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Modification of the R1 and R2 side chain structures has been used as the main strategy to expand the spectrum of cephalosporins and impart resistance to hydrolysis by β-lactamases. These structural modifications also result in a wide range of plasma protein binding, especially with human serum albumin (HSA). Here, we determined the crystal structures of the HSA complexes with two clinically important cephalosporins, ceftriaxone and cefazolin, and evaluated the binding of cephalosporin to HSA by susceptibility testing and competitive binding assay. Ceftriaxone and cefazolin bind to subdomain IB of HSA, and their cephem core structures are recognized by Arg117 of HSA. Tyr161 of HSA changes its rotamer depending on the cephalosporin, resulting in alterations of the cavity shape occupied by the R2 side chain of cephalosporins. These findings provide structural insight into the mechanisms underlying the HSA binding of cephalosporins.

Staphylococcus xylosus has emerged as a bovine mastitis pathogen with increasing drug resistance, resulting in substantial economic impacts. This study utilized iTRAQ analysis to investigate the mechanisms driving resistance evolution in S. xylosus under ceftiofur sodium stress. Findings revealed notable variations in the expression of 143 proteins, particularly glycolysis-related proteins (TpiA, Eno, GlpD, Ldh) and peptidoglycan (PG) hydrolase Atl. Following the induction of ceftiofur sodium resistance in S. xylosus, the emergence of resistant strains displaying characteristics of small colony variants (SCVs) was observed. The transcript levels of TpiA, Eno, GlpD and Ldh were up-regulated, TCA cycle proteins (ICDH, MDH) and Atl were down-regulated, lactate content was increased, and NADH concentration was decreased in SCV compared to the wild strain. That indicates a potential role of carbon metabolism, specifically PG hydrolysis, glycolysis, and the TCA cycle, in the development of resistance to ceftiofur sodium in S. xylosus., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: Stenotrophomonas spp. intrinsically resistant to many β-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A β-lactamase. The goal of this study was to reveal biochemical and structural characteristics of CESS-1., Methods: The hydrolytic activities of CESS-1 towards penicillins (penicillin G and ampicillin), cephalosporins (cephalexin, cefaclor, and cefotaxime), and carbapenems (imipenem and meropenem) was spectrophotometrically monitored. Structural information on E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin were determined by X-ray crystallography., Results: CESS-1 displayed hydrolytic activities toward penicillins and cephalosporins, with negligible activity toward carbapenems. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward them were distinct. The k cat values for cefaclor, cephalexin, and ampicillin were 1249.6 s -1 , 204.3 s -1 , and 69.8 s -1 , respectively, with the accompanying K M values of 287.6 μM, 236.7 μM, and 28.8 μM, respectively., Conclusions: CESS-1 was able to discriminate between cefaclor and cephalexin with a single structural difference at C3 position: -Cl (cefaclor) and -CH 3 (cephalexin). Structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin, revealed that cooperative positional changes in the R1 side chain of substrates and their interaction with the β5-β6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar β-lactam antibiotics., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Effective treatment of gonorrhea is threatened by the increasing prevalence of Neisseria gonorrhoeae strains resistant to the extended-spectrum cephalosporins (ESCs). Recently, we demonstrated the promise of the third-generation cephalosporin cefoperazone as an antigonococcal agent due to its rapid second-order rate of acylation against penicillin-binding protein 2 (PBP2) from the ESC-resistant strain H041 and robust antimicrobial activity against H041. Noting the presence of a ureido moiety in cefoperazone, we evaluated a subset of structurally similar ureido β-lactams, including piperacillin, azlocillin, and mezlocillin, for activity against PBP2 from H041 using biochemical and structural analyses. We found that the ureidopenicillin piperacillin has a second-order rate of acylation against PBP2 that is 12-fold higher than cefoperazone and 85-fold higher than ceftriaxone and a lower MIC against H041 than ceftriaxone. Surprisingly, the affinity of ureidopenicillins for PBP2 is minimal, indicating that their inhibitory potency is due to a higher rate of the acylation step of the reaction compared to cephalosporins. Enhanced acylation results from the combination of a penam scaffold with a 2,3-dioxopiperazine-containing R 1 group. Crystal structures show that the ureido β-lactams overcome the effects of resistance mutations present in PBP2 from H041 by eliciting conformational changes that are hindered when PBP2 interacts with the weaker inhibitor ceftriaxone. Overall, our results support the potential of piperacillin as a treatment for gonorrhea and provide a framework for the future design of β-lactams with improved activity against ESC-resistant N. gonorrhoeae .

