Β-lactam Inhibitors Research Articles

Efficacy and In Vitro Activity of Novel Antibiotics for Infections With Carbapenem-Resistant Gram-Negative Pathogens.

Infections by Gram-negative multi-drug resistant (MDR) bacterial species are difficult to treat using available antibiotics. Overuse of carbapenems has contributed to widespread resistance to these antibiotics; as a result, carbapenem-resistant Enterobacterales (CRE), A. baumannii (CRAB), and P. aeruginosa (CRPA) have become common causes of healthcare-associated infections. Carbapenems, tigecycline, and colistin are the last resource antibiotics currently used; however, multiple reports of resistance to these antimicrobial agents have been documented worldwide. Recently, new antibiotics have been evaluated against Gram-negatives, including plazomicin (a new aminoglycoside) to treat CRE infection, eravacycline (a novel tetracycline) with in vitro activity against CRAB, and cefiderocol (a synthetic conjugate) for the treatment of nosocomial pneumonia by carbapenem-non-susceptible Gram-negative isolates. Furthermore, combinations of known β-lactams with recently developed β-lactam inhibitors, such as ceftazidime-avibactam, ceftolozane-tazobactam, ceftazidime-tazobactam, and meropenem-vaborbactam, has been suggested for the treatment of infections by extended-spectrum β-lactamases, carbapenemases, and AmpC producer bacteria. Nonetheless, they are not active against all carbapenemases, and there are reports of resistance to these combinations in clinical isolates.This review summarizes and discusses the in vitro and clinical evidence of the recently approved antibiotics, β-lactam inhibitors, and those in advanced phases of development for treating MDR infections caused by Gram-negative multi-drug resistant (MDR) bacterial species.

β-Lactam Antibiotics and β-Lactamase Enzymes Inhibitors, Part 2: Our Limited Resources.

β-lactam antibiotics (BLAs) are crucial molecules among antibacterial drugs, but the increasing emergence of resistance to them, developed by bacteria producing β-lactamase enzymes (BLEs), is becoming one of the major warnings to the global public health. Since only a small number of novel antibiotics are in development, a current clinical approach to limit this phenomenon consists of administering proper combinations of β-lactam antibiotics (BLAs) and β-lactamase inhibitors (BLEsIs). Unfortunately, while few clinically approved BLEsIs are capable of inhibiting most class-A and -C serine β-lactamases (SBLEs) and some carbapenemases of class D, they are unable to inhibit most part of the carbapenem hydrolyzing enzymes of class D and the worrying metallo-β-lactamases (MBLEs) of class B. Particularly, MBLEs are a set of enzymes that catalyzes the hydrolysis of a broad range of BLAs by a zinc-mediated mechanism, and currently no clinically available molecule capable of inhibiting MBLEs exists. Additionally, new types of alarming “superbugs”, were found to produce the New Delhi metallo-β-lactamases (NDMs) encoded by increasing variants of a plasmid-mediated gene capable of rapidly spreading among bacteria of the same species and even among different species. Particularly, NDM-1 possesses a flexible hydrolysis mechanism that inactivates all BLAs, except for aztreonam. The present review provides first an overview of existing BLAs and the most clinically relevant BLEs detected so far. Then, the BLEsIs and their most common associations with BLAs already clinically applied and those still in development are reviewed.

The Spread of 𝛽-Lactamases and Extended Spectrum 𝛽-Lactamase Enzymes among Bacteria: A Threat to Effective Health Care Delivery

