Reduction In MIC Research Articles

Study on Desulfovibrio desulfuricans mediated copper corrosion and its inhibition using bio-wax of Colocasia esculenta

In the present study, the bio-wax was extracted from Colocasia esculenta and was tested for its corrosion inhibiting property against Desulfovibrio desulfuricans. Inhibition of copper corrosion by bio wax from Colocasia was observed using weight loss method, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Results obtained from the weight loss studies and impedance measurements provided similar trends of inhibition efficiency. Cu coupon coated with bio-wax showed significant reduction in corrosion rate i.e. Rcorr of 0.0011mmpy. This result gives an idea about the protective nature of thick bio-wax coated copper coupons from bacterial adherence and bacterial metabolites. The impedance value in the bio wax coated coupon was found to be from 188.67 to 781.25 ohms at 1 Volt. The wax FTIR analysis in a spectral region of 4000 cm- 1 - 400 cm -1 showed peaks at 2918.75 cm -1 and 2849.73 cm -1 confirming the presence of hydrophobic hydrocarbon. Morphological changes were observed after coating the bio wax as shown from SEM analysis. The incorporation of the hydrophobic property of bio-wax showed sufficient reduction in MIC of copper. Bio-wax acted as a protective covering inhibiting the adherence of corroding bacteria on the surface. Inhibition efficiency results are in good agreement to use Colocasia bio wax as a source of sustainable and renewable surface coating material. This property might be used in future as an ingredient in anti-rust paint used in coating metal surfaces like ship parts, pipelines systems of petroleum industry and other metal made machines etc.

Olive oil nanoemulsion containing curcumin: antimicrobial agent against multidrug-resistant bacteria.

The present work aimed to develop, characterize, and evaluate the antibacterial and antibiofilm activity of two nanoemulsions (NEs) containing 500µg/mL of curcumin from Curcuma longa (CUR). These NEs, produced with heating, contain olive oil (5%) and the surfactants tween 80 (5%) and span 80 (2.5%), water q.s. 100mL, and were stable for 120days. NE-2-CUR presented Ø of 165.40 ± 2.56nm, PDI of 0.254, ζ of - 33.20 ± 1.35mV, pH of 6.49, and Entrapment Drug Efficiency (EE) of 99%. The NE-4-CUR showed a Ø of 105.70 ± 4.13nm, PDI of 0.459, ζ of - 32.10 ± 1.45mV, pH of 6.40 and EE of 99.29%. Structural characterization was performed using DRX and FTIR, thermal characterization using DSC and TG, and morphological characterization using SEM, suggesting that there is no significant change in the CUR present in the NEs and that they remain stable. The MIC was performed by the broth microdilution method for nine gram-positive and gram-negative bacteria, as well as Klebsiella pneumoniae clinical isolates resistant to antibiotics and biofilm and efflux pump producers. The NEs mostly showed a bacteriostatic profile. The MIC varied between 125 and 250µg/mL. The most sensitive bacteria were Staphylococcus aureus and Enterococcus faecalis, for which NE-2-CUR showed a MIC of 125µg/mL. The NEs and ceftazidime (CAZ) interaction was also evaluated against the K. pneumoniae resistant clinical isolates using the Checkerboard method. NE-2-CUR and NE-4-CUR showed a synergistic or additive profile; there was a reduction in CAZ MICs between 256 times (K26-A2) and 2 times (K29-A2). Furthermore, the NEs inhibited these isolates biofilms formation. The NEs showed a MBIC ranging from 15.625 to 250µg/mL. Thus, the NEs showed physicochemical characteristics suitable for future clinical trials, enhancing the CAZ antibacterial and antibiofilm activity, thus becoming a promising strategy for the treatment of bacterial infections caused by multidrug-resistant K. pneumoniae. KEY POINTS: • The NEs showed physicochemical characteristics suitable for future clinical trials. • The NEs showed a synergistic/additive profile, when associated with ceftazidime. • The NEs inhibited biofilm formation of clinical isolates.

Inhibitors against New Delhi metallo-betalactamase-1 (NDM-1) and its variants endemic in Indian settings along with the laboratory functional gain mutant of NDM-1.

