Studies on enmetazobactam clarify mechanisms of widely used ß-lactamase inhibitors.

ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone­derived acyl­enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl­enzyme complexes.

What Contributes to the MIC? Beyond ß-Lactamase Gene Detection in Klebsiella pneumoniae.

BACKGROUND: K. pneumoniae is capable of resistance to ß-lactam antibiotics through expression of ß-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which ß-lactamases and outer membrane porins affected ß-lactam resistance. METHODS: MICs to ß-lactams and inhibitor combinations were determined by agar dilution or E-test. Outer membrane porin production was evaluated by western blot of outer membrane fractions. ß-lactamase carriage was determined by whole genome sequencing and expression evaluated by RT-qPCR. RESULTS: Plasmid-encoded ß--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane/tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. ESßLs and pAmpCs in addition to porin loss were sufficient to confer resistance to the third generation cephalosporins, pipercillin/tazobactam, ceftolozane/tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to pipercillin/tazobactam. DISCUSSION: Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. some ß-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal ß-lactamases and loss of porins.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Mutation Rate of AmpC ß-Lactamase-Producing Enterobacterales and Treatment in Clinical Practice: A Word of Caution.

In a retrospective multicenter study of 575 patients with bloodstream infections or pneumonia due to wild-type AmpC ß-lactamase-producing Enterobacterales, species with low in vitro mutation rates for AmpC derepression were associated with fewer treatment failures due to AmpC overproduction (adjusted hazard ratio, 0.5 [95% CI, .2-.9]). However, compared to cefepime/carbapenems, using third-generation cephalosporins as definitive therapy remained associated with this adverse outcome (15% vs 1%).

The mechanism of ceftazidime and cefiderocol hydrolysis by D179Y variants of KPC carbapenemases is similar and involves the formation of a long-lived covalent intermediate.

Klebsiella pneumoniae carbapenemase (KPC) variants have been described that confer resistance to both ceftazidime-avibactam and cefiderocol. Of these, KPC-33 and KPC-31 are D179Y-containing variants derived from KPC-2 and KPC-3, respectively. To better understand this atypical phenotype, the catalytic mechanism of ceftazidime and cefiderocol hydrolysis by KPC-33 and KPC-31 as well as the ancestral KPC-2 and KPC-3 enzymes was studied. Steady-state kinetics showed that the D179Y substitution in either KPC-2 or KPC-3 is associated with a large decrease in both kcat and KM such that kcat/KM values were largely unchanged for both ceftazidime and cefiderocol substrates. A decrease in both kcat and KM is consistent with a decreased and rate-limiting deacylation step. We explored this hypothesis by performing pre-steady-state kinetics and showed that the acylation step is rate-limiting for KPC-2 and KPC-3 for both ceftazidime and cefiderocol hydrolysis. In contrast, we observed a burst of acyl-enzyme formation followed by a slow steady-state rate for the D179Y variants of KPC-2 and KPC-3 with either ceftazidime or cefiderocol, indicating that deacylation of the covalent intermediate is the rate-limiting step for catalysis. Finally, we show that the low KM value for ceftazidime or cefiderocol hydrolysis of the D179Y variants is not an indication of tight binding affinity for the substrates but rather is a reflection of the deacylation reaction becoming rate-limiting. Thus, the hydrolysis mechanism of ceftazidime and cefiderocol by the D179Y variants is very similar and involves the formation of a long-lived covalent intermediate that is associated with resistance to the drugs.

Characterization of Acinetobacter baumannii-calcoaceticus complex isolates and microbiological outcome for patients treated with sulbactam-durlobactam in a phase 3 trial (ATTACK).

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe, difficult-to-treat infections that are frequently antibiotic resistant. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat ABC infections, including those caused by multidrug-resistant strains. In a global, pathogen-specific, randomized, controlled phase 3 trial (ATTACK), the efficacy and safety of SUL-DUR were compared to colistin, both dosed with imipenem-cilastatin as background therapy, in patients with serious infections caused by carbapenem-resistant ABC. Results from ATTACK showed that SUL-DUR met the criteria for non-inferiority to colistin for the primary efficacy endpoint of 28-day all-cause mortality with improved clinical and microbiological outcomes compared to colistin. This report describes the characterization of the baseline ABC isolates from patients enrolled in ATTACK, including an analysis of the correlation of microbiological outcomes with SUL-DUR MIC values and the molecular drivers of SUL-DUR resistance.

Meropenem-ANT3310, a unique ß-lactam-ß-lactamase inhibitor combination with expanded antibacterial spectrum against Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii.

