Minimal Inhibitory Concentration Of Rifampicin Research Articles

A Comparison of the Minimum Inhibitory Concentration of Antibiotics in Staphylococcus Species Isolated From Orthopedic and Respiratory Medicine Infections.

Antibiotic susceptibility is very important for the successful treatment of orthopedic infections, particularly for implant-related infections. While the minimum inhibitory concentrations (MICs) of Staphylococcus species were well investigated for the isolates from the respiratory tract, investigations for orthopedic pathogens are very limited. We investigated the antibiotic MIC values of Staphylococcus species isolated from orthopedic infections and compared them with those of respiratory medicine isolatesused as a control. The MICs of vancomycin (VCM), arbekacin (ABK), teicoplanin (TEIC), linezolid (LZD), and rifampicin (RFP) of a total of consecutive 259 (89 orthopedic and 170 respiratory) Staphylococcus speciesisolated in our laboratory from January 2013 to July 2016 were retrospectively reviewed. Differences between the MICs of each antibiotic in orthopedic and respiratory samples were determined. The number of methicillin-sensitive Staphylococcus aureus (MSSA) with a VCM MIC of <0.5 μg/mL among respiratory isolates was significantly higher than that among orthopedic isolates, while those with a MIC of 2 μg/mL were significantly lower (P = 0.0078). The proportion of methicillin-resistant coagulase-negative staphylococci (MRCNS) isolates with a VCM MIC of 2 μg/mL was significantly higher in orthopedic samples than that of methicillin-resistant Staphylococcus aureus (MRSA) (P < 0.001) in respiratory isolates. The proportion of MRCNS orthopedic isolates with an RFP MIC of >2 μg/mL was significantly higher (P = 0.0058) than that of other orthopedic staphylococci. The VCM MICs of Staphylococcus species from orthopedic infections were higher than those of respiratory samples, particularly MRCNS from implant-related samples.

Amphiphilic tribasic galactosamines potentiate rifampicin in Gram-negative bacteria at low Mg++/Ca++concentrations

Many antibiotics specific to Gram-positive bacteria like rifampicin (RIF) are inactive in Gram-negative bacteria because of outer membrane (OM) impermeability. Enhancing the OM permeability of these antibiotics with the help of OM perturbants is a promising strategy to develop new agents against Gram-negative bacteria. Here we report the synthesis and biological properties of amphiphilic tribasic galactosamines as potential RIF potentiators. Our results demonstrate that tribasic galactose-based amphiphiles potentiate RIF in multidrug-resistant Acinetobacter baumannii and Escherichia coli but not Pseudomonas aeruginosa in low salt-containing media. Under these conditions, lead compounds 20, 22 and 35 lowered the minimum inhibitory concentration of RIF by 64- to 256-fold against Gram-negative bacteria. However, the RIF-potentiating effect was reduced when bivalent Mg++ or Ca++ ions were added in the media at physiological concentrations. Overall, our results indicate that amphiphilic tribasic galactosamine-based compounds show reduced RIF-potentiating effects when compared to amphiphilic tobramycin antibiotics at physiological salt concentrations.

Efflux pump effects on Mycobacterium tuberculosis drug resistance

Resistance and tolerance to antituberculosis drugs have become serious problems in disease treatment. This multi-phase study investigated the contributions of efflux pumps to Mycobacterium tuberculosis drug resistance. In the first phase, the minimum inhibitory concentration (MIC) levels of antibiotics were determined. In the second phase, MIC levels were determined in the presence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl hydrazone (CCCP), verapamil, reserpine and thioridazine. In the third phase, MIC levels were reduced in 6 M. tuberculosis isolates in the presence of efflux pump inhibitors to determine the expression of putative efflux pump genes by reverse transcriptase-polymerase chain reaction (RT-PCR). MIC levels of fluoroquinolones decreased in 6 (6.52%) isolates, MIC of rifampicin in 4 (4.34%), and MIC of streptomycin in 3 (3.26%) in the presence of efflux pump inhibitors reserpine, CCCP and verapamil. The efflux pump inhibitors CCCP, verapamil, and reserpine changed MICs 2- to 16-fold. Overexpression of all 15 efflux pump genes was observed in 6 isolates with a reduction in MIC values in the presence of efflux pump inhibitors. The overexpression of efflux-related genes in resistant isolates suggests that efflux pumps are associated with resistance development.