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria., Competing Interests: No competing interests., (Copyright: © 2024 Djorić et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter P AngpdA . Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high-yielding strains AG-6, AG-18, and AG-41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac-∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression., (© 2024 Wiley-VCH GmbH.)

In this study, AmpC β-lactamase of Escherichia coli was expressed, and its intermolecular interaction mechanisms with 15 cephalosporins (CPs) were studied by using a molecular docking technique. Results showed that this enzyme mainly interacted with the β-lactam ring of these CPs, and the key contacting amino acids were Ser80 and Ser228. The AmpC β-lactamase was combined with 5 horseradish peroxidase-labeled conjugates to develop a direct competitive array on a microplate for determination of 15 drugs in milk. Due to the use of principal component analysis method to analyze the data, this method could discriminate the 15 drugs at the concentration as low as 10 ng/mL. The detection results for the unknown milk samples were consistent with those obtained by the liquid chromatography-mass spectrometry method. As a general comparison, this method is better than the previous antibody-based and receptor-based detection methods for CPs. This is the first paper reporting a competitive array for discriminative determination of a class of small-molecule substances.

Mycobacteria are intrinsically resistant to beta-lactams as they possess several putative penicillin-interactive enzymes (PIEs), some of those are with dual-activity, namely DD-carboxypeptidase and beta-lactamase. Here, with help of molecular approaches, we elucidated the nature of one such putative PIE, MSMEG_1586, in Mycobacterium smegmatis. The in vivo expression of the membrane-bound form of MSMEG_1586 enhanced the beta-lactam resistance of a beta-lactamase deleted host E. coli strain (AM1OC), particularly for aztreonam (eight-fold) and cephalosporins (8-16 fold). To understand the reason for such elevation of resistance, soluble-form of MSMEG_1586 (sMSMEG_1586) was created by removing signal peptides and partially eliminating the amphipathic helix, and finally, expressed and purified. The purified sMSMEG_1586 was active and manifested a strong penicillin-binding affinity as shown by its ability to bind to fluorescent penicillin (Bocillin-FL). Interestingly, the steady-state kinetics apparently confirmed the hydrolytic ability of sMSMEG_1586 towards cefotaxime and aztreonam where hydrolysing aztreonam is a unique and rare behaviour among the beta-lactamases. However, sMSMEG_1586 was devoid of exerting DD-carboxypeptidase like activity. Finally, in silico analysis of MSMEG_1586 revealed a special folding that resembles class C beta-lactamase, except for the absence of a characteristic R2 loop. Overall, MSMEG_1586 could be categorized as a cephalosporinase with the ability to hydrolyse aztreonam., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The structure and mechanism of the bacterial enzyme β-lactamase have been well-studied due to its clinical role in antibiotic resistance. β-Lactamase is known to hydrolyze the β-lactam ring of the cephalosporin scaffold, allowing a spontaneous self-immolation to occur. Previously, cephalosporin-based sensors have been developed to evaluate β-lactamase expression in both mammalian cells and zebrafish embryos. Here, we present a circular caged morpholino oligonucleotide (cMO) activated by β-lactamase-mediated cleavage of a cephalosporin motif capable of silencing the expression of T-box transcription factor Ta ( tbxta ), also referred to as no tail a ( ntla ), eliciting a distinct, observable phenotype. We explore the use of β-lactamase to elicit a biological response in aquatic embryos for the first time and expand the utility of cephalosporin as a cleavable linker beyond targeting antibiotic-resistant bacteria. The addition of β-lactamase to the current suite of enzymatic triggers presents unique opportunities for robust, orthogonal control over endogenous gene expression in a spatially resolved manner.