Antibiotic discovery continue to play a critical role in disease containment. Misuse and overuse of these drugs are the main drivers in the development of drug resistant pathogens. The World Health Organisation has declared that Antimicrobial resistance is one of the top 10 global public health threats facing humanity. There is the fears that if nothing is done, it might end the antibiotic era soon. In 2017 alone, over 9000 human deaths were caused by ESBL-producing Enterobacteriaceae in the USA. The ability of bacteria to develop newer strategies to acquire and disseminate resistance, can be traced as far back to 1940s when (R-factor) plasmid mediated antibiotic resistance was observed. They become resistant via the production of 𝛽-Lactamases and ESBLs enzymes which inactivate or modify antibiotics. Extended Spectrum 𝛽-Lactamases are enzymes whose rates of hydrolysis of the extended-spectrum 𝛽-Lactam antibiotics are >10 % than that for benzylpenicillin. Some bacteria may produce multiple ß-lactamases, which may reduce the effectiveness of ß-lactam/ ß-lactamase inhibitor combinations. These enzymes are susceptible to inhibition by 𝛽-Lactam inhibitors such as clavulanic acid, tazobactam, or sulbactam but have no hydrolytic activity against cephamycins and carbapenems. The production of acquired 𝛽-Lactamase and ESBL makes the choice of antibiotic treatment of infections caused by Gram-negative bacteria very limited, these have been the major causes of treatment failures, outbreaks of both community and hospital acquired infections, surgical failures, long hospital stay and huge economic losses, which continue to claim uncountable lives, especially in Nigeria and Africa where the health system are weak. The emergence of drug resistannt strains may be minimized by maintaining high levels of the drug in the tissues to inhibit mutants, administering two drugs that do not give cross-resistance, and by limiting the use of valuable second line ‘reserve drugs’ such .....

A novel combination therapy for multidrug resistant pathogens using chitosan nanoparticles loaded with β-lactam antibiotics and β-lactamase inhibitors

Antimicrobial resistance is one of the greatest global threats. Particularly, multidrug resistant extended-spectrum β-lactamase (ESBL)-producing pathogens confer resistance to many commonly used medically important antibiotics, especially beta-lactam antibiotics. Here, we developed an innovative combination approach to therapy for multidrug resistant pathogens by encapsulating cephalosporin antibiotics and β-lactamase inhibitors with chitosan nanoparticles (CNAIs). The four combinations of CNAIs including two cephalosporin antibiotics (cefotaxime and ceftiofur) with two β-lactamase inhibitors (tazobactam and clavulanate) were engineered as water-oil-water emulsions. Four combinations of CNAIs showed efficient antimicrobial activity against multidrug resistant ESBL-producing Enterobacteriaceae. The CNAIs showed enhanced antimicrobial activity compared to naïve chitosan nanoparticles and to the combination of cephalosporin antibiotics and β-lactamase inhibitors. Furthermore, CNAIs attached on the bacterial surface changed the permeability to the outer membrane, resulting in cell damage that leads to cell death. Taken together, CNAIs have provided promising potential for treatment of diseases caused by critically important ESBL-producing multidrug resistant pathogens.

172. Inpatient Antibiotic Prescribing Patterns Using the WHO Access Watch and Reserve (AWaRe) Classification in Okinawa, Japan: A Point Prevalence Survey

BackgroundFew studies have been done on inpatient antibiotic use in Japan and antibiotic stewardship programs with dedicated full-time equivalents are rare. We sought to better understand inpatient antibiotic use in Okinawa, Japan. We applied the World Health Organization (WHO) Access, Watch and Reserve (AWaRe) Classification to compare our findings to international literature. Access antibiotics are common front-line antibiotics, Watch antibiotics are high-priority antibiotics with toxicity or resistance concerns, and Reserve antibiotics are last-line treatments for multi-drug resistant infections.MethodsA point prevalence study was conducted in five hospitals in Okinawa, Japan on Oct 1, 2020. Physicians conducted chart reviews of all patients receiving intravenous antibiotics. Type of antibiotic, reason for use, duration, and microbiologic data was collected. The primary aim was to evaluate the proportion of patients who received antibiotics on the assessment date; secondary aims were to categorize antibiotics according to indication, class and AWaRe classification. Descriptive statistics were used to derive the distribution of AWaRe Classifications and drug class.Results1,728 unique patients were included and 504 (29%) received ≥1 antibiotic on the assessment date. A total of 559 antibiotics were used for 504 patients and 22.0% (n=123) were for prophylaxis. Of those receiving antibiotics for treatment (N=436), 385 (88.3%) patients had a documented infection source. The most common indications for antibiotic use were pneumonia (24.2% n=93), urinary tract infection (19.7% n=76), and intraabdominal (17.9% n=69). Overall, 43.1% (n=241) of the antibiotics were categorized Access and 54.4% (n=304) Watch [Figure 1]. Cephalosporins were the most common antibiotic class (56% n=313), followed by β-lactam inhibitors (18% n=106) and narrow penicillins (8.2% n=46) [Figure 2].Conclusion29% of inpatients in these 5 Okinawan hospitals were prescribed an antibiotic on the survey date. A majority of antibiotics used fall under the WHO AWaRe Watch classification which are antibiotics that may be more likely to cause resistance. Understanding appropriateness of antibiotics used in this population could inform antibiotic stewardship strategies and reduce antibiotic resistance.Figure 1. Antibiotic Distribution According to World Health Organization (WHO) Access, Watch and Reserve (AWaRe) Classification Figure 2. Antibiotic Distribution by Class in Okinawan Hospitals Disclosures All Authors: No reported disclosures