The emergence of NDM-1 producing bacteria has become common in both hospital and community settings, but no inhibitor has yet been available for clinical treatment. Hence, demanding the urgent need of NDM-1 inhibitors, we initiated to screen broad spectrum inhibitors against NDM natural variants and laboratory mutant. We used docking and molecular dynamics simulations, in silico pharmacokinetic investigations, and density functional theory calculation to characterize molecules. Furthermore, an in vitro study, including MIC, kinetics, and fluorescence study were carried out to confirm the efficacies of the selected compounds. According to the findings of the computational studies, three compounds were effective against NDM variants. Fourfold reduction in MIC of imipenem and meropenem was observed when combined with inhibitors (D2573, D2148, and D63) against blaNDM-1, blaNDM-4, blaNDM-6, and blaNDM-1Q123A, while twofold reduction in MIC of imipenem and meropenem was observed against blaNDM-5 and blaNDM-7. Similarly in the presence of inhibitors (D2573, D2148, and D63) the efficiency of nitrocefin hydrolysis by NDM-4, NDM-6, and Q123A decreases to much more extent as compared to NDM-5 and NDM-7. These results showed that the efficacy of these broad spectrum inhibitors decreases with increasing resistance of NDM variants. This is the first time inhibitors were tested against different NDM natural variants which are endemic in Indian settings. Moreover, a functional gain laboratory mutant was also checked for their efficacies. We may propose these molecules for the pre-clinical trial to further translate.

Chlorinephenyl‐Substituted Sulfonylurea Confer Inhibitory Efficacy against Metallo‐β‐lactamase ImiS

AbstractSuper bacterial infection caused by metallo‐β‐lactamases (MβLs) has posed a serious public health threat, while developing inhibitors of MβLs is an ideal strategy to combat drug‐resistance. Here, seventeen sulfonylureas were synthesized and confirmed. Biological assay shown that these molecules effectively inhibited MβL ImiS, with an IC50 value in the range of 29.5–188.2 μM, and the best inhibitor competitively and irreversibly inhibited ImiS with a Ki value of 15.2 μM. Structure‐activity relationship analysis revealed that the chlorine‐substituted phenyl significantly raised inhibitory activity of sulfonylureas on ImiS. Antibacterial and chessboard dilution assays shown that the most sulfonylureas tested enhanced antimicrobial effect of meropenem (MER) on E. coli‐ImiS, and the two inhibitors with best effect resulted in an 8‐fold reduction in MIC of MER. Mice tests shown that the best inhibitor synergizing MER virtually diminished the load of E. coli‐ImiS in spleen and liver. Docking study revealed that the sulfonylurea reacts with ImiS mainly through the sulfonyl group, in which one of oxygen atoms coordinates to Zn(II), and forms two hydrogen bonds with Lys224 (2.1 Å) and His263 (3.1 Å). This work revealed the sulfonylureas are promising candidates for the development of ImiS inhibitors.

Zeolitic imidazolate framework-8 encapsulated with Mo-based polyoxometalates as surfaces with antibacterial activity against Escherichia coli.

Bacterial infections represent a major global health concern, causing millions of deaths and a significant economic burden. The development of antibacterial nanoporous surfaces with potential mechano-bactericidal effects can revolutionize infection control practices. In this study, a hybrid material of zeolitic imidazolate framework-8 (ZIF-8) doped with phosphomolybdic acid (PMA) was synthesized and characterized by field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and N2 sorption isotherms. PMA@ZIF-8 performance as an antibacterial agent against E. coli was superior to that of its individual constituents, suggesting a synergistic effect of PMA and ZIF-8. The incorporation of PMA into ZIF-8 significantly enhanced its antibacterial efficacy, as evidenced by a twofold reduction in MIC (375 μg mL-1 vs. 750 μg mL-1) and a 4.35 times increase in the bactericidal kinetics rate constant. The time-kill curve experiment revealed that PMA@ZIF-8 achieved a 3-log reduction within 7 hours, whereas ZIF-8 required 24 hours to reach the same level of reduction. The density functional theory (DFT) calculated bandgap of PMA@ZIF-8 was significantly less than that of ZIF-8. Also, PMA@ZIF-8 has caused the elimination of 56.72% of the thiol group as detected by Ellman's assay. Accordingly, PMA@ZIF-8 can be both computationally and experimentally demonstrated as an oxidative nanozyme. PMA@ZIF-8's surface topology revealed nanorod protrusions, suggesting a potential mechano-bactericidal effect, which was confirmed by live/dead assay on PMA@ZIF-8-coated glass. This study highlights the potential of the PMA@ZIF-8 hybrid as a highly effective antibacterial agent, holding promise for creating multifunctional antibacterial surfaces.