The β-lactamase BlaC conveys resistance to a broad spectrum of β-lactam antibiotics to its host Mycobacterium tuberculosis but poorly hydrolyzes third-generation cephalosporins, such as ceftazidime. Variants of other β-lactamases have been reported to gain activity against ceftazidime at the cost of the native activity. To understand this trade-off, laboratory evolution was performed, screening for enhanced ceftazidime activity. The variant BlaC Pro167Ser shows faster breakdown of ceftazidime, poor hydrolysis of ampicillin and only moderately reduced activity against nitrocefin. NMR spectroscopy, crystallography and kinetic assays demonstrate that the resting state of BlaC P167S exists in an open and a closed state. The open state is more active in the hydrolysis of ceftazidime. In this state the catalytic residue Glu166, generally believed to be involved in the activation of the water molecule required for deacylation, is rotated away from the active site, suggesting it plays no role in the hydrolysis of ceftazidime. In the closed state, deacylation of the BlaC-ceftazidime adduct is slow, while hydrolysis of nitrocefin, which requires the presence of Glu166 in the active site, is barely affected, providing a structural explanation for the trade-off in activities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

CTX-M β-lactamases are a widespread source of resistance to β-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 β-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A β-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both k cat and k cat /K M . A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only k cat /K M . Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Following the invasion of eukaryotic cells, Salmonella enterica serovar Typhimurium replaces PBP2/PBP3, main targets of β-lactam antibiotics, with PBP2SAL/PBP3SAL, two homologue peptidoglycan synthases absent in Escherichia coli. PBP3SAL promotes pathogen cell division in acidic environments independently of PBP3 and shows low affinity for β-lactams that bind to PBP3 such as aztreonam, cefepime, cefotaxime, ceftazidime, ceftriaxone, cefuroxime and cefalotin., Objectives: To find compounds with high affinity for PBP3SAL to control Salmonella intracellular infections., Methods: An S. Typhimurium ΔPBP3 mutant that divides using PBP3SAL and its parental wild-type strain, were exposed to a library of 1520 approved drugs in acidified (pH 4.6) nutrient-rich LB medium. Changes in optical density associated with cell filamentation, a read-out of blockage in cell division, were monitored. Compounds causing filamentation in the ΔPBP3 mutant but not in wild-type strain-the latter strain expressing both PBP3 and PBP3SAL in LB pH 4.6-were selected for further study. The bactericidal effect due to PBP3SAL inhibition was evaluated in vitro using a bacterial infection model of cultured fibroblasts., Results: The cephalosporin cefotiam exhibited higher affinity for PBP3SAL than for PBP3 in bacteria growing in acidified LB pH 4.6 medium. Cefotiam also proved to be effective against intracellular Salmonella in a PBP3SAL-dependent manner. Conversely, cefuroxime, which has higher affinity for PBP3, showed decreased effectiveness in killing intracellular Salmonella., Conclusions: Antibiotics with affinity for PBP3SAL, like the cephalosporin cefotiam, have therapeutic value for treating Salmonella intracellular infections., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