ANT3310 is a novel broad-spectrum diazabicyclooctane serine ß-lactamase inhibitor being developed in combination with meropenem (MEM) for the treatment of serious infections in hospitalized patients where carbapenem-resistant Gram-negative pathogens are expected. In this study, we evaluated the in vitro antibacterial activity of MEM in the presence of ANT3310 at 8 µg/mL against global clinical isolates that included Acinetobacter baumannii (n = 905), carbapenem-resistant Enterobacterales (CRE), carrying either oxacillinase (OXA) (n = 252) or Klebsiella pneumoniae carbapenemase (KPC) (n = 180) carbapenemases, and Pseudomonas aeruginosa (n = 502). MEM was poorly active against A. baumannii, as were MEM-vaborbactam, ceftazidime-avibactam, aztreonam-avibactam, cefepime-taniborbactam, cefepime-zidebactam, and imipenem-relebactam (MIC90 values of ≥32 µg/mL). On the other hand, MEM-ANT3310 displayed an MIC90 value of 4 µg/mL, similar to that observed with sulbactam-durlobactam, a drug developed to specifically treat A. baumannii infections. ANT3310 (8 µg/mL) additionally restored the activity of MEM against OXA- and KPC-producing CREs decreasing MEM MIC90 values from >32 µg/mL to 0.25 and 0.5 µg/mL, respectively. The combination of 8 µg/mL of both MEM and ANT3310 prevented growth of 97.5% of A. baumannii and 100% of OXA- and KPC-positive CREs, with ~90% of P. aeruginosa isolates also displaying MEM MICs ≤8 µg/mL. Furthermore, MEM-ANT3310 was efficacious in both thigh and lung murine infection models with OXA-23 A. baumannii. This study demonstrates the potent in vitro activity of the MEM-ANT3310 combination against both carbapenem-resistant A. baumannii and Enterobacterales clinical isolates, a key differentiator to other ß-lactam/ß-lactamase combinations.

The metallo-ß-lactamases strike back: emergence of taniborbactam escape variants.

Metallo-ß-lactamases (MBLs) have evolved relatively rapidly to become an international public health threat. There are no clinically available ß-lactamase inhibitors with activity against MBLs. This may change with the introduction of cefepime-taniborbactam. Herein, we review three manuscripts (S. I. Drusin, C. Le Terrier, L. Poirel, R. A. Bonomo, et al., Antimicrob Agents Chemother 68:e01168-23, 2024, https://doi.org/10.1128/aac.01168-23; C. Le Terrier, C. Viguier, P. Nordmann, A. J. Vila, and L. Poirel, Antimicrob Agents Chemother 68:e00991-23, 2024, https://doi.org/10.1128/aac.00991-23; D. Ono, M. F. Mojica, C. R. Bethel, Y. Ishii, et al., Antimicrob Agents Chemother 68:e01332-23, 2024, https://doi.org/10.1128/aac.01332-23) in which investigators describe elegant experiments to explore MBL/taniborbactam interactions and modifications to MBLs, in response, to reduce the affinity of taniborbactam. Challenges with MBL inhibition will not disappear; rather, they will evolve commensurate with advancements in medicinal chemistry.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid ß-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-ß-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Patient outcomes by baseline pathogen resistance phenotype and genotype in CERTAIN-1, a Phase 3 study of cefepime-taniborbactam versus meropenem in adults with complicated urinary tract infection.

CERTAIN-1 was a Phase 3, double-blind, randomized, parallel group study of the efficacy and safety of cefepime-taniborbactam versus meropenem in the treatment of adults with complicated urinary tract infection (cUTI), including acute pyelonephritis. We determined susceptibility of Enterobacterales and Pseudomonas aeruginosa baseline pathogens to cefepime-taniborbactam and comparators and characterized ß-lactam resistance mechanisms. Microbiologic response and clinical response were assessed in patient subsets defined by baseline pathogens that were of cefepime-, multidrug-, or carbapenem-resistant phenotype or that carried ß-lactamase genes. Among Enterobacterales baseline pathogens, 26.8%, 4.1%, and 3.0% carried genes for extended-spectrum ß-lactamases (ESBLs), AmpC, and carbapenemases, respectively. Within each treatment group, while composite success rates at Test of Cure in resistant subsets by pathogen species were similar to those by pathogen overall, composite success rates in meropenem patients were numerically lower for cefepime-resistant Escherichia coli (9/19; 47.4%) and ESBL E. coli (13/25; 52.0%) compared with E. coli overall (62/100; 62.0%). Cefepime-taniborbactam achieved composite success in 7/8 (87.5%) patients with carbapenem-resistant Enterobacterales and 8/9 (88.9%) patients with Enterobacterales with a carbapenemase gene (5 OXA-48-group; 2 KPC-3; 2 NDM-1). Cefepime-taniborbactam also achieved composite success in 8/16 (50.0%) patients and clinical success in 13/16 (81.3%) patients with P. aeruginosa; corresponding rates were 4/7 (57.1%) and 6/7 (85.7%) for meropenem. Cefepime-taniborbactam demonstrated efficacy in adult cUTI patients with cefepime-, multidrug-, and carbapenem-resistant pathogens including pathogens with ESBL, AmpC, and carbapenemase genes. CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT03840148.