Discovery of hydroxamate as a promising scaffold dually inhibiting metallo- and serine-β-lactamases

The bacterial infection mediated by β-lactamases MβLs and SβLs has grown into an emergent health threat, however, development of a molecule that dual inhibits both MβLs and SβLs is challenging. In this work, a series of hydroxamates 1a-g, 2a-e, 3a-c, 4a-c were synthesized, characterized by 1H and 13C NMR and confirmed by HRMS. Biochemical assays revealed that these molecules dually inhibited MβLs (NDM-1, IMP-1) and SβLs (KPC-2, OXA-48), with an IC50 value in the range of 0.64–41.08 and 1.01–41.91 μM (except 1a and 1d on SβLs, IC50 > 50 μM), and 1f was found to be the best inhibitor with an IC50 value in the range of 0.64–1.32 and 0.57–1.01 μM, respectively. Mechanism evaluation indicated that 1f noncompetitively and irreversibly inhibited NDM-1 and KPC-2, with Ki value of 2.5 and 0.55 μM, is a time- and dose-dependent inhibitor of both MβLs and SβLs.MIC tests shown that all hydroxamates increased the antimicrobial effect of MER on E. coli-NDM-1 and E. coli-IMP-1 (expect 1b, 1d, 1g and 2d), resulting in a 2-8-fold reduction in MICs of MER, 1e-g, 2b–d, 3a-c and 4b-c decreased 2-4-fold MICs of MER on E. coli-KPC-2, and 1c, 1f-g, 2a–c, 3b, 4a and 4c decreased 2-16-fold MICs of MER on E. coli-OXA-48. Most importantly, 1f-g, 2b–c, 3b and 4c exhibited the dual synergizing inhibition against both E. coli-MβLs and E. coli-SβLs tested, resulting in a 2-8-fold reduction in MICs of MER, and 1f was found to have the best effect on the drug-resistant bacteria tested. Also, 1f shown synergizing antimicrobial effect on five clinical isolates EC04, EC06, EC08, EC10 and EC24 that produce NDM-1, resulting in a 2-8-fold reduction in MIC of MER, but its effect on E. coli and K. pneumonia-KPC-NDM was not to be observed using the same dose of inhibitor. Mice tests shown that the monotherapy of 1f or 4a in combination with MER significantly reduced the bacterial load of E. coli-NDM-1 and E. coli-OXA-48 cells in liver and spleen, respectively. The discovery in this work offered a promising bifunctional scaffold for creating the specific molecules that dually inhibit MβLs and MβLs, in combating antibiotic-resistant bacteria.

2747. Aztreonam-Avibactam Susceptibility Testing Program for Metallo-β-Lactamase-Producing Enterobacterales collected through the Antimicrobial Resistance Laboratory Network, March 2019 to April 2023

Abstract Background Metallo-β-lactamase-producing Enterobacterales (MBL-E) are a major public health concern as they cause infections with few effective treatment options. Aztreonam-avibactam (AZA) is a β-lactam/β-lactamase inhibitor combination agent in the drug development pipeline that is active against MBL-E. Though clinical studies are ongoing, the effective components can be administered by combining two FDA-approved drugs: aztreonam and ceftazidime-avibactam. In 2019, CDC initiated a program in the Antimicrobial Resistance Laboratory Network (AR Lab Network), the Expanded Antimicrobial Susceptibility Testing Program for Hard-to-Treat Infections, to provide antimicrobial susceptibility testing (AST) of MBL-E for AZA. We provide a summary of the program’s results. Methods Seven AR Lab Network laboratories validated the use of a digital dispenser to create custom broth microdilution panels for AZA AST. Testing requests must be for clinical decision-making purposes. Isolates must be an Enterobacterales and have laboratory results positive for an MBL gene (i.e., blaNDM, blaVIM, or blaIMP), or be not susceptible to all β-lactams tested. AZA testing results for confirmed MBL-E were reported back to submitters within 3 working days from receipt of the isolate and shared with CDC. Results From March 2019 through April 2023, AZA AST was performed for 250 isolates: 103 Escherichia coli, 86 Klebsiella pneumoniae, 46 Enterobacter cloacae complex, 8 K. oxytoca, 2 Providencia rettgeri, 1 K. aerogenes, 1 Morganella morganii, 1 Proteus mirabilis, 1 E. hormaechei, and 1 Citrobacter amalonaticus. Carbapenemase genes detected were: 206 blaNDM, 24 blaNDM/blaOXA-48-like, 14 blaKPC/blaNDM, 2 blaIMP, 2 blaKPC/blaIMP, and 2 blaNDM/blaIMP. All isolates were resistant to ceftazidime-avibactam; 227/250 (90.8%) were not susceptible to aztreonam (≥8 µg/mL). For isolates not susceptible to aztreonam, the addition of avibactam resulted in a median 128-fold MIC reduction of aztreonam. The MIC range of AZA was ≤0.03/4 - >64/4 µg/mL. Overall, the MIC50 and MIC90 of AZA were 0.5/4 µg/mL and 8/4 µg/mL, respectively; the MIC50 and MIC90 for E. coli were higher than other species, 4/4 µg/mL and 16/4 µg/mL. Conclusion Our data suggest that AZA has potent in vitro activity against MBL-E collected in the United States. Disclosures All Authors: No reported disclosures