Myoinositol and methyl stearate increases rifampicin susceptibility among drug-resistant Mycobacterium tuberculosis expressing Rv1819c.

The alarming increase in multidrug resistance, which includes Bedaquiline and Delamanid, stumbles success in Tuberculosis treatment outcome. Mycobacterium tuberculosis gains resistance to rifampicin, which is one of the less toxic and potent anti-TB drugs, through genetic mutations predominantly besides efflux pump mediated drug resistance. In recent decades, scientific interventions are being carried out to overcome this hurdle using novel approaches to save this drug by combining it with other drugs/molecules or by use of high dose rifampicin. This study reports five small molecules namely Ellagic acid, Methyl Stearate, Myoinositol, Rutin, and Shikimic acid that exhibit synergistic inhibitory activity with rifampicin against resistant TB isolates. In-silico examinations revealed possible blocking of Rv1819c-an ABC transporter efflux pump that was known to confer resistance in M. tuberculosis to rifampicin. The synergistic anti-TB activity was assessed using a drug combination checkerboard assay. Efflux pump inhibition activity of ellagic acid, myoinositol, and methyl stearate was observed through ethidium bromide accumulation assay in the drug-resistant M. tuberculosis clinical strains and recombinant Mycobacterium smegmatis expressing Rv1819c in coherence with the significant reduction in the minimum inhibitory concentration of rifampicin. Cytotoxicity of the active efflux inhibitors was tested using in silico and ex vivo methods. Myoinositol and methyl stearate were completely non-toxic to the hematological and epithelial cells of different organs under ex vivo conditions. Based on these findings, these molecules can be considered for adjunct TB therapy; however, their impact on other drugs of anti-TB regimen needs to be tested.

The Inappropriateness of Using Rifampicin E-Test to Predict Rifabutin Resistance in Helicobacter pylori

The aim of this study was to evaluate the rifamycin cross-resistance in Helicobacter pylori, and whether the use of rifampicin E-test strips to screen H. pylori rifabutin resistance is appropriate. A total of 89 H. pylori isolates were included. Rifampicin minimum inhibitory concentrations (MICs) were obtained by E-test, while the MICs for rifapentine, rifaximin, and rifabutin were determined by agar dilution method. The rifamycin resistance rates based on different breakpoints were compared. Isolates with high-level rifampicin resistance were subjected to whole-genome sequencing. A wide distribution of MICs (mostly in the range 0.125-8 mg/L) was observed for rifampicin, rifapentine, and rifaximin. Using MIC >1, ≥ 4, and > 4 mg/L as the breakpoints, resistance rates to rifampicin/rifapentine/rifaximin were 60.4%/48.3%/38.2%, 28.1%/25.8%/23.6%, and 15.7%/16.9%/7.9%, respectively. However, the rifabutin MICs of all the tested H. pylori isolates were extremely low (≤0.016 mg/L). Applying MIC ≥ 0.125 mg/L as the breakpoint, rifabutin resistance was nil. No mutation was found in the rpoB gene sequences of the 2 isolates with high-level rifampicin resistance. There is a lack of cross-resistance between rifabutin and other rifamycins in H. pylori. The use of rifampicin E-test to predict H. pylori rifabutin resistance is inappropriate.

The RNA Polymerase Inhibitor Corallopyronin A Has a Lower Frequency of Resistance Than Rifampicin in Staphylococcus aureus.