In vitro activity of cefoxitin, imipenem, meropenem, and ceftaroline in combination with vaborbactam against Mycobacterium abscessus.

Mycobacterium abscessus (MAB) infections pose a growing public health threat. Here, we assessed the in vitro activity of the boronic acid-based ß-lactamase inhibitor, vaborbactam, with different ß-lactams against 100 clinical MAB isolates. Enhanced activity was observed with meropenem and ceftaroline with vaborbactam (1- and >4-fold MIC50/90 reduction). CRISPRi-mediated blaMAB gene knockdown showed a fourfold MIC reduction to ceftaroline but not the other ß-lactams. Our findings demonstrate vaborbactam's potential in combination therapy against MAB infections.

Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum ß-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to ß-lactam/ß-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum ß-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

First-in-human study to evaluate the safety, tolerability, and population pharmacokinetic/pharmacodynamic target attainment analysis of FL058 alone and in combination with meropenem in healthy subjects.

FL058 is a novel diazabicyclooctane ß-lactamase inhibitor. This first-in-human study evaluated the safety, tolerability, and population pharmacokinetic (PK)/pharmacodynamic target attainment analysis of FL058 alone and in combination with meropenem in healthy subjects. The results showed that the maximum tolerated dose of FL058 was 3,000 mg after single-dose infusion. FL058 in combination with meropenem did not cause any grade 3 or higher adverse event when the dose was escalated up to 1,000 mg/2,000 mg. FL058 exposure PK parameters showed dose proportionality. FL058 was excreted primarily in urine. No significant PK interaction was found between FL058 and meropenem. Population PK model analysis indicated that the PK profiles of FL058 and meropenem were consistent with the two-compartment model. The impact of covariates, creatinine clearance, concomitant use of meropenem, body weight, sex, and FL058 dose, on FL058 exposure was less than 10%. FL058/meropenem combination was safe and well tolerated up to a 1,000-mg/2,000-mg dose in healthy adults. The recommended minimum dose of FL058/meropenem combination was 500 mg/1,000 mg by intravenous infusion over 2 h every 8 h based on target attainment analysis. The good safety, tolerability, and satisfactory PK profiles of FL058 alone and in combination with meropenem in this first-in-human study will support further clinical development of FL058 in combination with meropenem in patients with target infections (ClinicalTrials.gov identifiers: NCT05055687, NCT05058118, and NCT05058105).

Plasmid-mediated acquisition and chromosomal integration of blaCTX-M-14 in a subclade of Escherichia coli ST131-H30 clade C1.

Escherichia coli ST131 is a multidrug-resistant lineage associated with the global spread of extended-spectrum ß-lactamase-producing organisms. Particularly, ST131 clade C1 is the most predominant clade in Japan, harboring blaCTX-M-14 at a high frequency. However, the process of resistance gene acquisition and spread remains unclear. Here, we performed whole-genome sequencing of 19 E. coli strains belonging to 12 STs and 12 fimH types collected between 1997 and 2016. Additionally, we analyzed the full-length genome sequences of 96 ST131-H30 clade C0 and C1 strains, including those obtained from this study and those registered in public databases, to understand how ST131 clade C1 acquired and spread blaCTX-M-14. We detected conjugative IncFII plasmids and IncB/O/K/Z plasmids carrying blaCTX-M-14 in diverse genetic lineages of E. coli strains from the 1990s to the 2010s, suggesting that these plasmids played an important role in the spread of blaCTX-M-14. Molecular phylogenetic and molecular clock analyses of the 96 ST131-H30 clade C0 and C1 strains identified 8 subclades. Strains harboring blaCTX-M-14 were clustered in subclades 4 and 5, and it was inferred that clade C1 acquired blaCTX-M-14 around 1993. All 34 strains belonging to subclade 5 possessed blaCTX-M-14 with ISEcp1 upstream at the same chromosomal position, indicating their common ancestor acquired blaCTX-M-14 in a single ISEcp1-mediated transposition event during the early formation of the subclade around 1999. Therefore, both the horizontal transfer of plasmids carrying blaCTX-M-14 to diverse genetic lineages and chromosomal integration in the predominant genetic lineage have contributed to the spread of blaCTX-M-14.