171. A Multicenter Analysis of Inpatient Antibiotic Use During the 2015-2019 Influenza Season in the US: Untapped Opportunities for Antimicrobial Stewardship

BackgroundInappropriate antibiotic (AB) use for viral respiratory illnesses remains widespread in the United States (US) with strong seasonal fluctuations. In contrast to outpatient AB use, the seasonality inpatient AB utilization (IAU) and its correlation with the influenza season are not well understood. We sought to describe trends, seasonality, and the association between IAU use and the 2015-2019 influenza seasons.MethodsWe used the BD Insights Research Database (Franklin Lakes, NJ USA) to identify IAU that were prescribed in patients >17 years old from up to 236 US acute care facilities from July 2015 to December 2019. We included the following AB categories: extended spectrum cephalosporins (ESCs), macrolides, β-lactam inhibitor combination (BLIC), fluoroquinolones, carbapenems, glycopeptides, lipopeptide, tetracyclines, and others. We defined IAU use as days of therapy (DOT) per 1000 patient days present. We used influenza laboratory data to identify facility-level positivity ratio per 100 tests. We used random effect models to estimate IAU: 1) trends overtime, 2) seasonality, and 3) association with influenza positivity rate.ResultsFor IAU from 2015 to 2019, BLICs, ESCs, and glycopeptides were the most used [average 91, 107, and 96 DOT/1000 days presents, respectively]. Visually, we observed strong seasonality that matches the influenza season for macrolide, ESC, and quinolone use (See Figure). Unadjusted bivariate results showed ascending trends over time for BLICs [β= 3.8, p= .003], ESCs [β= 11.0, p= .005], and macrolides [β=1.5, p= .005]. Unadjusted bivariate results showed descending trends with quinolones [β= -10.9, p< .001] and others [β= -2.060, p< .001]. In the adjusted analysis, increased influenza positivity rate was associated with use of ESCs, glycopeptides, lipopeptides, macrolides, fluoroquinolone, and tetracyclines (see Table). No correlation was observed with BLICs, carbapenems, lipopeptides, and Others.IAU (DOT/1000 days presents) and Flu Rate (% Positive) Trends Over Time ConclusionOur study shows that IAU is on the rise for the ESC and BLIC classes. ESC and macrolide use was strongly correlated with influenza season. Monitoring influenza signals may provide more insights that can inform the interpretation of IAU trends and be incorporated into antimicrobial stewardship programs.Disclosures Amine Amiche, PhD, Sanofi (Employee, Shareholder) Heidi Kabler, MD, Sanofi Pasteur (Employee) Janet Weeks, PhD, Becton, Dickinson and Company (Employee) Kalvin Yu, MD, BD (Employee) Vikas Gupta, PharmD, BCPS, Becton, Dickinson and Company (Employee, Shareholder)

Utilization of Colistin Versus β-Lactam and β-Lactamase Inhibitor Agents in Relation to Acute Kidney Injury in Patients with Severe Gram-Negative Infections.