Corallopyronin A (CorA) is active against Gram-positive bacteria and targets the switch region of RNA polymerase. Because of the high frequency of mutation (FoM) leading to rifampicin resistance, we determined the CorA FoM in S. aureus using fluctuation analysis at 4 × minimum inhibitory concentration (MIC). Resistant mutants were characterized. S. aureus strains HG001, Mu50, N315, and USA300 had an MIC of 0.25 mg/L. The median FoM for CorA resistance was 1.5 × 10−8, 4.5-fold lower than the median FoM of 6.7 × 10−8 for rifampicin, and was reflected in a 4-fold lower mutation rate for CorA than rifampicin (6 × 10−9 for CorA vs. 2.5 × 10−8 for rifampicin). In CorA-resistant/rifampicin-sensitive strains, the majority of amino acid exchanges were S1127L in RpoB or K334N in RpoC. S. aureus Mu50, a rifampicin-resistant clinical isolate, yielded two further exchanges targeting amino acids L1131 and E1048 of the RpoB subunit. The plating of >1011 cells on agar containing a combination of 4 × MIC of rifampicin and 4 × MIC of CorA did not yield any growth. In conclusion, with proper usage, e.g., in combination therapy and good antibiotic stewardship, CorA is a potential antibiotic for treating S. aureus infections.

Inclusion Complexes of Rifampicin with Native and Derivatized Cyclodextrins: In Silico Modeling, Formulation, and Characterization.

Inclusion complexation of rifampicin (RIF) with several types of cyclodextrins (βCD, hydroxypropyl-βCD, γCD, hydroxypropyl-γCD) in aqueous solutions at different pH values was investigated to assess the interactions between RIF and cyclodextrins (CDs). Molecular modeling was performed to determine the possible interactions between RIF and CDs at several pH values. The inclusion complexes were characterized by differential scanning calorimetry, Fourier transform infrared spectroscopy, powder X-ray diffractometry, and scanning electron microscopy. Moreover, this study evaluated the dissolution profile and antibacterial activity of the formed complexes. Phase solubility analysis suggested the formation of RIF-CD affirmed 1:1 stoichiometry at all pH values (except RIF-βCD at pH 4.0 and both βCD and γCD at pH 9.0). The inclusion complexation of RIF with CD successfully increased the percentage of RIF released in in vitro studies. The inclusion complexes of RIF exhibited more than 60% of RIF released in 2 h which was significantly higher (p < 0.05) than release of pure RIF, which was only less than 10%. Antibacterial activity of RIF-CD complexes (measured by the minimum inhibitory concentration of RIF against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus) was lower for both RIF-βCD and RIF-HPγCD at pH 7.0 to pure RIF suspension. In conclusion, this work reports that both βCD and γCD can be used to enhance the solubility of RIF and thus, improve the effectivity of RIF by decreasing the required daily dose of RIF for the treatment of bacterial infections.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

Effects of cigarette smoke on the administration of isoniazid and rifampicin to macrophages infected with Mycobacterium tuberculosis

Background Smoking is a cause behind many diseases, including tuberculosis, and it is a risk factor for tuberculosis infection and mortality. Moreover, smoking is associated with a poor tuberculosis treatment outcome. Objectives In this study, we focus on the effects of cigarette smoke on an infected cell culture treated with anti-tuberculosis drugs. Materials and methods Cytotoxicity on THP-1, J774A.1 and MH-S cell lines and growth of Mycobacterium tuberculosis exposed to a reference or a commercial cigarette was evaluated. THP-1 cell line was exposed to cigarette smoke, infected with Mycobacterium tuberculosis and treated with anti-tuberculosis drugs. Apoptosis and death cell were also tested on M. bovis BCG infected cells. Minimal inhibitory concentrations of anti-tuberculosis drugs were analyzed. Results All cells lines showed viability values higher than 80% when exposed to cigarette smoke extract. However, THP-1 cell line infected with M. bovis BCG and exposed to Marlboro cigarette smoke showed up to a 54% reduction of apoptotic cells than cells unexposed to smoke. M. tuberculosis exposed to Marlboro cigarette smoke for 11 days had an optical density 16% lower than unexposed bacteria. When cells were infected with M. tuberculosis, the intracellular recovery of CFUs showed up to a 0.66 log reduction in cells exposed to cigarette smoke extract because of a potential impairment in the phagocytosis. Macrophages treated with drugs showed up to a 2.55 log reduction in the intracellular load burden compared with non-treated ones. Despite poor treatment outcome on TB smoker patients, minimal inhibitory concentration of rifampicin increased only 2-fold in M. tuberculosis exposed to cigarette smoke. Conclusion Smoking interferes with tuberculosis treatment impairing the immunity of the host.