Synthesis of a tetralone derivative of ampicillin to control ampicillin-resistant Staphylococcusaureus.

Due to the rapid emergence of antibiotic resistant new bacterial strains and new infections, there is an urgent need for novel or newly modified and efficient alternatives of treatment. However, conventional antibiotics are still used in therapeutic settings but their efficacy is uncertain due to the rapid evolution of drug resistance. In the present study, we have synthesized a new derivative of conventional antibiotic ampicillin using SN2-type substitution reaction. NMR and mass analysis of the newly synthesized derivative of ampicillin confirmed it as ampicillin-bromo-methoxy-tetralone (ABMT). Importantly, ABMT is revealed to have efficient activity against Staphylococcus aureus (S. aureus) with a MIC value of 32 µg ml-1 while ampicillin was not effective, even at 64 µg ml-1 of concentration. Electron microscopy results confirmed the membrane-specific killing of S. aureus at 1 h of treatment. Additionally, molecular docking analysis revealed a strong binding affinity of ABMT with ß-lactamase via the formation of a closed compact bridge. Our findings, avail a new derivative of ampicillin that could be a potential alternative to fight ampicillin-resistant bacteria possibly by neutralizing the ß-lactamase action.

Costs of antibiotic resistance genes depend on host strain and environment and can influence community composition.

Antibiotic resistance genes (ARGs) benefit host bacteria in environments containing corresponding antibiotics, but it is less clear how they are maintained in environments where antibiotic selection is weak or sporadic. In particular, few studies have measured if the direct effect of ARGs on host fitness is fixed or if it depends on the host strain, perhaps marking some ARG-host combinations as selective refuges that can maintain ARGs in the absence of antibiotic selection. We quantified the fitness effects of six ARGs in 11 diverse Escherichia spp. strains. Three ARGs (blaTEM-116, cat and dfrA5, encoding resistance to ß-lactams, chloramphenicol, and trimethoprim, respectively) imposed an overall cost, but all ARGs had an effect in at least one host strain, reflecting a significant strain interaction effect. A simulation predicts these interactions can cause the success of ARGs to depend on available host strains, and, to a lesser extent, can cause host strain success to depend on the ARGs present in a community. These results indicate the importance of considering ARG effects across different host strains, and especially the potential of refuge strains to allow resistance to persist in the absence of direct selection, in efforts to understand resistance dynamics.

Genomic characterization of multi drug resistant ESBL-producing Escherichia coli isolates from patients and patient environments in a teaching hospital in Ghana.

BACKGROUND: ESBL-producing Escherichia coli pose a growing health risk in community and healthcare settings. We investigated the resistome, virulome, mobilome, and genetic relatedness of multidrug-resistant (MDR) E. coli isolates from patients and their environment in a Ghanaian teaching hospital. MATERIALS AND METHODS: Twenty-three MDR ESBL-producing or carbapenem-resistant E. coli isolates from a collection of MDR Gram-negative bacteria (GNB) from patients and environments were selected for genomic analyses. Whole genome sequencing and bioinformatics tools were used to analyze genomic characteristics and phylogeny. RESULTS: The prevalence and incidence of rectal carriage of ESBL E. coli among patients were 13.65% and 11.32% respectively. The ß-lactamase genes, blaTEM-1B (10 isolates) and blaCTX-M-15 (12 isolates) were commonly associated with IncFIB plasmid replicons and co-occurred with aminoglycoside, macrolide, and sulfamethoxazole/trimethoprim resistance. Insertion sequences, transposons, and class I integrons were found with blaCTX-M-15. Carriage and environmental isolates carried multiple virulence genes, with terC being the most prevalent in 21 isolates. Seventeen sequence types (STs) were identified, including a novel ST (ST13846). Phylogenetic analysis grouped the isolates into four main clusters, with one outlier. High genetic relatedness was observed between two carriage isolates of ST940 and between a carriage isolate and an environmental isolate of ST648. Isolates with different STs, collected at different times and locations, also showed genetic similarities. CONCLUSION: We identified ESBL-producing E. coli with diverse genomic characteristics circulating in different hospital directorates. Clonal relatedness was observed among isolates from patients and the environment, as well as between different patients, suggesting transmission within and between sources.

Solid waste dumpsite leachate and contiguous surface water contain multidrug-resistant ESBL-producing Escherichia coli carrying Extended Spectrum ß-Lactamase (ESBL) genes.