High efficacy and enhanced synergistic activity of the novel siderophore-cephalosporin cefiderocol against multidrug-resistant and extensively drug-resistant Klebsiella pneumoniae from inpatients attending a single hospital in the United Arab Emirates

BackgroundCefiderocol (CFDC) is a novel siderophore-cephalosporin, which usually penetrates the bacteria through the iron-uptake pathways. Data is limited on the factors affecting CFDC activity and methods for overcoming resistance development. Synergistic approaches are needed to tackle antimicrobial resistance. This study aimed to determine CFDC activity on Klebsiella pneumoniae isolates from patients attending a single hospital in the United Arab Emirates (UAE), to explore the effect of β-lactamases on CFDC activity and to enhance CFDC susceptibility in both iron-depleted and iron-enriched conditions. MethodsWe investigated 238 K. pneumoniae strains from diverse clinical sources. β-lactamase genes were detected by PCR. Susceptibility to CFDC and 12 comparator antibiotics were tested. Combinations of CFDC with β-lactamase inhibitors (BLIs) and/or an outer membrane (OM) permeabilizer (polymyxin B nonapeptide) were tested in iron-depleted and iron-enriched conditions. ResultsCFDC exhibited efficacy of 97.9%, against multidrug-resistant (MDR), and extensively drug-resistant (XDR) strains, in addition to strains resistant to the last resort drugs such as colistin and tigecycline, including dual carbapenemase-producers (blaNDM and blaOXA-48-like) with MIC ≤ 0.06–8 µg/ml. It was effective in killing strains with single and multiple β-lactamases; however, it lost activity in iron-enriched conditions. Synergy was achieved with dual combination of CFDC and BLIs, especially avibactam, which caused a significant reduction in MICs even in iron-enriched conditions. A significant reduction was seen with the triple combination including an OM permeabilizer plus avibactam. Killing-kinetic studies proved that the combination therapy caused dose reduction and faster killing by CFDC than the monotherapy. ConclusionsCFDC was deemed effective against MDR and XDR K. pneumoniae. Synergistic combination of CFDC with BLIs and OM permeabilizers could be effective to treat infections in iron-rich sites, but this should be investigated in vivo.

Exploring synergy between azole antifungal drugs and statins for Candida auris.

Global emergence of rapidly developing resistance to multiple antifungal drugs and high mortality pose challenges to the treatment of invasive Candida auris infections. New therapeutic approaches are needed, such as repurposing drugs including combination with antifungals. Statins have been reported to exert antifungal effects against various Candida species. Our study investigated potential synergy between the statins (rosuvastatin and fluvastatin) and azoles (voriconazole, posaconazole and isavuconazole) on clinical isolates of C. auris. Twenty-one clinical isolates of C. auris were obtained. Chequerboard assays based on the CLSI broth microdilution method were used to assess synergy based on FIC index (FICI) calculations of MICs of individual drugs and in combinations. Single drug geometric mean (GM) MICs of fluvastatin and rosuvastatin were ≥128 mg/L in all 21 isolates. GM (range) MICs of posaconazole, voriconazole and isavuconazole were 0.259 (0.016-1 mg/L), 0.469 (0.016-2 mg/L) and 0.085 (0.004-1 mg/L), respectively. Combination of azoles with fluvastatin showed synergy in 70%-90% of C. auris isolates. In particular, voriconazole/fluvastatin resulted in 16-fold reduction in voriconazole MIC and synergy in 14/21 (67%) isolates. Posaconazole/fluvastatin resulted in 8-fold reduction in posaconazole MIC and synergy in 19/21 (90%) isolates.Combining rosuvastatin with the azoles also showed synergy against C. auris in 40%-60% of the isolates and additive effect in 40%-50%. None of the combinations was antagonistic. Our results provide a rationale for pursuing in vivo synergy tests as well as clinical studies to explore tolerability, treatment outcomes, optimal dose and exposure targets.