Local and Sustained Delivery of Rifampicin from a Bioactive Ceramic Carrier Treats Bone Infection in Rat Tibia.

Next-generation treatment strategies to treat osteomyelitis with complete eradication of pathogen at the bone nidus and prevention of emergence of drug resistance is a real challenge in orthopedics. Conventional treatment strategies including long-term adherence of patients to systemic antibiotic delivery, local delivery using nondegradable vehicles, and surgical debridement are not completely effective in achieving successful results. In this study, a broad-spectrum antibiotic, rifampicin (RFP), was incorporated into a biphasic nanohydroxyapatite (nHAP)/calcium sulfate ceramic carrier (NC) system. In vivo release and distribution of rifampicin was evaluated for a period of one month by implanting NC and NC + RFP in a subcutaneous pouch in a rat model. We detected the RFP in bone and implanted NC scaffolds even after day 28 and the concentration was still higher than the minimal inhibitory concentration of RFP when it was implanted with NC in an abdominal subcutaneous pouch. Moreover, we also observed the accumulation of RFP in bone and NC when administered orally, showing strong binding between RFP and nHAP. Additionally, we generated an osteomyelitis bone infection model in the rat tibia using Staphylococcus aureus as an infective agent to evaluate the antibacterial and osteogenic efficiency of RFP containing NC as a delivery system. S. aureus mediated implant infection is a major problem in orthopedics. The results suggested that NC loaded with RFP could eradicate the pathogen completely in the bone nidus. Further, defect healing and bone formation were also evaluated by micro-CT and histological analysis demonstrating proper trabecular-type bone formation at the debridement site and complete healing of the defect when NC + RFP was implanted. Our findings provide an insight into the use of an nHAP based ceramic matrix as a carrier of rifampicin to eradicate the bone infection and simultaneously promote bone healing at the bone nidus.

Wild-Type MIC Distribution for Re-evaluating the Critical Concentration of Anti-TB Drugs and Pharmacodynamics Among Tuberculosis Patients From South India.

The World Health Organization (WHO) has developed specific guidelines for critical concentrations (CCs) of antibiotics used for tuberculosis (TB) treatment, which is universally followed for drug susceptibility testing (DST) of clinical specimens. However, the CC of drugs can differ significantly among the mycobacterial species based on the population, geographic location, and the prevalence of the infecting strain in a particular area. The association between CC and the minimal inhibitory concentration (MIC) of anti-TB drugs is poorly understood. In this study, we assessed the MICs of anti-TB drugs, including isoniazid (INH), rifampicin (RMP), moxifloxacin (MXF), ethambutol (ETH), and p-aminosalicylic acid (PAS) on drug-sensitive Mtb isolates from pulmonary TB patients in South India. The MIC assays performed using solid- and liquid-growth media showed changes in the CC of a few of the tested antibiotics compared with the WHO-recommended levels. Our observation suggests that the WHO guidelines could potentially lead to overdiagnosis of drug-resistant cases, which can result in inappropriate therapeutic decisions. To evaluate the correlation between drug-resistance and CC, we performed the whole-genome sequencing for 16 mycobacterial isolates, including two wild-type and 14 resistant isolates. Our results showed that two of the isolates belonged to the W-Beijing lineage, while the rest were of the East-African–Indian type. We identified a total of 74 mutations, including five novel mutations, which are known to be associated with resistance to anti-TB drugs in these isolates. In our previous study, we determined the serum levels of INH and RMP among the same patients recruited in the current study and estimated the MICs of the corresponding infected isolates in these cases. Using these data and the CCs for INH and RMP from the present study, we performed pharmacodynamics (PD) evaluation. The results show that the PD of RMP was subtherapeutic. Together, these observations emphasize the need for optimizing the drug dosage based on the PD of large-scale studies conducted in different geographical settings.

