Β-lactamase Inhibitor Research Articles

P19 The role of policy and key stakeholder decision-making in the propagation, and deceleration, of resistance evolution

Abstract Background Antimicrobials have an intangible value on reducing global health burden of infection, yet current valuation methods do not holistically capture the enablement of healthcare as a result of antimicrobials. A lack of incentive and subsequent investment into the novel antimicrobial pipeline demonstrates the urgent need for collaboration amongst those influencing the decision-making process, namely key stakeholders such as governmental bodies, industry and academia. Studies into resistance evolution focus almost entirely on proximate causes, such as the number of antibiotics used in clinical settings, or the understanding of evolutionary pathways to resistance under controlled laboratory conditions. Comparatively, the association between resistance evolution and policy decisions from governments, industry and regulatory bodies is poorly understood. Objectives The primary aim was to evaluate the multifactorial associations between β-lactam development and evolution of β-lactam resistance to inform the current and future antibiotic development pipeline. Methods To undertake this narrative report, we focussed on β-lactam antibiotics, given their clear development timeline and continuous molecular refinement to meet arising clinical and microbiological needs. As such, the evolution of resistance to β-lactams poses a significant threat to mortality, quality of life, and societal function. This report consists of six historical case studies, all of which have significantly influenced the antimicrobial resistance landscape since Fleming’s discovery of penicillin in 1928. We include lesser-known events in our narrative, such as the establishment of the National Research and Development Corporation (NRDC), which propelled the mass commercialization of penicillin, and the subsequent emergence of penicillinases. Pivotal events such as the emergence of ESBL and MBLs into clinical settings also provide insight into the historical decision-making process by key stakeholders such as governmental bodies, industry and academia. Literature reviews were conducted with a focus on the initial introduction of a particular β-lactam into the clinic and when resistance arose. Key stakeholder activities and policy decisions made during these time periods influenced the formation of case studies. Results The case studies are as follows: (i) the influence of the NRDC in accelerated antimicrobial development; (ii) the introduction of the β-lactamase inhibitor—the influence of short-term clinical and financial benefit on prescribing behaviour; (iii) the ‘golden era’ of antibiotics—cephalosporin development and the consequences of short-term market forces; (iv) knowledge of MBL resistance prior to introduction of carbapenems in clinical settings; (v) consequences of controlled manufacturing for the global supply of piperacillin/tazobactam; and (vi) early surveillance studies and the beginning of the antimicrobial stewardship agenda. Conclusions In each case study we provide insight into how key stakeholders can improve practices by learning from past experiences, to inform the future stewardship agenda. We built a case for the need to incorporate evolutionary microbiology into policy and industrial decision-making in order to improve the mitigation of antimicrobial resistance.

In vitro activity of ceftazidime/avibactam, cefiderocol, meropenem/vaborbactam and imipenem/relebactam against clinical strains of the Stenotrophomonas maltophilia complex.

Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 μg/mL (4.95%). The β-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 μg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type β-lactamases, iron transporters and type-1 fimbriae. Antimicrobial resistance to first-line treatment is low. The in vitro activity of new β-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.

Unraveling the complexity of Stenotrophomonas maltophilia – a comprehensive review of current knowledge

Abstract Stenotrophomonas maltophilia is a Gram-negative (G−) opportunistic pathogen, prevalent and concerning for susceptible patients. It primarily causes nosocomial and some community-acquired respiratory and bloodstream infections. Reservoirs include water, plant rhizospheres, food, and animals. Virulence factors include motility, biofilm formation, and quorum sensing, with ongoing research on these mechanisms. A major concern is its multidrug resistance to antimicrobial agents like trimethoprim-sulfamethoxazole (TMP-SMX), β-lactams, macrolides, fluoroquinolones, aminoglycosides, chloramphenicol, tetracyclines, and polymyxins. Effective treatments include TMP-SMX, quinolones, penicillins, and penicillins with β-lactamase inhibitors. Rapid diagnosis is crucial for successful treatment, leading to the development of new detection methods such as selective steno medium agar (SMA) and molecular techniques for nucleic acid detection. Patients with S. maltophilia are prone to co-infections with other G− bacteria. A study by Safdar et al. on cancer patients from 1986 to 2002 showed an increase in S. maltophilia infections from 2% to 7%, making it the 5th most commonly isolated G-bacterium in this group. It commonly infects ICU patients and individuals with SARS-CoV-2, with pneumonia being the most frequent clinical manifestation. The COVID-19 pandemic increased intensive care unit admissions and mechanical ventilation use, leading to higher infection rates of S. maltophilia.