IntroductionColistin is used to treat severe antibiotic-resistant Gram-negative infections (GNIs). With the rise of antibiotic resistance, colistin has been used increasingly as a ‘last-line’ therapy for multidrug-resistant GNIs. We evaluated the incidence of acute kidney injury (AKI) and mortality among patients receiving colistin or one of the new β-lactam/β-lactamase inhibitors (βL + βLI) (ceftazidime/avibactam, ceftolozane/tazobactam, or meropenem/vaborbactam).MethodsThis retrospective cohort study used data from the Premier Healthcare Database. The cohort included propensity score-matched adults with an inpatient stay between January 2016 and December 2018. Patients given both colistin and BL + BLI as treatment for ≥ 72 h were excluded. AKI was defined as acute renal failure or dialysis during hospitalization with antibiotic administration. Propensity score matching was used to control for selection bias and confounding. Logistic regression evaluated associations between treatment, AKI, and in-hospital mortality.ResultsThe total number of patients in the matched cohorts were 256 in each. Overall, 23.8% and 13.3% of patients receiving colistin or new βL + βLI agents, respectively, experienced AKI during hospitalization (p = 0.002); odds of AKI for colistin were 3.0 (95% CI 1.71, 5.21). Following propensity score-matching, patients without baseline renal disease experienced AKI during hospitalization to a higher degree in the colistin group compared to the βL + βLI group (17.1% vs. 6.8%); colistin use was associated with 3.7 times higher odds (95% CI 1.84, 7.42) of AKI compared to βL + βLI agents. The odds of mortality in patients on colistin developing AKI were more than three times that of patients receiving a BL + BLI agent who developed AKI. Among patients receiving colistin, incident AKI was associated with 6.1 times higher odds (95% CI 2.53, 14.71) of mortality.ConclusionsPatients receiving colistin for GNIs had significantly higher odds of AKI and mortality than those receiving βL + βLI.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-021-00556-x.

Sepsis-associated hospitalizations and antimicrobial use in Hong Kong

Our study was conducted to assess the sepsis-associated hospitalisations and antimicrobials prescribed for sepsis inpatients in Hong Kong. Demographic, diagnostic and antimicrobial prescription data were analysed for patients admitted to public hospitals with a diagnosis of septicaemia from 2000 to 2015. A total of 223 250 sepsis hospitalisations were recorded in Hong Kong from 2000 to 2015 during which the hospitalisation rate increased by 85.6%. The majority of the sepsis hospitalisations occurred in adults ≥65 years and children aged 0–4 years. Adults with a secondary diagnosis of sepsis were often admitted with a primary diagnosis of urological conditions or pneumonia; whereas diabetes mellitus was the most common secondary diagnosis among those with primary sepsis. Paediatric sepsis patients aged 0–4 years were often diagnosed with disorders relating to short gestation and low birthweight. Antimicrobial prescriptions increased by 51.1% and 34.4% for primary and secondary sepsis patients, respectively. β-Lactam and β-lactamase inhibitor combinations were the most used antibiotics whereas the usage of carbapenems increased more than 10 times over the study period. A substantial burden of hospitalisations was attributable to sepsis in Hong Kong, particularly in the extremes of age. Broad-spectrum and last-resort antibiotics had been increasingly dispensed for sepsis inpatients.

Modeling of Clavulanic Acid Production from Streptomyces clavuligerus using a Continuous Operation Mode

Clavulanic acid is a β-lactam inhibitor produced by fermentation with Streptomyces clavuligerus cells, and it is usually used to prevent resistance to certain antibiotics. However, CA production is limited at the bioreactor level due to its low performance. The latter generates expensive processes and challenging to operate on a large scale. In this research, a mathematical model is proposed to simulate the clavulanic acid production from an operation strategy based on continuous mode. The preceding, to identify trends allowing to improve the productivity of the mentioned metabolite. Results are compared to the traditional operating batch mode. According to the results found, the final concentration of the β-lactam inhibitor could be increased by up to 60% regarding the simulated data in batch mode. Results obtained demonstrate the importance of computational techniques in bioprocess engineering since bioprocess simulation focuses on identifying critical operating parameters as a starting point in antibiotic production optimization.

Genetic and Phenotypic Characterization of the Novel Metallo-β-Lactamase NDM-29 From Escherichia coli.