ZnO Nanoparticles and Rifampicin Synergistically Damage the Membrane of Mycobacteria

The emergence of drug resistance in tuberculosis requires alternative strategies to combat the resistance. To this end, we have synthesized zinc oxide nanoparticles (ZnO NPs) of 11 nm diameter and evaluated its effect against mycobacteria alone, as well as in combination with the first-line anti-TB drug, rifampicin. These ZnO NPs themselves are not inhibitory against the wild-type (WT) Mycobacterium smegmatis, up to a concentration as high as 256 μg/mL. However, a subinhibitory concentration of only 32 μg/mL of ZnO NPs in combination with rifampicin, showed a 4-fold reduction in the minimum inhibitory concentration of rifampicin, against WT M. smegmatis. Thus, the fractional inhibitory concentration index for the combination was found to be 0.375, which proves the effect to be synergistic. This is explained by high membrane permeability observed in presence of the combination of ZnO NPs and rifampicin, which increased with time. High membrane permeability, in turn, increased rifampicin uptake in cells, which subsequently enhanced the bactericidal effect of the drug. The resultant morphology of the bacteria, from Cryo-SEM, showed membrane damage, following treatment with a combination of ZnO NPs and rifampicin. However, this combination showed very less reactive oxygen species (ROS) production, compared to ZnO NPs alone. Hence, we can conclude that membrane damage is the dominant mechanism behind synergism of the combination, rather than that of ROS generation. This combination also proved to be successful against our laboratory-generated, drug resistant strains of M. smegmatis, as well as against vaccine strain- M. bovis BCG, which proves the general applicability of this synergism for different strains. We hence propose that our developed ZnO NPs and drug combination can be promising for overcoming mycobacterial drug resistance.

Detection of High-Level Rifaximin Resistance in Enteric Bacteria by Agar Screen.

Objectives: This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was developed to detect high-level resistance. Methods: A total of 401 isolates nonsusceptible to third-generation cephalosporins (including 342 Escherichia coli and 39 Klebsiella spp. and 20 Enterobacter spp.) were tested by microdilution for their MICs of rifaximin and rifampicin. Isolates with a confirmed rifaximin minimal inhibitory concentration (MIC) of >64 mg/L and a number of high-level resistant, and susceptible control isolates were tested for growth on Mueller-Hinton agar supplemented with rifaximin or rifampicin at a concentration of 256 mg/L. Amino acid mutations in rpoB and the presence of rifaximin resistance-associated genes arabidopsis response regulator (arr) 2/3 were investigated. Results: Microdilution assays identified rifaximin resistance in nine E. coli and three Klebsiella spp. isolates with complete cross-resistance to rifampicin (MICs of both >64 mg/L). The rifaximin agar screen correctly identified 9/9 clinical E. coli isolates, 2/2 E. coli controls, and 3/3 Klebsiella spp. with high-level rifaximin resistance, and was negative in 45 control clinical isolates with rifaximin MICs ranging between 2 and 32 mg/L according to broth microdilution. All nine high-level rifaximin agar screen-positive E. coli clinical isolates (vs. none of the tested controls) had rpoB mutations or carried arr2/3. Conclusions: Our agar screen test has the potential to detect high-level rifaximin-resistant Enterobacterales. Such strains remain rare among extended spectrum beta-lactamase (ESBL)-positive enteric bacteria, but may emerge among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.

RpoB Mutations Causing Discordant Rifampicin Susceptibility in Mycobacterium tuberculosis: Retrospective Analysis of Prevalence, Phenotypic, Genotypic, and Treatment Outcomes.