DOCKING, SYNTHESIS AND β-LACTAMASE INHIBITORY ACTIVITY EVALUATION FOR NEW AMIDE COMPOUNDS

The beta-lactams are recognized as effective antibiotics for treating infections, yet bacterial production of β-lactamase, which hydrolyzes the beta-lactam ring, can render these drugs inactive. Combining these antibiotics with a β-lactamase inhibitor, such as clavulanic acid, mitigates this resistance. In a docking study involving Temoniera-1 (TEM-1), 1pzp, we induced the synthesis of eight amide compounds by reacting acid chloride derivatives with sulphathiazol or oxadiazol amine, forming an amide bond. The newly synthesized compounds were differentiated using physical and spectroscopic methods and verified biologically by estimating their minimum inhibitory concentration (MIC) against four strains of βlactamase G(+)ve and G(-)ve bacteria. Their anti β-lactamase behaviors were then compared with that of clavulanic acid as a co-inhibitor with amoxicillin against the same four strains of bacteria. The results indicate that four of the new amides exhibit excellent anti-β-lactamase activity and contain one or more hydrophobic residues in their structures. Halogen atoms enhance the selectivity of tower β-lactamases. Derivatives of both oxadiazole and sulfathiazole scaffolds show promising anti-β-lactamase activity as nonβ-lactam inhibitors.

Retrospective Analysis of Risk Factors for Cefoperazone/Sulbactam-Induced Thrombocytopenia in Adult Chinese Patients: A Six-Year Real-World Study.

Drug-induced thrombocytopenia is a rare adverse reaction of drug therapy and usually underdiagnosed. Cefoperazone/sulbactam is a compound preparation composed of the third generation of cephalosporin and β-lactamase inhibitor, of which thrombocytopenia is an uncommon but serious adverse reaction. However, the existing literature on cefoperazone/sulbactam-induced thrombocytopenia remains limited, and the specific risk factors associated with this adverse effect have not been thoroughly elucidated. Consequently, this study aims to investigate the clinical characteristics and identify the risk factors for thrombocytopenia in adult patients undergoing cefoperazone/sulbactam therapy. In this retrospective study, we reviewed patients treated with cefoperazone/sulbactam at Beijing Hospital between January 2017 and June 2023. Patients were categorized into two groups based on the presence or absence of thrombocytopenia: the thrombocytopenia group and the non-thrombocytopenia group. We collected data on demographic features, clinical characteristics, laboratory parameters, treatments, and outcomes. Subsequently, univariate and multivariate logistic regression analyses were performed to identify potential risk factors for cefoperazone/sulbactam-induced thrombocytopenia. In total, 6489 patients were included in this study, and 2.4% (155/6489) developed thrombocytopenia. The results of multivariate analysis showed that cefoperazone/sulbactam therapy duration (d) >14, PLT (109/L) <200, daily dose of cefoperazone/sulbactam (g) ≥6, TBil (μmoL/L) >21, AST (U/L) >35, and use of non-invasive ventilator were risk factors for cefoperazone/sulbactam-induced thrombocytopenia. Despite the low incidence (2.4%), cefoperazone/sulbactam could cause serious thrombocytopenia sometimes accompanied with hemorrhage. In clinical therapy, clinicians should be vigilant in monitoring platelet count, especially for patients with risk factors of cefoperazone/sulbactam-induced thrombocytopenia.