Objectives: The New Delhi metallo-β-lactamase (NDM) can hydrolyze almost all clinically available β-lactam antibiotics and has widely spread all over the world. NDM-29, a novel carbapenemase, was discovered in an Escherichia coli (19NC225) isolated from a patient with biliary tract infection in 2019 in China.Methods: Conjugation, transformation, cloning test, fitness cost, PacBio Sequel, and Illumina sequencing were performed to analyze the genetic and phenotypic characterization of blaNDM–29.Results: The susceptibility testing results showed 19NC225 was resistant to cephalosporins, carbapenems, combinations of β-lactam and β-lactamase inhibitors, and levofloxacin. Conjugation and transformation were performed to verify the transferability of NDM-29-encoding plasmid, and cloning test was conducted to prove the function of blaNDM–29 to increase carbapenem resistance. Furthermore, fitness cost test confirmed that the presence of NDM-29 exerts no survival pressure on bacteria. PacBio Sequel and Illumina sequencing were performed to analyze the genetic characterization of 19NC225, which contains two plasmids (pNC225-TEM1B and pNC225-NDM-29). pNC225-NDM-29, exhibiting 99.96% identity and 100% coverage with pNDM-BTR (an IncN1 plasmid from an E. coli in urine specimen from Beijing in 2013), showed responsibility for the multidrug-resistant (MDR) phenotype. Compared with blaNDM–1, blaNDM–29, located on pNC225-NDM-29, carries a G388A (D130N) mutation. The region harboring blaNDM–29 is located in an ISKpn19-based transposon, and two Tn6292 remnants are symmetrically located upstream and downstream of the transposon. The sequence results also indicated several important virulence genes.Conclusion: The findings of the novel carbapenemase NDM-29 could pose a threat to the control of antimicrobial resistance and arouse attention about the mutation of bacteria.

Non-β-Lactam Allosteric Inhibitors Target Methicillin-Resistant Staphylococcus aureus: An In Silico Drug Discovery Study.

Penicillin-binding proteins (PBPs) catalyze the final stages for peptidoglycan cell-wall bio-synthesis. Mutations in the PBP2a subunit can attenuate β-lactam antibiotic activity, resulting in unimpeded cell-wall formation and methicillin-resistant Staphylococcus aureus (MRSA). A double mutation in PBP2a (i.e., N146K and E150K) is resistant to β-lactam inhibitors; however, (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl) benzoic acid (QNZ), a heterocyclic antibiotic devoid of a β-lactam ring, interacts non-covalently with PBP2a allosteric site and inhibits PBP enzymatic activity. In the search for novel inhibitors that target this PBP2a allosteric site in acidic medium, an in silico screening was performed. Chemical databases including eMolecules, ChEMBL, and ChEBI were virtually screened for candidate inhibitors with a physicochemical similarity to QNZ. PBP2a binding affinities from the screening were calculated based on molecular docking with co-crystallized ligand QNZ serving as a reference. Molecular minimization calculations were performed for inhibitors with docking scores lower than QNZ (calc. −8.3 kcal/mol) followed by combined MD simulations and MM-GBSA binding energy calculations. Compounds eMol26313223 and eMol26314565 exhibited promising inhibitor activities based on binding affinities (ΔGbinding) that were twice that of QNZ (−38.5, −34.5, and −15.4 kcal/mol, respectively). Structural and energetic analyses over a 50 ns MD simulation revealed high stability for the inhibitors when complexed with the double mutated PBP2a. The pharmacokinetic properties of the two inhibitors were predicted using an in silico ADMET analysis. Calculated binding affinities hold promise for eMol26313223 and eMol26314565 as allosteric inhibitors of PBP2a in acidic medium and establish that further in vitro and in vivo inhibition experimentation is warranted.

Discovery of 2-Sulfinyl-Diazabicyclooctane Derivatives, Potential Oral β-Lactamase Inhibitors for Infections Caused by Serine β-Lactamase-Producing Enterobacterales.

Coadministration of β-lactam and β-lactamase inhibitor (BLI) is one of the well-established therapeutic measures for bacterial infections caused by β-lactam-resistant Gram-negative bacteria, whereas we have only two options for orally active BLI, clavulanic acid and sulbactam. Furthermore, these BLIs are losing their clinical usefulness because of the spread of new β-lactamases, including extended-spectrum β-lactamases (ESBLs) belonging to class A β-lactamases, class C and D β-lactamases, and carbapenemases, which are hardly or not inhibited by these classical BLIs. From the viewpoints of medical cost and burden of healthcare personnel, oral therapy offers many advantages. In our search for novel diazabicyclooctane (DBO) BLIs possessing a thio-functional group at the C2 position, we discovered a 2-sulfinyl-DBO derivative (2), which restores the antibacterial activities of an orally available third-generation cephalosporin, ceftibuten (CTB), against various serine β-lactamase-producing strains including carbapenem-resistant Enterobacteriaceae (CRE). It can be orally absorbed via the ester prodrug modification and exhibits in vivo efficacy in a combination with CTB.