BackgroundDiscordant genotypic/phenotypic rifampicin susceptibility testing in Mycobacterium tuberculosis is a significant challenge, yet there are limited data on its prevalence and how best to manage such patients. Whether to treat isolates with rpoB mutations not conferring phenotypic resistance as susceptible or multidrug-resistant tuberculosis (MDR-TB) is unknown. We describe phenotypic and genotypic characteristics of discordant isolates and clinical characteristics and treatment outcomes of affected patients in KwaZulu-Natal, South Africa.MethodsWe analyzed clinical isolates showing rifampicin resistance on GenoType MTBDRplus while susceptible on 1% agar proportion method. We measured rifampicin minimum inhibitory concentrations (MICs) using Middlebrook 7H10 agar dilution and BACTEC MGIT 960. Sensititre MYCOTB plates were used for drug-susceptibility testing, and rpoB gene sequencing was performed on all isolates. Local MDR-TB program data were reviewed for clinical information and patient outcomes.ResultsDiscordant isolates constituted 4.6% (60) of 1302 rifampicin-resistant cases over the study period. Of these, 62% remained susceptible to isoniazid and 98% remained susceptible to rifabutin. Rifampicin MICs were close to the critical concentration of 1 µg/mL (0.5–2 µg/mL) for 83% of isolates. The most frequent rpoB mutations were Q513P (25.3%), D516V (19.2%), and D516Y (13.3%). Whereas 70% were human immunodeficiency virus infected, the mean CD4 count was 289 cells/mm3 and 87% were receiving antiretroviral therapy. Standard therapy for MDR-TB was used and 53% achieved successful treatment outcomes.ConclusionsRifampicin-discordant TB is not uncommon and sequencing is required to confirm results. The high susceptibility to rifabutin and isoniazid and poor treatment outcomes with the current regimen suggest a potential utility for rifabutin-based therapy.

Staphylococcus aureus at an Indian tertiary hospital: Antimicrobial susceptibility and minimum inhibitory concentration (MIC) creep of antimicrobial agents.

Staphylococcus aureus causes a variety of symptoms and diseases and has been associated with high morbidity and mortality. A global population drift in clinical S. aureus isolates towards reduced antimicrobial susceptibility is being reported. In this study, the antimicrobial susceptibility profile and minimum inhibitory concentration (MIC) creep of vancomycin, linezolid, teicoplanin and rifampicin against clinical S. aureus isolates at an Indian tertiary centre from January 2012 to December 2016 were investigated. All consecutive, non-duplicate S. aureus isolates (n=1466) recovered from hospitalised patients identified by VITEK®2 were included in the study. Clinical isolates were tested against 20 antibiotics and were evaluated according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Statistical significance of the MIC creeps of four antimicrobials (vancomycin, linezolid, teicoplanin and rifampicin) was ascertained. S. aureus isolates recovered from all clinical samples demonstrated high resistance to ampicillin, ciprofloxacin and penicillin (75-100%) and low resistance to amikacin, linezolid, netilmicin, nitrofurantoin, teicoplanin and vancomycin (0-13%). The MIC90 values (MIC required to inhibit 90% of the isolates) for vancomycin, linezolid and rifampicin decreased, whereas the MIC90 for teicoplanin increased. The change in the geometric mean MIC of rifampicin was found to be statistically significant. A statistically significant and progressive MIC creep was observed for teicoplanin and rifampicin. Despite remaining susceptible, S. aureus is not inert to antibiotic pressure. Implementation of preventive measures in healthcare settings is required worldwide to combat the increasing number of infections by this pathogen.

Minimum inhibitory concentration and killing properties of rifampicin against canine Staphylococcus pseudintermedius isolates from dogs in the southeast USA.

Meticillin-resistant (MR) staphylococcal pyoderma in dogs has led to increased use of alternate antibiotics such as rifampicin (RFP). However, little information exists regarding its pharmacodynamics in MR Staphylococcus pseudintermedius. To determine the minimum inhibitory concentration (MIC) and killing properties of RFP for canine Staphylococcus pseudintermedius isolates. The MIC of RFP was determined using the ETEST® for 50 meticillin-susceptible (MS) and 50 MR S.pseudintermedius isolates collected from dogs. From these isolates, two MS isolates (RFP MIC of 0.003 and 0.008μg/mL, respectively) and two MR isolates (RFP MIC of 0.003 and 0.012μg/mL, respectively) were subjected to time-kill studies. Mueller-Hinton broth was supplemented with RFP at 0, 0.5, 1, 2, 4, 8, 16 and 32 times the MIC for 0, 2, 4, 10, 16 and 24h. The number of viable colony forming units in each sample was determined using a commercial luciferase assay kit. The MIC50 and MIC90 were the same for MS and MR isolates, at 0.004μg/mL and 0.008μg/mL, respectively. Rifampicin kill curves were not indicative of concentration-dependency, suggesting time-dependent activity. Two isolates (MS 0.003 and 0.008μg/mL) exhibited bacteriostatic activity, whereas two others (MR 0.003 and 0.012μg/mL) exhibited bactericidal activity. This study demonstrated that MS and MR S.pseudintermedius isolates were equally susceptible to rifampicin and that dosing intervals should be designed for time-dependent efficacy. These data can support pharmacokinetic studies of RFP in dogs with susceptible infections caused by S.pseudintermedius.