Mutations in TAC1 and ERG11 are major drivers of triazole antifungal resistance in clinical isolates of Candida parapsilosis

ObjectivesThe aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates. MethodsSequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR. ResultsSix isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 μg/mL vs. 0.5 μg/mL, 0.125 μg/mL vs. 0.5 μg/mL, and 0.5 μg/mL vs. 4 μg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 μg/mL vs. 2 μg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 μg/mL vs. 2 μg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 μg/mL vs. 8 μg/mL). DiscussionThese results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis.

SYNERGISTIC EFFECTS OF RUTA GRAVEOLENS L. EXTRACTS WITH ERYTHROMYCIN AGAINST SKIN ISOLATES OF MLS-RESISTANT STAPHYLOCOCCI

The aim: to research the synergistic interaction of garden ruta ethanolic extracts with erythromycin against skin isolates of MLS-resistant staphylococci.
 Materials and methods. The study of the synergistic interaction of 5 water-ethanol extracts of the garden ruta herb (extractants - 40%, 50%, 70%, 90% and 96% ethanol) with erythromycin was performed on 11 clinical strains of skin isolates of MLS-resistant staphylococci. Determination of MLS-resistance was carried out by the disc-diffusion method. MIC of erythromycin of staphylococcal strains was determined by the method of two-fold serial dilutions in Muller-Hinton broth. A screening analysis of the potential ability for synergistic interaction of ruta herb extracts with erythromycin was carried out by the method of microdiffusion in agar. The validity of the synergistic interaction of the studied ruta herb extracts with erythromycin was confirmed by the сheckerboard assay with the calculation of the Fractional Inhibition Concentration Index (FICI).
 Results. Using the qualitative method of microdiffusion in agar, it was established that subbacteriostatic concentrations of erythromycin increased the diameters of the zones of inhibition of the bacterial growth around the wells with all the studied extracts in 50±1.3% of the test strains. The best results were shown by the 96% extract of ruta herb, which increased the diameters of zones by 49.7-75.5%. Around the wells with 70% and 90% extracts, the diameters of zones increased by 32.4-48.3% and 34.6-52.4%, respectively. A decrease in MIC of erythromycin in the presence of efflux pump blockers - reserpine and potassium arsenate was observed in 2-4 times at four strains with R-phenotype, twice - at one strain with Neg-phenotype and one strain with D-phenotype of MLS-resistance. 90% and 96% ruta herb extracts showed a synergistic interaction with erythromycin among the largest number of researched strains, regardless phenotype of MLS-resistance (4-128-fold decrease in MIC of ERY). The least antibiotic potentiating ability was shown by 40% and 50% ruta herb extracts, under the influence of which the MIC of erythromycin decreased by 2-32 and 2-64 times, respectively.
 Ruta herb extracts in 90% and 96% ethanol showed a synergistic interaction with erythromycin (average FICI values 0.49±0.42 and 0.42±0.25, respectively). Extracts in 40%, 50% and 70% ethanol were generally characterized by an additive effect (FICI values of 0.72±0.47, 0.63±0.24 and 0.68±0.38, respectively).
 It should be noticed, that the synergistic interaction of erythromycin and subbacteriostatic concentrations (1/4, 1/8, 1/16 IPC) of ethanolic ruta herb extracts against the studied skin isolates of MLS-resistant staphylococci had a dose-dependent nature (F=6,9812; F>Fcrit.= 2,8916; p=0,000917).
 Conclusions. Therefore, water-ethanol extracts of garden ruta herb demonstrate the ability to 4-128-fold reduction of MIC of erythromycin in MLS-resistant strains of S. epidermidis and S. aureus; extracts on 90% and 96% ethanol have more pronounced erythromycin-potentiating properties compared to extracts made on 40%, 50% and 70% ethanol. The synergistic interaction of erythromycin with ruta herb extracts was manifested on staphylococcal strains with efflux and combined mechanisms of MLS-resistance. The obtained experimental data suggest the presence of compounds in the garden ruta extracts which are capable of blocking the MrsA-mediated efflux of macrolides from staphylococcal cells.