Trends of Antimicrobial Resistance and Combination Susceptibility Testing of Multidrug-Resistant Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients: a 10-Year Update.

Antimicrobial combination therapy is a time/resource-intensive procedure commonly employed in the treatment of cystic fibrosis (CF) pulmonary exacerbations caused by Pseudomonas aeruginosa Ten years ago, the most promising antimicrobial combinations were proposed, but there has since been the introduction of new β-lactam plus β-lactamase inhibitor antimicrobial combinations. The aims of this study were to (i) compare in vitro activity of these new antimicrobials with other antipseudomonal agents and suggest their most synergistic antimicrobial combinations and (ii) determine antimicrobial resistance rates and study inherent trends of antimicrobials over 10 years. A total of 721 multidrug-resistant P. aeruginosa isolates from 183 patients were collated over the study period. Antimicrobial susceptibility and combination testing were carried out using the Etest method. The results were further assessed using the fractional inhibitory concentration index (FICI) and the susceptible breakpoint index (SBPI). Resistance to almost all antimicrobial agents maintained a similar level during the studied period. Colistin (P < 0.001) and tobramycin (P = 0.001) were the only antimicrobials with significant increasing isolate susceptibility, while an increasing resistance trend was observed for levofloxacin. The most active antimicrobials were colistin, ceftolozane-tazobactam, ceftazidime-avibactam, and gentamicin. All combinations with β-lactam plus β-lactamase inhibitors produced some synergistic results. Ciprofloxacin plus ceftolozane-tazobactam (40%) and amikacin plus ceftazidime (36.7%) were the most synergistic combinations, while colistin combinations gave the best median SBPI (50.11). This study suggests that effective fluoroquinolone stewardship should be employed for CF patients. It also presents in vitro data to support the efficacy of novel combinations for use in the treatment of chronic P. aeruginosa infections.

Ab Initio Spectroscopic Investigation of Pharmacologically Relevant Chiral Molecules: The Cases of Avibactam, Cephems, and Idelalisib as Benchmarks for Antibiotics and Anticancer Drugs

The ability to accurately measure or predict several physicochemical properties of molecules which play a role as active substances in drugs can be of strategic importance for pharmacological applications, in addition to its possible interest in fundamental research. Chirality is a relevant feature in the characterization of drug molecules: enantiomers can show different pharmacological activity and adverse effects. The ability to separate stereoisomers and to assign their absolute configuration can thus be crucial. Circular dichroism (CD) spectra are a useful tool to distinguish between enantiomers. In this work we apply an in-house developed code, based on an efficient DFT approach for circular dichroism, to fully characterize the molecular optical properties in the case of few selected fundamental molecules for current medical and pharmaceutical research, namely avibactam, as representative of non β-lactam inhibitors, two cephems (cefepime and cefoxitin), as examples of β-lactam antibiotics, and idelalisib, as a recent relevant anticancer active substance to treat major leukemias. For the above molecules, in addition to their optical absorption spectra, we calculate their CD spectra within state-of-the-art computational techniques. We then investigate both the conformational and chemical sensitivity of absorption and CD spectra for the chosen molecules. The outcomes of the present research could be of fundamental importance to gain additional information on molecules involved in therapeutic protocols for severe diseases or in drug design.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of β-lactam antibiotics and β-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While β-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

Activities of imipenem-relebactam combination against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