Management of antimicrobial therapy in emergency department admissions and hospitalizations for firearm injuries: A single-center experience.

Firearm injuries (FI) remain a significant cause of morbidity and mortality globally. Antibiotic use, supported by guideline recommendations for preventing post-injury infections in FI cases, encounters uncertainties regarding the selection of anti-microbial agents and associated outcomes. This study aimed to investigate the relationship between Injury Severity Scores (ISS) for FI cases presented to the emergency department. We empirically revised antimicrobial treatment protocols based on culture results and mortality rates. In the study, 164 firearm injury cases, admitted to the emergency department in 2022 and subsequently hospitalized in clinics and intensive care units (ICU), were evaluated. Cases included in the study were categorized into four groups based on ISS: mild, moderate, severe, and profound injury severity. The study compared the timing of hospital presentation following the injury, hospital length of stay, tissue or blood culture positivity, empirical treatment administered, antimicrobial revision based on culture results, need for ICU admission, mortality status, and ISS among the cases. Data were analyzed using IBM SPSS Statistics 22.0 (SPSS Inc., Chicago, IL). Variables in trauma patients were compared among various groups using Pearson Chi-Square tests. Binary logistic regression tests were performed to identify independent risk factors. A significance level of p<0.05 was considered statistically significant. The study included 164 patients, all of whom were male. The mean age was calculated as 28.9±4.51 years. The average hospital length of stay was 25.54±21.81 days. Eighty-three patients (50.6%) required intensive care. Tissue cultures were obtained from 79 patients (48%). Bacterial growth was observed in 45 of these 79 patients (57%). The appropriate empirical antibiotic treatment rate, assessed among patients who received empirical treatment followed by culture-based antibiotic sensitivity testing, was 48.9%. It was observed that empirical antibiotic regimens were appropriate in 80% of cases in the mild group and 16.7% in the profound severe group (p=0.005). Our study compared the relationship between hospitalization duration and ISS groups. It was observed that hospitalization duration was significantly shorter in the mild group compared to the other groups (p=0.003, p=0.000, p=0.000). It was also observed that the need for ICU admission was higher in groups with higher ISS, indicating a correlation between higher ISS and increased ICU requirements (p=0.000). In conclusion, for cases of firearm injuries, we believe empirical antimicrobial therapy should be initiated with narrow-spectrum agents such as beta-lactam + beta-lactamase inhibitor or third-generation cephalosporin + nitroimidazole in the mild group, considering the lack of Pseudomonal activity.

Flux dynamics of C-5 amino acid precursors in fed-batch cultures of Streptomyces clavuligerus during clavulanic acid biosynthesis

Clavulanic acid (CA) is a well-known β-lactamase inhibitor that is mainly obtained from submerged cultures of Streptomyces clavuligerus. Dynamic genome-scale in silico studies were performed to gain insights into the intracellular metabolic fluxes of C-5 precursors during the cultivation of wild-type S. clavuligerus in batch and fed-batch operation. A preliminary literature screening was conducted for media selection and determination of culture conditions for further cultivation in a 5-L bioreactor. Simulations provided insights into the metabolism of C-5 precursors in CA biosynthesis. In addition, carbon utilization for CA biosynthesis was assessed in terms of the ratio of total carbon used in CA biosynthesis to the total carbon influx in batch and feed media. Based on simulation results, we proposed a modification of the glycerol-sucrose-proline-glutamate medium for fed-batch cultivation to improve the carbon utilization for CA biosynthesis. The proposed fed-batch scenario achieved higher specific CA concentration at lower biomass production, indicating better carbon utilization for its synthesis. The dynamic in silico fluxes suggests that metabolic fluxes in this scenario would be stable, favoring a longer stage of continued antibiotic secretion.