Role of the NaHCO3 Transporter MpsABC in the NaHCO3-β-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are an increasing concern due to their intrinsic resistance to most standard-of-care β-lactam antibiotics. Recent studies of clinical isolates have documented a novel phenotype, termed NaHCO3 responsiveness, in which a substantial proportion of MRSA strains exhibit enhanced susceptibility to β-lactams such as cefazolin and oxacillin in the presence of NaHCO3. A bicarbonate transporter, MpsAB (membrane potential-generating system), was recently found in S. aureus, where it plays a role in concentrating NaHCO3 for anaplerotic pathways. Here, we investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype. Radiolabeled NaH14CO3 uptake profiling revealed significantly higher accumulation in NaHCO3-responsive vs nonresponsive MRSA strains when grown in ambient air. In contrast, under 5% CO2 conditions, NaHCO3-responsive (but not nonresponsive) strains exhibited repressed uptake. Oxacillin MICs were measured in four prototype strains and their mpsABC deletion mutants in the presence of NaHCO3 supplementation under 5% CO2 conditions. NaHCO3-mediated reductions in oxacillin MICs were observed in the responsive parental strains but not in mpsABC deletion mutants. No significant impact on oxacillin MICs was observed in the nonresponsive strains under the same conditions. Transcriptional and translational studies were carried out using both quantitative reverse transcription-PCR (qRT-PCR) and mpsA-green fluorescent protein (GFP) fusion constructs; these investigations showed that mpsA expression and translation were significantly upregulated during mid-exponential-phase growth in oxacillin-NaHCO3-supplemented medium in responsive versus nonresponsive strains. Taken together, these data show that the NaHCO3 transporter MpsABC is a key contributor to the NaHCO3-β-lactam responsiveness phenotype in MRSA. IMPORTANCE MRSA infections are increasingly difficult to treat, due in part to their resistance to most β-lactam antibiotics. A novel and relatively common phenotype, termed NaHCO3 responsiveness, has been identified in which MRSA strains show increased susceptibility in vitro and in vivo to β-lactams in the presence of NaHCO3. A recently described S. aureus NaHCO3 transporter, MpsAB, is involved in intracellular NaHCO3 concentration for anaplerotic pathways. We investigated the role of MpsAB in mediating the NaHCO3 responsiveness phenotype in four prototype MRSA strains (two responsive and two nonresponsive). We demonstrated that MpsABC is an important contributor to the NaHCO3-β-lactam responsiveness phenotype. Our study adds to the growing body of well-defined characteristics of this novel phenotype, which could potentially translate to alternative targets for MRSA treatment using β-lactams.

Modification of the drug resistance of emerging milk-borne pathogens through sodium alginate-based antibiotics and nanoparticles.

Streptococcus agalactiae and Klebsiella pneumoniae are emerging as major milk-borne pathogens. Additionally, resistance to antibiotics of pathogens is of concern. Therefore, this study investigated the prevalence and drug resistance of S. agalactiae and K. pneumoniae in mastitis milk samples and assessed the antimicrobial potential of sodium alginate (G)-stabilized MgO nanoparticles (M) and antibiotics (tylosin [T] and ampicillin [A]) against both of these pathogens. A total of n = 200 milk samples from cattle were collected using purposive sampling, and standard microbiological approaches were adopted to isolate target bacteria. Parametric and non-parametric statistical tests were used to analyze the obtained data. Four preparations, GT (gel-stabilized tylosin), GA (gel-stabilized ampicillin), GTM (tylosin and MgO nanoparticles stabilized in gel), and GAM (ampicillin and MgO nanoparticles stabilized in gel), were evaluated against both bacteria through well diffusion and broth microdilution method. The analysis revealed that 45.24% (95/210) of the milk samples were positive for mastitis, of which 11.58% (11/95) were positive for S. agalactiae and 9.47% (9/95) were positive for K. pneumoniae. S. agalactiae had a significantly higher zone of inhibition (ZOI) than K. pneumoniae against penicillin, tetracycline, and amoxicillin, whereas the opposite was observed against imipenem and erythromycin. All gel (G)-based preparations showed an increase in the percentage of ZOI compared with antibiotics alone, with GTM presenting the highest of all, i.e., 59.09 and 56.25% ZOI compared with tylosin alone against S. agalactiae and K. pneumoniae, respectively. Similarly, in a broth microdilution assay, the lowest MIC was found for K. pneumoniae (9.766 ± 0.0 μg/mL) against GTM, followed by GT, GAM, and GA after incubation for 24 h. A similar response was noted for preparations against S. agalactiae but with a comparatively higher MIC. A significant reduction in MIC with respect to incubation time was found at 8 h and remained until at 20 h against both pathogens. The cytotoxicity of the MgO nanoparticles used in this study was significantly lower than that of the positive control. Overall, this study found that K. pneumoniae and S. agalactiae appeared higher in prevalence and antimicrobial resistance, and sodium alginate-based antibiotics and MgO nanoparticles were effective alternative approaches for tackling antimicrobial resistance.