Imipenem-relebactam is a new β-lactam and β-lactamase inhibitor combination to treat carbapenem-resistant gram-negative bacteria infections. However, difference in carbapenem resistant mechanisms existed with geographic variations. To evaluate the susceptibility of imipenem-relebactam to 660 carbapenem-nonsusceptible Enterobacteriaceae isolates in Taiwan and to identify the invivo efficacy with a Caenorhabditis elegans model. 188 carbapenem-nonsusceptible Escherichia coli isolates and 472 carbapenem-nonsusceptible Klebsiella pneumoniae isolates were collected from a national surveillance study in Taiwan. The antimicrobial susceptibility profiles and carbapenemase distributions were determined. An agar dilution method was performed to evaluate the invitro activities of imipenem monotherapy and imipenem-relebactam combination. Contributions of metallo-carbapenemase to imipenem-relebactam susceptibility was investigated via EDTA treatment. A C. elegans model was used to evaluate the invivo efficacy of imipenem-relebactam combination. 87.8% and 82.2% susceptibility to imipenem-relebactam was observed for 188 carbapenem-nonsusceptible E. coli and 472 carbapenem-nonsusceptible K. pneumoniae, respectively. However, poor activities of imipenem-relebactam was observed against 23 metallo-carbapenemase producers tested in this study. In the invivo C. elegans model, imipenem-relebactam significantly rescued nematodes from the infection of a blaKPC-producing K. pneumoniae isolate. Our study supports that imipenem-relebactam is a potential therapy against carbapenem-nonsusceptible Enterobacteriaceae, and to our knowledge, this is the first report of evaluation for imipenem-relebactam efficacy against carbapenem-nonsusceptible Enterobacteriaceae in Taiwan.

β-Lactam antibiotic targets and resistance mechanisms: from covalent inhibitors to substrates.

The β-lactams are the most widely used antibacterial agents worldwide. These antibiotics, a group that includes the penicillins and cephalosporins, are covalent inhibitors that target bacterial penicillin-binding proteins and disrupt peptidoglycan synthesis. Bacteria can achieve resistance to β-lactams in several ways, including the production of serine β-lactamase enzymes. While β-lactams also covalently interact with serine β-lactamases, these enzymes are capable of deacylating this complex, treating the antibiotic as a substrate. In this tutorial-style review, we provide an overview of the β-lactam antibiotics, focusing on their covalent interactions with their target proteins and resistance mechanisms. We begin by describing the structurally diverse range of β-lactam antibiotics and β-lactamase inhibitors that are currently used as therapeutics. Then, we introduce the penicillin-binding proteins, describing their functions and structures, and highlighting their interactions with β-lactam antibiotics. We next describe the classes of serine β-lactamases, exploring some of the mechanisms by which they achieve the ability to degrade β-lactams. Finally, we introduce the l,d-transpeptidases, a group of bacterial enzymes involved in peptidoglycan synthesis which are also targeted by β-lactam antibiotics. Although resistance mechanisms are now prevalent for all antibiotics in this class, past successes in antibiotic development have at least delayed this onset of resistance. The β-lactams continue to be an essential tool for the treatment of infectious disease, and recent advances (e.g., β-lactamase inhibitor development) will continue to support their future use.

ANTIBIOTICOTERAPIA COM INIBIDORES β-LACTÂMICOS PARA RESSENSIBILIZAÇÃO DE BACTÉRIAS MULTIRRESISTENTES

O uso indiscriminado de antibióticos de um modo geral proporcionou o desenvolvimento de várias bactérias multirresistentes, as quais são responsáveis por infecções sistêmicas de grande importância clínica. O objetivo deste estudo foi descrever de forma narrativa a resistência antibiótica aos β-lactâmicos e sua administração combinada com outros fármacos visando a ressensibilização bacteriana de cepas multirresistentes. Trata-se de um estudo qualitativo de revisão narrativa, constituída por análise da literatura com busca de dados publicados na base de dados ScienceDirect sobre o tema “uso de inibidores β-lactâmicos para o tratamento de infecções causadas por bactérias multirresistentes”. Este estudo mostrou que há possibilidades de tratamento das infecções bacterianas causadas por cepas multirresistentes por meio de antibioticoterapia de inibidores β-lactâmicos. As infecções bacterianas multirresistentes continuam sendo um grande desafio para medicina, uma vez que existem enzimas que conferem resistência bacteriana contra todos os antibióticos. Mesmo que atualmente exista inibidores de β-lactamases disponíveis e com atividade antimicrobiana, é necessário que o esforço pela busca de novos antibióticos seja contínuo para o controle de bactérias multirresistentes.