Emerging Coproduction of AmpC Beta-lactamase in Extended-spectrum Beta-lactamase-producing Escherichia coli Clinical Isolates in Indonesia

ABSTRACT Introduction: Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC-BL) in Enterobacteriaceae are a global threat. Coproduction of AmpC-BL and ESBL reduces therapeutic options, with more resistance against cephamycin and beta-lactamase inhibitor combinations. This study determines the proportion, incidence, and distribution of AmpC-BL-resistant genes in clinically isolated ESBL-producing Escherichia coli, completing its scarce data in Indonesia. Methods: The samples in this study were ESBL-producing E. coli from blood and urine specimens, confirmed by BD Phoenix semiautomatic examination combined with cefoxitin disk screening method. Confirmation tests of AmpC-BL used the AmpC-disk method and the polymerase chain reaction method. Results: Thirteen (27.8%) of 108 E. coli isolates were nonsusceptible to cefoxitin, and 3 (11.5%) isolates were confirmed to produce AmpC-BL and contained AmpC-BL CITM gene. Of the 3 AmpC-BL and ESBL coproduction isolates, one isolate was a copresentation of ESBL and AmpC-BL genes, namely blaTEM and CITM. Fisher’s exact test showed that the coproduction of AmpC-BL in ESBL-producing isolates was associated with reduced susceptibility to cefoxitin (P = 0.020) and amoxicillin-clavulanate (P = 0.048) compared to isolates producing ESBL alone. The effect of ESBL and AmpC-BL coproduction on reducing susceptibility to carbapenems needs further investigation. Conclusions: AmpC coproduction was found in 3 (2.8%) of 108 ESBL-producing E. coli isolates, and one isolate copresented AmpC-BL and ESBL coding genes blaTEM and CITM. These three isolates were associated with widened antibiotic resistance to cefoxitin and amoxicillin--clavulanate compared to isolates producing ESBL alone.

Activity of imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae and imipenem-resistant Pseudomonas aeruginosa

PurposeRelebactam is a novel β-lactamase inhibitor, which, when combined with imipenem/cilastatin, is active against both class A and class C β-lactamases. To evaluate in vitro antimicrobial activity of imipenem/relebactam against a collection of recent clinical isolates of carbapenem-non-susceptible P. aeruginosa and K. pneumoniae ST258 and ST512 KPC producers belonging to different lineages from hospitals in Southern Spain.MethodsSix hundred and seventy-eight isolates were tested: 265 K. pneumoniae (230 ST512/KPC-3 and 35 ST258/KPC-3) and 413 carbapenem-non-susceptible P. aeruginosa. Imipenem, piperacillin/tazobactam, ceftazidime, cefepime, aztreonam, ceftolozane/tazobactam, meropenem, amikacin, ciprofloxacin, colistin, and ceftazidime/avibactam were used as comparators against P. aeruginosa. Against K. pneumoniae ceftazidime, cefepime, aztreonam, and ceftolozane/tazobactam were not tested, and tigecycline was studied instead. MICs were determined in duplicate by broth microdilution according to EUCAST guidelines.ResultsImipenem/relebactam displayed potent in vitro activity against both sequence types of KPC-3-producing K. pneumoniae. MIC50 and MIC90 values were 0.25 mg/L and 1 mg/L, respectively, with percent of susceptible isolates >97%. Only three K. pneumoniae ST512/KPC-3 isolates and one ST258/KPC-3 were resistant to imipenem/relebactam. Relebactam sensitized 98.5% of K. pneumoniae isolates resistant to imipenem. The activity of imipenem/relebactam against P. aeruginosa was moderate (susceptibility rate: 62.7%). Analysis of the acquired and mutational resistome of isolates with high levels of resistance to imipenem/relebactam has not shown a clear association between them.ConclusionImipenem/relebactam showed excellent activity against K. pneumoniae KPC-3. The activity of imipenem/relebactam against imipenem-resistant P. aeruginosa was moderate.

Evaluation of selected natural sesquiterpenes as sensitizing agents of β-lactam-resistant bacterial strains.