Monitoring of multiresistant community-associated MRSA strains from patients with pathological processes of different localization

The therapy of infections, caused by methicillin-resistant Staphylococcus aureus (MRSA) with multiple resistance to antibiotics remains one of the most acute problems all over the world. It is all the more complicated since a priori the MSRA strains are not sensitive to the group of β-lactam antibiotics and multiresistant isolates are resistant to other groups of antimicrobial preparations, including antibiotics of choice (rifampicin, vancomycin, fusidic acid, co-trimoxazole and linezolid). From the samples of biomaterials of patients with pathological processes of different localization, we isolated 335 strains of bacteria, which were identified as Staphylococcus aureus, 169 (50.4%) of which were methicillin-resistant variants: 57.5% cultures were isolated from the nasal discharge; 50.7% – from faeces at intestinal dysbioses; by 40.0% – from conjunctival discharge, pharyngeal swab, outer ear swab and sputum; 33.3% – from urine samples. Antibiotic susceptibility of the isolated cultures was estimated by the disc-diffusion method and the method of serial dilution. The MRSA strains appeared to be most resistant to gentamycin, erythromycin (by 59.5% of cultures) and ciprofloxacin (53.3% of isolates), most sensitive – to vancomycin, co-trimoxazole and fusidic acid. The frequency of isolation of the cultures that are resistant to antibiotics did not exceed 4.1%. Rifampicin suppressed the growth of 75.8% and linezolid – of 100.0% of strains. Depending on the kind of biomaterial taken, MRSA strains, isolated from the nasal cavity, outer ear, urine samples, samples of sputum and faeces at intestinal dysbioses proved to be most resistant to the tested antimicrobial preparations. Rifampicin- and vancomycin-resistant strains of methicillin-resistant staphylococci made up 21.3% of the total number of the detected MRSA. They were most often isolated from the clinical samples taken from the nasal cavity and faeces. When determining minimal inhibitory concentration (MIC) of rifampicin and vancomycin, which are antibiotics of choice for treatment of infections caused by multiresistant MRSA, it was found that for 55.5% of the MRSA strains isolated from faeces, MIC of rifampicin coincided with the threshold value for this antibiotic and for 44.5%, it exceeded the threshold value by 2 times (4 µg/ml). 22.2% of them were characterized by the critical value of susceptibility to vancomycin (MIC ≥ 2 µg/ml). From rifampicin- and vancomycin-resistant MRSA stains, isolated from the nasal cavity, MIC of rifampicin coincided with the threshold value for this antibiotic for 66.7% of cultures, and exceeded it at least by 2 times for 33.3%. 11.1% of them were characterized by the critical level of susceptibility to vancomycin (MIC ≥ 2 µg/ml) and by 3.7% of strains exceeded MIC by 2 and 4 times respectively (4 and 8 µg/ml).

Synergistic Response of Rifampicin with Hydroperoxides on Mycobacterium: A Mechanistic Study.