Prevalence and coexistence of mcr-1 and ESBL producing Escherichia coli isolated from poultry and environmental water from Pakistan

Background: Emergence of colistin resistance and extended-spectrum-β-lactamase (ESBL) producing Escherichia coli is a serious threat to public health sector around the globe particularly in developing countries like Pakistan. ESBL producing bacteria display resistance against the β-lactam drugs except carbapenem. However, mcr-1 is a novel plasmid-mediated gene conferring resistance to colistin which is considering a last resort to treat clinical infection caused by multidrug resistant pathogens. The aim of the study was to determine the prevalence of both mcr-1 and ESBL producing E. coli in different settings. Methods and materials: A total of 100 poultry cloacal swabs and 100 environmental water samples were collected from Faisalabad metropolitan during October 2018–April 2019. Samples were screened for ESBL and colistin producing E. coli using colistin (4 μg/mL) containing MacConkey agar and cefotaxime (4 mg/L) containing ChromeID ESBL agar. Further, isolates were confirmed using UTI ChromoSelect agar and API 20E. Antibiogram and phenotypic confirmation of ESBL was carried out as per CLSI 2018 guidelines. Molecular identification of mcr-1and ESBL genes (blaCTX-M, blaTEM, blaSHV, blaOXA) was performed using PCR. Results: Of 100 poultry samples; 22 E. coli were positive for both mcr-1 and ESBL. Among ESBL producing E. coli, 22 (100%) carried blaCTX-M and 11 (50%) blaTEM. Of 100 water samples, 59 E. coli were ESBL producers and 4 were positive for mcr-1. Among ESBL producing E. coli, 32 (54.2%) were positive for blaTEM followed by 29 (49.1%) blaCTX-M and 1 (1.6%) blaSHV. E. coli from poultry displayed 100% resistance to β-lactam, β-lactam inhibitors and colistin while 63% to ciprofloxacin and 18% to meropenem. However, E. coli from water samples also displayed 100% resistance to β-lactam, β-lactam inhibitors followed by 52.5% to ciprofloxacin, 47.4% to amikacin, 16.9% to meropenem and 6.7% to colistin. Conclusion: Occurrence of mcr-1 and ESBL producing E. coli from poultry and environmental water is a matter of great concern for both livestock and public health. A One Health approach is necessary to further explicate the variability of these high-risk genes.

Antibiotic Resistance Crisis in Urinary Tract Infections Utis in Egypt

Abstract Introduction/Objective Bacterial adaptation to antibiotics has been very successful. Over the past decade, the increase in antibiotic resistance has generated considerable medical problems. UTIs are one of the most common community-onset infections. The emergence of resistance to all classes of antibiotics in previously susceptible bacterial pathogens is a major challenge to infectious disease medicine. Thus, it is important that we forcefully implement strategies to reduce the rate of appearance and spread of resistant bacteria to allow new drug discovery to catch up with bacterial resistance development. Objectives: Methods The aim of this study is to predict the percentage of urine cultures antibiotic resistance in patients with UTIs in Egypt. Subjects and methods: A total of 200 subjects were enrolled in this study from The Delta region in Egypt; 38% males and 62% females with a median age of 39 years and 35 years respectively. Identification of the isolated organisms was identified by the cultural characters (Morphological characters of the purified bacterial isolates were carried out according to the colony characters, Gram stain reaction and biochemical tests for each organism using a standard semi-automated technique Results 98 cultures (49%) were observed to have resistance to 3- 5 classes of antibiotics including, 98% Cephalosporins, 85% β-lactam inhibitors and 71% fluoroquinolones; 66 females (57 gram -ve stain and 9 gram +ve stain) and 33 males (27gm -ve stain, 6gm +ve stain). 82 cultures (41%) were not resistant to any classes.; 32 males (28 gram -ve stain and 3 gram +ve stain) and 50 females (45 gram -ve stain and 5 gram +ve stain). 19 cultures (9.5%) were observed to be resistant to all antibiotics.; 12 males (11 gram -ve stain and 1 gram +ve stain) and 7 females (2 gram -ve stain and 5 gram +ve stain). Conclusion This study highlights the highest antibiotic resistance associated with urinary tract infection in Egypt as well as the classification of the organisms. The high resistance percentage is due to the overuse of antibiotics by public and inappropriate antibiotic prescribing. Antibiotics are unregulated and available over the counter without a prescription in Egypt which makes it easily accessible. We recommend regulating the use of antibiotics and lunch antimicrobial stewardship programs in Egypt.