A novel method to evaluate ceftazidime/avibactam therapy in patients with carbapenemase-producing Enterobactericeae (CPE)bloodstream infections.

This study reports experience managing eight patients with bloodstream infections treated with a continuous infusion of ceftazidime-avibactam. Patients who were treated for documented CPE BSIs susceptible to CAZ-AVI and who underwent real-time therapeutic drug monitoring were retrospectively assessed. Ceftazidime MICs were assessed in presence of increasing concentrations of avibactam by the broth microdilution method. An inhibitory sigmoid Emax model was used to characterize ceftazidime MIC reduction as a function of avibactam concentration, and the MICi was derived by conditioning the best-fit model using steady-state avibactam concentrations (Css). Ceftazidime fCss/MICi ratio was calculated for each patient and correlated to microbiological outcome. By adopting the innovative concept of effective MIC with an inhibitor (MICi), a trend towards higher microbiological failure and resistance development was found in patients with a lower ceftazidime fCss/MICi ratio (2/3 vs. 0/5). Assessment of changes in the ceftazidime MIC in relation to increasing avibactam concentration could represent a more robust pharmacokinetic/pharmacodynamic method for predicting microbiological failure given beta-lactam/beta-lactamase inhibitor combinations.

CRISPRi-mediated characterization of novel anti-tuberculosis targets: Mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survival.

The lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications.

Critical role of the RpoE stress response pathway in polymyxin resistance of Escherichia coli.

Polymyxins, including colistin, are the drugs of last resort to treat MDR bacterial infections in humans. In-depth understanding of the molecular basis and regulation of polymyxin resistance would provide new therapeutic opportunities to combat increasing polymyxin resistance. Here we aimed to identify novel targets that are crucial for polymyxin resistance using Escherichia coli BL21(DE3), a unique colistin-resistant model strain. BL21(DE3) was subjected to random transposon mutagenesis for screening colistin-susceptible mutants. The insertion sites of desired mutants were mapped; the key genes of interest were also inactivated in different strains to examine functional conservation. Specific genes in the known PmrAB and PhoPQ regulatory network were inactivated to examine crosstalk among different pathways. Lipid A species and membrane phospholipids were analysed by normal phase LC/MS. Among eight mutants with increased susceptibility to colistin, five mutants contained different mutations in three genes (rseP, degS and surA) that belong to the RpoE stress response pathway. Inactivation of rpoE, pmrB, eptA or pmrD led to significantly increased susceptibility to colistin; however, inactivation of phoQ or eptB did not change colistin MIC. RpoE mutation in different E. coli and Salmonella resistant strains all led to significant reduction in colistin MIC (16-32-fold). Inactivation of rpoE did not change the lipid A profile but significantly altered the phospholipid profile. Inactivation of the important members of the RpoE regulon in polymyxin-resistant strains led to a drastic reduction in polymyxin MIC and an increase of lysophospholipids with no change in lipid A modifications.

Synergistic Effect of Clinically Available Beta-Lactamase Inhibitors Combined with Cefiderocol against Carbapenemase-Producing Gram-Negative Organisms.

The role of β-lactamases in reduced susceptibility or resistance to cefiderocol has been supported by recent reports. The purpose of this study was to investigate the in vitro impact of clinically available β-lactamase inhibitors on cefiderocol activity against characterized carbapenemase-producing Gram-negative isolates. A collection of 39 well-characterized Gram-negative isolates obtained from various clinical sources and countries were included. Cefiderocol antimicrobial susceptibility was evaluated via reference broth microdilution. The chequerboard microdilution method and time-kill assays were used to determine the synergy of tazobactam, avibactam, vaborbactam and relebactam in combination with cefiderocol. MICs of cefiderocol presented a 4- to 256-fold reduction against Klebsiella pneumoniae carbapenemase (KPC)-producing Gram-negative isolates (predominantly K. pneumoniae) when avibactam, vaborbactam and relebactam were combined individually. Notably, the KPC-inhibitors led to a 4- to 32-fold reduction in cefiderocol MICs in the four cefiderocol-resistant KPC-producing K. pneumoniae isolates, showing restoration of cefiderocol susceptibility (MIC ≤ 2 mg/L) in ten out of twelve cases. Tazobactam led to a 4- to 64-fold decrease in cefiderocol MICs only in K. pneumoniae strains harbouring blaKPC-41, blaKPC-31, blaKPC-53 and blaKPC-66. The synergistic effect of all serine-β-lactamase inhibitors on cefiderocol activity was also shown in OXA-48-like-producing Enterobacterales strains. Conversely, a combination of β-lactamases inhibitors with cefiderocol was not synergistic with all OXA-23-like-producing strains and most metallo-β-lactamases producers. In conclusion, the addition of clinically available serine β-lactamase inhibitors to cefiderocol might represent an important development in the formulation to increase its spectrum and therapeutic efficacy, and to limit in vivo resistance emergence.