To evaluate the capacity of fourteen sesquiterpenes to enhance the action of known antibiotics against two β-lactam resistant strains, and to determine a possible mechanism of antibiotic sensitization by assessing their ability to inhibit a β-lactamase enzyme. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of β-lactams cefuroxime (CEFM) and cefepime (CPM) against Staphylococcus aureus 23MR and Escherichia coli 82MR strains in the absence and presence of subinhibitory concentrations of fourteen natural sesquiterpenes. (1R,4R)-4H-1,2,3,4-tetrahydro-1-hydroxycadalen-15-oic acid (5), xerantolide (8), estafiatin (11), and ambrosin (12) exhibited the best sensitizing effects in both strains. These compounds were able to reduce the MIC of CEFM by 2-fold (from 15.0 to 7.5µg/mL) and CPM by 15-fold (from 0.9 to 0.06µg/mL) in S. aureus 23MR. For E. coli 82MR, the MIC of CEFM was reduced up to 8-fold (from 120.0 to 15.0µg/mL). In this strain, the activity of 8 and 11 surpassed that of clavulanic acid (positive reference), which reduced the MIC of CEFM from 120.0 to 60.0µg/mL. To elucidate a possible mechanism of antibiotic sensitization, molecular docking studies were conducted with β-lactamases. These studies revealed an affinity with the enzymes (energies > -4.93kcal/mol) by the formation of hydrogen bonds with certain conserved amino acid residues within the active sites. However, the in vitro results indicated only marginal inhibition, with percentages <50%. The bioevaluations indicate that nine of fourteen sesquiterpenes enhance the action of CEFM and CPM against the β-lactam resistant strains, and these compounds displayed moderate activity as inhibitors of β-lactamase.

Efficacy and safety of novel carbapenem-β-lactamase inhibitor combinations: imipenem-cilastatin/relebactam results from randomized controlled trials.

Gram-negative bacteria is a global public health problem. Treatment options include novel beta-lactamase inhibitors. The objective of this study was to collect information on the efficacy and safety of novel β-lactamase inhibitor combinations such as imipenem-cilastatin/relebactam (IMI/REL). In order to comprehensively evaluate the clinical, microbiological, and adverse events outcomes, a meta-analysis was conducted on clinical trials comparing novel β-lactamase inhibitor combinations with existing comparator therapies. Four studies comprising 948 patients were included in the analysis. IMI/REL therapy demonstrated similar clinical responses to comparators across various treatment visits, including discontinuation of intravenously administered therapy visits [DCIV, RR = 1.00 (0.88, 1.12)], early follow-up visits [EFU, RR = 1.00 (0.89, 1.14)], late follow-up visits [LFU, RR = 1.00 (0.88, 1.13)]. Moreover, no significant difference in the microbiologic response of MITT patients was observed between IMI/REL and comparators across DCIV [RR = 0.99 (0.89, 1.11)], EFU [RR = 1.01 (0.95, 1.07)], and LFU visits [RR = 1.00 (90.94, 1.07)]. In terms of safety, therapy with IMI/REL and comparators exhibited similar risks of at least one adverse event (AE), drug-related AEs, and discontinuation due to AEs. The incidence of serious AEs (SAEs) was significantly lower in the IMI/REL group compared to the comparison groups. The predominant AEs were gastrointestinal disorders, with no significant difference observed between the IMI/REL group and comparators. The clinical and microbiologic response to IMI/REL in the treatment of bacterial infection was comparable to that of the comparator. Furthermore, the incidence of AEs and the tolerability of IMI/REL were similar among the comparators. Based on these findings, IMI/REL can be considered as a viable alternative treatment option.

Exploiting the Carboxylate-Binding Pocket of β-Lactamase Enzymes Using a Focused DNA-Encoded Chemical Library.

