique. The biofilm was evaluated using the microtiter plate method in the presence and absence of gentamicin at sub-MIC. The results showed that 18 (31%) and 40 (69%) S. aureus isolates were sensitive and resistant to gentamicin, respectively. All S. aureus isolates succeeded in forming biofilm. However, eight (13.79%), 28 (48.28%) and 22 (37.93%) isolates produced weak, moderate and strong biofilms respectively. In most isolates, gentamicin at sub-MIC decreased biofilm intensity. Due to gentamicin stress, the hla and nuc genes were upregulated in S. aureus biofilm.