Efficiency of Rifampin-loaded chitosan NPs in Killing of Brucella melitensis Isolated From Aborted Sheep in Wasit Province, Iraq

Brucellosis is consider one of the utmost common bacterial infectious illness that infect man and animals. unfortunately, A worldwide community in the developing country didn't provide active assistance for reducing it spread out globally. Therefore, the aim of this study is the synthesis of chitosan nanoparticles loaded with rifampin to overcome bacterial resistance and provide high intracellular penetration and the potential for potent intracellular antibacterial activity. The study was conducted from October 2019 to September 2021, Of 57 positive clinical specimens identified in the present study, 25 (65.78%), 20 (74.07%), and 12 (66.66%) were isolated from vaginal swabs, fetal membranes, and fetal abomasum contents, respectively. From the results of SEM images of NPs, it was clear that RIF-loaded CHNPs presented irregular surfaces or rough with spherical shape and presence of some particle aggregates. The RIF-CHNPs particle size observed using SEM images was 260.54 nm, while the diameter was 150.23 nm. In addition, the antibacterial activity were also measured and the results shows that the MIC values for RIF, CHNPs and RIF-CHNPs and they were 4, 2.5 and 0.625 ug/ml, respectively. While the EE % and DL % of RIF were measured spectrophotometrically. Loaded antibiotic formulation gave good percentage of entrapment efficiency and drug loading at concentration 50 mg/ml. Then the EE % at concentration 50 mg/ml was re-estimated in different pH values and the best EE% was at pH 5. In conclusion, the antibacterial activity of drug nanocarrier against B. melitensis found that it had a relatively low MIC 0.625 ug/ml. whereas the inhibitory concentrations were about 4 μg/ml and 2.5 μg/ml for the RIF and CHTNPs, respectively. Indicating there were 6.4 times reduction in MIC for RIF-CHTNPs and 1.6 time reduction for CHTNPs comparing to MIC of antibiotic alone.

Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene blaNDM-1, with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes blaNDM-1 and blaSHV-12, with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the blaNDM-1 promoter region can silence the entire blaNDM-1-bleMBL-trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene blaKPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.

Carbapenem resistance in non-carbapenemase-producing Pseudomonas aeruginosa strains: the role importance of OprD and AmpC

Carbapenem-resistance is a great challenge for antimicrobial therapy in Pseudomonas aeruginosa multidrug-resistant infections, as it reduces therapeutic options. This study investigated carbapenem-resistance mechanisms in six strains of non-carbapenemase-producing P. aeruginosa. Minimal inhibitory concentrations for imipenem and meropenem were determined by epsilometric test and broth microdilution. Mutations in the oprD gene were investigated by PCR, followed by sequencing. Transcriptional levels of oprD, ampC, and efflux pumps genes were analysed through RT-qPCR. Detection of efflux and AmpC activity was assessed by MIC reduction in the presence of the inhibitors: PAβN and cloxacillin, respectively. Resistant strains showed moderate levels of resistance for the evaluated carbapenems. Sequencing of oprD gene revealed similar mutation patterns in strains of the same Sequence Type -ST. A premature stop codon was detected only in the resistant strains of ST2236. Indel mutations were found in the oprD gene of ST2237 strains. Failure to detect oprD transcripts by RT-qPCR further confirms the absence of porin on ST2237 strains. ST2236 strains showed low transcriptional levels for oprD. MexXY-OprM was the only efflux system overexpressed in resistant strains. However, no efflux and AmpC activity was detected. High transcriptional levels of ampC were found in 50% of non-induced resistant strains. All imipem-induced resistant strains showed an increase in ampC expression (>102 - 103 X). It was concluded that the reduction and/or loss of OprD associated with AmpC overexpression is probably the main carbapenem-resistance mechanism in the evaluated strains.