Dumpsites generate leachates containing bacteria that may carry antibiotic resistance genes, such as extended spectrum ß-lactamase (ESBL). However, the contribution of dumpsite leachates in the environmental spread of ESBL genes has not been investigated in greater detail. This study aimed to quantify the impact of Ajakanga dumpsite leachate on the spread of ESBL genes through surface water. The susceptibility of Escherichia coli isolated from dumpsite leachate and the accompanying surface water to selected antibiotics was assessed by the standardized disc diffusion method. The isolates were evaluated for phenotypic ESBL production using the double disc synergy test (DDST). The detection of ESBL genes in the isolates was carried out using a primer-specific polymerase chain reaction (PCR). Escherichia coli isolates from leachate (n = 26/32) and surface water (n = 9/12) expressed ESBL phenotype. The ESBL-producing isolates showed the highest level of resistance to the 3rd generation cephalosporin antibiotics: cefotaxime (100%), cefpodoxime (97%), ceftazidime (97%), with low resistance observed to imipenem (6%) and azithromycin (3%). All the isolates were multidrug-resistant, showing resistance to three or more classes of antibiotics. All the ESBL-producing E. coli obtained carried blaCTX-M, 21/35 (60%) carried blaTEM while none of the isolates bore blaSHV. This study found that ESBL-producing Escherichia coli from dumpsite leachate and nearby surface water had identical resistance signatures indicating the relatedness of the isolates, and that dumpsite leachate could contribute to the transfer of ESBL-producing bacteria and their genes to receiving surface water. This study has necessitated the need for a review of the guidelines and operational procedures of dumpsites to forestall a potential public health challenge.

Characterization of GQA as a novel ß-lactamase inhibitor of CTX-M-15 and KPC-2 enzymes.

ß-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum ß-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine ß­lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential ß-lactamase inhibitor.In the present study, blaCTX-M-15 and blaKPC-2 represented genes encoding for serine ß-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested ß-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-ß-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a ß-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC50 values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-ß-lactam inhibitor, which interacts with the crucial residues involved in ß-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of ß-lactams against carbapenemase and extended-spectrum ß-lactamase-producing strains, reducing the MICs of ß-lactams to the susceptible range. The ß-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent ß-lactamase inhibitors.

Multidrug-resistant Gram-negative clinical isolates with reduced susceptibility/resistance to cefiderocol: which are the best present and future therapeutic alternatives?

PURPOSE: To evaluate the different present and future therapeutic ß-lactam/ß-lactamase inhibitor (BL/BLI) alternatives, namely aztreonam-avibactam, imipenem-relebactam, meropenem-vaborbactam, cefepime-zidebactam, cefepime-taniborbactam, meropenem-nacubactam, and sulbactam-durlobactam against clinical isolates showing reduced susceptibility or resistance to cefiderocol in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa. METHODS: MIC values of aztreonam, aztreonam-avibactam, cefepime, cefepime-taniborbactam, cefepime-zidebactam, imipenem, imipenem-relebactam, meropenem, meropenem-vaborbactam, meropenem-nacubactam, sulbactam-durlobactam, and cefiderocol combined with a BLI were determined for 67, 9, and 11 clinical Enterobacterales, P. aeruginosa or A. baumannii isolates, respectively, showing MIC values of cefiderocol being ≥1 mg/L. If unavailable, the respective ß-lactam breakpoints according to EUCAST were used for BL/BLI combinations. RESULTS: For Enterobacterales, the susceptibility rates for aztreonam, cefepime, imipenem, and meropenem were 7.5%, 0%, 10.4%, and 10.4%, respectively, while they were much higher for cefepime-zidebactam (91%), cefiderocol-zidebactam (91%), meropenem-nacubactam (71.6%), cefiderocol-nacubactam (74.6%), and cefiderocol-taniborbactam (76.1%), as expected. For P. aeruginosa isolates, the higher susceptibility rates were observed for imipenem-relebactam, cefiderocol-zidebactam, and meropenem-vaborbactam (56% for all combinations). For A. baumannii isolates, lower susceptibility rates were observed with commercially or under development BL/BLI combos; however, a high susceptibility rate (70%) was found for sulbactam-durlobactam and when cefiderocol was associated to some BLIs. CONCLUSIONS: Zidebactam- and nacubactam-containing combinations showed a significant in vitro activity against multidrug-resistant Enterobacterales clinical isolates with reduced susceptibility to cefiderocol. On the other hand, imipenem-relebactam and meropenem-vaborbactam showed the highest susceptibility rates against P. aeruginosa isolates. Finally, sulbactam-durlobactam and cefiderocol combined with a BLI were the only effective options against A. baumannii tested isolates.