β-Lactamase enzymes hydrolyze and thereby provide bacterial resistance to the important β-lactam class of antibiotics. The OXA-48 and NDM-1 β-lactamases cause resistance to the last-resort β-lactams, carbapenems, leading to a serious public health threat. Here, we utilized DNA-encoded chemical library (DECL) technology to discover novel β-lactamase inhibitors. We exploited the β-lactamase enzyme-substrate binding interactions and created a DECL targeting the carboxylate-binding pocket present in all β-lactamases. A library of 106 compounds, each containing a carboxylic acid or a tetrazole as an enzyme recognition element, was designed, constructed, and used to identify OXA-48 and NDM-1 inhibitors with micromolar to nanomolar potency. Further optimization led to NDM-1 inhibitors with increased potencies and biological activities. This work demonstrates that the carboxylate-binding pocket-targeting DECL, designed based on substrate binding information, aids in inhibitor identification and led to the discovery of novel non-β-lactam pharmacophores for the development of β-lactamase inhibitors for enzymes of different structural and mechanistic classes.

Low Occurrence of Salmonella spp. in Wild Animals in Bahia, Brazil-Population Assessment and Characterization in the Caatinga and Atlantic Forest Biomes.

Salmonella spp. are known to persist in the environment. Wild animals are believed to act as important reservoirs, with antimicrobial resistance frequently occurring in the environment. However, little is known about the role of the wildlife in Bahia as a reservoir for Salmonella in Brazil. This study aimed to isolate and characterize Salmonella spp. from wildlife in the Atlantic Forest and Caatinga biomes considering indicators such as the animal species, degree of anthropization, sampling area, and feeding habits. Convenience wildlife sampling and characterization were conducted, followed by microbiological and molecular identification of Salmonella isolates, serotyping, and antimicrobial susceptibility testing. A total of 674 fecal samples were collected from 12 municipalities during 2015-2021, and 4 were positive for the following Salmonella species: Salmonella enterica subspecies enterica serovar Agona (n = 1), Salmonella enterica subsp. enterica serogroup O:16 (n = 2), and Salmonella enterica subsp. enterica serovar Muenchen (n = 1). Antimicrobial susceptibility analysis revealed that one isolate was resistant to six antibiotics, including extended-spectrum penicillins and beta-lactamase inhibitors. These results indicated a low frequency of Salmonella spp. in the sampled forest fragments. The presence of Salmonella in wild animals increases the risk to public health and biodiversity and indicates that they can act as sentinels of environmental contamination or indicators of preservation.

Mechanistic Approach of Effective Combination of Antibiotics Against Clinical Bacterial Strains Having New Delhi Metallo-Beta-Lactamase Variants and Functional Gain Laboratory Mutant.

Antimicrobial resistance has emerged as a serious issue for physicians and health-care workers treating infections that could lead to the next pandemic. One of the key resistance mechanisms is beta-lactamases. Although several beta-lactamase inhibitors in combination with antibiotics have been created and are being utilized in clinical settings, resistance to these formulations has also been evolving in the bacterial population due to their distinct targets. In this study we used effective combination of antibiotic as an approach to inhibit multidrug resistance bacteria. We used four combinations and checked its efficacy against NDM (New Delhi Metallo-beta-lactamase) variants and functional gain laboratory mutant by employing FICI, enzyme kinetics, fluorescence and computational biology approaches (Docking and Molecular Dynamics Simulation). FICI values of all the combinations were either less than 0.5 or equal to 0.5. Binding features acquired by spectroscopic techniques showed important interaction and complex formation between drugs and enzymes with decreased ksv and kq values. In steady-state kinetics, a reduction in hydrolytic efficiency of enzymes was shown by cooperative binding behaviour when they were treated with different drugs. We have also tested functional gain laboratory mutant developed in our lab, keeping in view that if in future upcoming variants of this kind be emerged then these mutants could also be subsided by combinational therapy. This study identifies three other combinations better than fluoroquinolones effective against NDM variants and laboratory mutant.

Solid-Phase Extraction at High pH as a Promising Tool for Targeted Isolation of Biologically Active Fractions of Humic Acids.