Prolonged chemotherapy as well as rapid development of antimicrobial resistance are two of the major concerns for treatment of mycobacterial infections. To enhance the effectiveness of current drug regimens, search for compounds having synergistic interaction with anti-mycobacterial drugs has become indispensable. Here, we have investigated the intervention by oxidative stress, a major factor in mycobacterial pathogenesis, in combination with rifampicin (RIF), a first-line drug used against Mycobacterium tuberculosis. We have observed that a sub-inhibitory concentration of cumene hydroperoxide (CHP), a hydrophobic oxidant, synergistically reduced the minimum inhibitory concentration of RIF by fourfold, with a Fractional Inhibitory Concentration Index (FICI) of 0.45. Also, this interaction was found to be robust and synergistic against different strains of M. smegmatis as well as on M. bovis BCG, with FICI ranging from 0.3 to 0.6. Various physiological, biochemical and molecular parameters were explored to understand the mechanism of synergy. It was observed that increased membrane permeability owing to the presence of the oxidant, led to higher uptake of the drug. Moreover, downregulation of the hydroperoxide reductases by RIF, a transcriptional inhibitor, prevented quenching of the reactive oxygen species produced in the presence of CHP. The lipid soluble reactive species triggered autocatalytic lipid peroxidation (LPO), observed here as extensive membrane damage eventually leading to growth inhibition. Furthermore, it was seen that in combination with hydrogen peroxide (H2O2), the effect was only additive, establishing LPO as a key aspect leading toward synergism. To conclude, this work suggests that targeting the bacterial membrane by a radical species can have a significant impact on the treatment of tuberculosis.

In vitro stepwise selection of reduced susceptibility to lipoglycopeptides in enterococci

The propensity of oritavancin to select for stably elevated oritavancin minimum inhibitory concentrations (MICs) was studied by serial passaging of strains in broth containing oritavancin for 20days. Seven clinical strains of Enterococcus faecalis and E. faecium were studied; they included vancomycin-susceptible and both VanA and VanB vancomycin-resistant isolates. Stepwise oritavancin selection yielded stably elevated oritavancin MICs in six of the seven strains, with MIC increases ranging from 4–32-fold. By comparison, stepwise selection with comparator agents dalbavancin (4- to >128-fold MIC increases), telavancin (4–8-fold MIC increases) and daptomycin (4–32-fold MIC increases) also yielded selectants with elevated MICs of the respective agents. Oritavancin selectants retained parental MICs of vancomycin, daptomycin, linezolid and rifampicin. Some, but not all of the oritavancin selectants also showed MIC increases to the lipoglycopeptides telavancin, dalbavancin and teicoplanin, suggesting that within the lipoglycopeptide class, different mechanisms of action may be elucidated.

In vitro antimicrobial synergy of colistin with rifampicin and carbapenems against colistin-resistant Acinetobacter baumannii clinical isolates

Increased use of colistin in a clinical setting had resulted in the emergence of colistin-resistant (CoR) Acinetobacter baumannii. Combination therapy has been studied as a new approach to treat infections caused by A. baumannii. Here, we investigated the in vitro antimicrobial synergistic activities of several antimicrobial agent combinations against CoR A. baumannii. A total of 41 non-duplicate clinical isolates of CoR A. baumannii from a tertiary care hospital in Korea were prospectively collected from April 2012 to December 2014. As a control group, 41 carbapenem-resistant but colistin-susceptible (CoS) A. baumannii strains were also evaluated. Minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by Etest in triplicate, and in vitro synergy tests were performed by the Etest MIC:MIC ratio method. Synergistic activity was determined as the sum of each antimicrobial agent's fractional inhibitory concentration evaluated (ΣFIC): synergy, ≤0.5; indifference, >0.5–4; and antagonism, >4. Synergistic activities were more frequently observed in the CoR group than the CoS group for combinations of colistin-rifampicin (80.5% vs. 14.6%, P< 0.0001), colistin-meropenem (85.4% vs. 4.9%, P< 0.0001), and colistin-imipenem (46.3% vs. 2.4%, P< 0.0001). Combination with rifampicin or meropenem lowered colistin MICs against CoR A. baumannii clinical isolates to the susceptible range (≤ 2 μg/mL) more frequently (61.0%, 25/41, both) than combination with imipenem (29.3%, 12/41). Clinical trials are needed to prove the in vivo efficacy of those antimicrobial combinations that exhibited significant in vitro antimicrobial synergistic effects against CoR A. baumannii.