A search for novel sources of biologically active compounds is at the top of the agenda for biomedical technologies. Natural humic substances (HSs) contain a large variety of different chemotypes, such as condensed tannins, hydrolyzable tannins, terpenoids, lignins, etc. The goal of this work was to develop an efficient separation technique based on solid-phase extraction (SPE) for the isolation of narrow fractions of HS with higher biological activity compared to the initial material. We used lignite humic acid as the parent humic material, which showed moderate inhibition activity toward beta-lactamase TEM 1 and antioxidant activity. We applied two different SPE techniques: the first one was based on a gradient elution with water/methanol mixtures of the humic material sorbed at pH 2, and the second one implied separation by a difference in the pKa value by the use of sequential sorption of HS at pH from 8 to 3. SPE cartridges Bond Elute PPL (Agilent) were used in the fractionation experiments. The first and second techniques yielded 9 and 7 fractions, respectively. All fractions were characterized using high-resolution mass spectrometry and biological assays, including the determination of beta-lactamase (TEM 1) inhibition activity and antioxidant activity. The acidity-based separation technique demonstrated substantial advantages: it enabled the isolation of components, outcompeting the initial material at the first step of separation (sorption at pH 8). It showed moderate orthogonality in separation with regard to the polarity-based technique. Good perspectives are shown for developing a 2D separation scheme using a combination of polarity and acidity-based approaches to reduce structural heterogeneity of the narrow fractions of HS.

Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a β-lactam/β-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane β-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine β-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of β-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

I n vitro pharmacokinetics/pharmacodynamics of the β-lactamase inhibitor, durlobactam, in combination with sulbactam against Acinetobacter baumannii-calcoaceticus complex.

Infections caused by Acinetobacter baumannii are increasingly multidrug resistant and associated with high rates of morbidity and mortality. Sulbactam is a β-lactamase inhibitor with intrinsic antibacterial activity against A. baumannii. Durlobactam is a non-β-lactam β-lactamase inhibitor with an extended spectrum of activity compared to other inhibitors of its class. In vitro pharmacodynamic infection models were undertaken to establish the pharmacokinetic/pharmacodynamic (PK/PD) index and magnitudes associated with sulbactam and durlobactam efficacy and to simulate epithelial lining fluid (ELF) exposures at clinical doses to understand sulbactam-durlobactam activity with and without co-administration of a carbapenem. Hollow fiber infection models (HFIMs) and one-compartment systems were used to identify the PK/PD indices and exposure magnitudes associated of 1-log10 and 2-log10 colony-forming unit (CFU)/mL reductions. Sulbactam and durlobactam demonstrated PK/PD drivers of % time above the minimum inhibition concentration (%T > MIC) and area under the plasma concentration-time curve from time 0 to 24 h (AUC0-24)/MIC, respectively. Against a sulbactam-susceptible strain, sulbactam %T > MIC of 71.5 and 82.0 were associated with 1-log10 and 2-log10 CFU/mL reductions, respectively, in the HFIM. Against a non-susceptible strain, durlobactam restored the activity of sulbactam with an AUC0-24/MICs of 34.0 and 46.8 using a polysulfone cartridge to achieve a 1-log10 and 2-log10 CFU/mL reduction. These magnitudes were reduced to 13.8 and 24.2, respectively, using a polyvinylidene fluoride cartridge with a membrane pore size of 0.1 μm. In the one-compartment model, durlobactam AUC0-24/MIC to achieve 1-log10 and 2-log10 CFU/mL reduction were 7.6 and 33.4, respectively. Simulations of clinical ELF exposures in the HFIM showed cidal activity at MICs ≤4 µg/mL. Penicillin binding protein 3 mutant strains with MICs of 8 μg/mL may benefit from the addition of a carbapenem at clinical exposures.

In vivo dose response and efficacy of the β-lactamase inhibitor, durlobactam, in combination with sulbactam against the Acinetobacter baumannii-calcoaceticus complex.

Multi-drug resistant (MDR) Acinetobacter baumannii is emerging as a pathogen of increasing prevalence and concern. Infections associated with this Gram-negative pathogen are often associated with increased morbidity and mortality and few therapeutic options. The β-lactamase inhibitor sulbactam used commonly in combination with ampicillin demonstrates intrinsic antibacterial activity against A. baumannii acting as an inhibitor of PBP1 and PBP3, which participate in cell wall biosynthesis. The production of β-lactamases, particularly class D oxacillinases, however, has limited the utility of sulbactam resorting to increased doses and the need for alternate therapies. Durlobactam is a non-β-lactam β-lactamase inhibitor that demonstrates broad β-lactamase inhibition including class D enzymes produced by A. baumannii and has shown potent in vitro activity against MDR A. baumannii, particularly carbapenem-resistant isolates in susceptibility and pharmacodynamic model systems. The objective of this study is to evaluate the exposure-response relationship of sulbactam and durlobactam in combination using in vivo neutropenic thigh and lung models to establish PK/PD exposure magnitudes to project clinically effective doses. Utilizing established PK/PD determinants of %T>MIC and AUC/MIC for sulbactam and durlobactam, respectively, non-linear regressional analysis of drug exposure was evaluated relative to the 24-hour change in bacterial burden (log10 CFU/g). Co-modeling of the data across multiple strains exhibiting a broad range of MIC susceptibility suggested net 1-log10 CFU/g0 reduction can be achieved when sulbactam T>MIC exceeds 50% of the dosing interval and durlobactam AUC/MIC is 10. These data were ultimately used to support sulbactam-durlobactam dose selection for Phase 3 clinical trials.

Mutations in V84I & A184V of blaTEM cluster plays a pivotal role in the dynamics of Ω-loop leading to genesis of IR-TEM

The Enterobacteriaceae family exhibits resistance to antibiotics by producing β-Lactamase. Mutations in β-Lactamase, have led to the generation of inhibitor resistant variants known as IR-TEM. In the present study, IR-TEM β-Lactamase of Enterobacter hormaechei and Enterobacter asburiae was compared. To investigate the mechanism behind the conferred mutation, we studied its interaction with Clavulanic acid, (β-Lactamase inhibitor) with different lineages of TEM and IR-TEM. We found that Clavulanic acid quickly left the binding pockets of both variants using molecular dynamics (MD) simulations. Interestingly, mutations at the V84I and A184V positions were found to drastically influence the protein dynamics. Mutating the residues at V84I and A184V positions by computational mutagenesis in Enterobacter hormaechei, it was observed that the residues on the Ω-loop as well as a few downstream residues were primarily involved in generating resistance towards inhibitors by conferring increased flexibility to the loop. This further strongly supports the notion that inhibitor resistance in β-Lactamase is conferred through allosteric regulation, wherein mutations in positions 84 and 184 may play an important role in regulating the movement of the Ω-loop. These two positions determine the lineage pattern between two clusters in TEM-1 and TEM-116. To date, no reports have been made regarding the importance of these mutations and their dynamics in Ω-loop.Communicated by Ramaswamy H. Sarma

Various types of extended spectrum β-lactamases: a literature review

The inappropriate use of antibacterials causes the spread of resistance in bacteria and increases the health burden of infection due to the nature of resistance to many classes of antibiotics which are referred to as multi-resistant (multidrug resistant). Extended spectrum β-lactamase (ESBL) is one of the enzymes that cause multi-resistance, where bacteria with this enzyme become resistant to third-generation cephalosporin antibiotics which are often used to treat gram-positive and gram-negative bacterial infections in humans with minimal side effects. β -lactamase is an enzyme capable of hydrolyzing the β-lactam ring in β -lactam class antibiotics so that antibiotics become inactive. Mutations in the gene that encodes this enzyme produce a β-lactamase enzyme which can break down the β-lactam ring in all penicillin and cephalosporin antibiotics. The classification of β-lactamases is complex due to genetic, biochemical properties, and substrate affinity for β-lactamase inhibitors. There are also various types of ESBL enzyme-coding genes including TEM, SHV, CTX-M, VEB, PER, OXA, SFO-1, BEL-1, BES-1, TLA-1, and GES. This study aims to review various types of extended-spectrum β-lactamases.