The mechanism(s) of acquisition of extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESCRE) on inpatient hospital units dedicated to hematopoietic stem cell transplantation (HSCT) is unclear. The objectives of this study were to determine whether ESCRE organisms are transmitted among patients housed on a HSCT unit, clarify the mechanisms involved, and determine whether routine surveillance for ESCRE carriage and contact isolation for ESCRE carriers is beneficial. The study was conducted on a 30-bed inpatient unit dedicated to the care of patients with hematologic malignancies and HSCT recipients. To investigate whether ESCRE organisms may be transmitted vertically to subsequent room occupants, presumably through contamination of room surfaces, we (1) cultured 6 high touch areas in 10 rooms before and 9 rooms after terminal cleaning that had been occupied by patients with ESCRE carriage, (2) determined the in vitro survivals of our most common clinical ESCRE species, and (3) followed the subsequent room occupants of 54 consecutive ESCRE colonized patients for the development of inpatient acquired ESCRE carriage. To investigate whether ESCRE organisms are transmitted horizontally among inpatients we (1) sequenced 60 available ESCRE Escherichia coli isolates obtained from unit inpatients and searched for identities using complete-genome multisequence locus typing (cgMLST) and (2) retrospectively tabulated the cumulative rates of acquired ESCRE carriage in 356 patients admitted for a first HSCT before (200 patients) or after (156 patients) institution of universal ESCRE stool surveillance and contact isolation for carriers. No ESCRE organisms were cultured from patient rooms before or after terminal cleaning. In vitro, few, if any, ESCRE organisms survived longer than 2 hours. Nine of the subsequent occupants of a room in which a patient with ESCRE carriage had resided were detected with ESCRE carriage, only 2 of whom carried the same species as that of the prior occupant. DNA sequencing and cgMLST determination of the 60 E. coli isolates showed 53 cgMLST strains. Seven of the 53 strains were shared by 2 patients. After institution of universal ESCRE surveillance/isolation there was a significant decline in acquired ESCRE carriage among HSCT recipients. We conclude that vertical transmission of ESCRE organisms through room contamination appears to be uncommon on modern HSCT units. Conversely, our results are consistent with the horizontal spread of ESCRE organisms, probably mediated by intermediate vectors such as personnel or shared equipment. Further studies are needed to better define the magnitude of and risk factors for ESCRE horizontal transfers and the benefits of ESCRE surveillance/isolation., (Copyright © 2022 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

The addition of exogenous polyamines increases the production of antibiotic cephalosporin C (CPC) in Acremonium chrysogenum high-yielding (HY) strain during fermentation on a complex medium. However, the molecular basis of this phenomenon is still unknown. In the current study, we developed a special synthetic medium on which we revealed the opposite effect of polyamines. The addition of 1,3-diaminopropane resulted in an increase in the yield of CPC by 12-15%. However, the addition of spermidine resulted in a decrease in the yield of CPC by 14-15% and accumulation of its metabolic pathway precursor, deacetylcephalosporin C (DAC); the total amount of cephems (DAC and CPC) was the same as after the addition of DAP. This indicates that spermidine, but not 1,3-diaminopropane, affects the final stage of CPC biosynthesis, associated with the acetylation of its precursor. In both cases, upregulation of biosynthetic genes from beta-lactam BGCs occurred at the same level as compared to the control; expression of transport genes was at the control level. The opposite effect may be due to the fact that N 1 -acetylation is much more efficient during spermidine catabolism than for 1,3-diaminopropane. The addition of spermidine, but not 1,3-diaminopropane, depleted the pool of acetyl coenzyme A by more than two-fold compared to control, which could lead to the accumulation of DAC.

Iron is an essential element for survival of most organisms. One mechanism of host defense is to tightly chelate iron to several proteins to limit its extracellular availability. This has forced pathogens such as Acinetobacter baumannii to adapt mechanisms for the acquisition and utilization of iron even in iron-limiting conditions. A. baumannii uses a variety of iron acquisition strategies to meet its iron requirements. It can lyse erythrocytes to harvest the heme molecules, use iron-chelating siderophores, and use outer membrane vesicles to acquire iron. Iron acquisition pathways, in general, have been seen to affect many other virulence factors such as cell adherence, cell motility, and biofilm formation. The knowledge gained from research on iron acquisition led to the synthesis of the antibiotic cefiderocol, which uses iron uptake pathways for entry into the cell with some success as a novel cephalosporin. Understanding the mechanisms of iron acquisition of A. baumannii allows for insight into clinical infections and offer potential targets for novel antibiotics or potentiators of current drugs.

Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired mechanisms of resistance to commonly used antibiotics, including cephalosporins. The transmembrane serine/threonine PASTA kinase, IreK, is an important determinant of enterococcal cephalosporin resistance. Upon exposure to cephalosporins, IreK becomes autophosphorylated, which stimulates its kinase activity to phosphorylate downstream substrates and drive cephalosporin resistance. However, the molecular mechanisms that modulate IreK autophosphorylation in response to cell wall stress, such as that induced by cephalosporins, remain unknown. A cytoplasmic protein, GpsB, promotes signaling by PASTA kinase homologs in other bacterial species, but the function of enterococcal GpsB has not been previously investigated. We used in vitro and in vivo approaches to test the hypothesis that enterococcal GpsB promotes IreK signaling in response to cephalosporins to drive cephalosporin resistance. We found that GpsB promotes IreK activity both in vivo and in vitro . This effect is required for cephalosporins to trigger IreK autophosphorylation and activation of an IreK-dependent signaling pathway, and thereby is also required for enterococcal intrinsic cephalosporin resistance. Moreover, analyses of GpsB mutants and a Δ ireK gpsB double mutant suggest that GpsB has an additional function, beyond regulation of IreK activity, which is required for optimal growth and full cephalosporin resistance. Collectively, our data provide new insights into the mechanism of signal transduction by the PASTA kinase IreK and the mechanism of enterococcal intrinsic cephalosporin resistance. IMPORTANCE Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired resistance to commonly used antibiotics. In particular, enterococci are intrinsically resistant to cephalosporin antibiotics, a trait that requires the activity of a transmembrane serine/threonine kinase, IreK, which belongs to the bacterial PASTA kinase family. The mechanisms by which PASTA kinases are regulated in cells are poorly understood. Here, we report that the cytoplasmic protein GpsB directly promotes IreK signaling in enterococci to drive cephalosporin resistance. Thus, we provide new insights into PASTA kinase regulation and control of enterococcal cephalosporin resistance, and suggest that GpsB could be a promising target for new therapeutics to disable cephalosporin resistance.

Objective: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum., Result: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed., Conclusion: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Klebsiella pneumoniae is an opportunistic bacterium that causes many infections, including septicemia, pneumonia, urinary tract infection, and liver abscesses. There are many mechanisms for antibiotic resistance and K. pneumonia is considered a multidrug-resistant pathogen. This study aimed to find the correlation between the susceptibility of K. pneumonia to certain antibiotics with the porin-related resistance and pumps mechanisms. In total, two genes that are responsible for porin formation were considered in the current study OmpK-35 gene and OmpK-36 gene, in addition to other four genes ( CfiaS , CfiaL , MFS , and MdtK genes) related to an efflux pump mechanism of antibiotic resistance. The bacterial resistance was investigated towards five cephalosporins (Cefazolin, Cefoxitin, Ceftazidime, Ceftriaxone, and Cefepime) and two carbapenems (imipenem and ertapenem). Clinical samples, including blood, swabs, and urine, consisting of 20 specimens for each group, were collected from patients who attended three hospitals in Baghdad. The VITEK-2 system and genetic tests (polymerase chain reaction and sequencing) of bacterial isolates were applied to confirm the diagnosis of K. pneumoniae and detect the antibiotic sensitivity profile. The results showed that 51 (85%) and 15 (25%) of the total 60 isolates had positive results for OmpK-35 and Omp-K36 genes, respectively. The MFS and MdtK genes were observed (70-88.3%) in cephalosporin-resistant isolates of K. pneumoniae. There were no significant variations of bacterial resistance genes of antibiotics within the specimen groups. It was concluded that the bacterial resistance of the selected antibiotics was elevated markedly with the loss of the OmpK-36 gene with a high expression of MFS and MdtK genes and a slight minimal occurrence in the new generation of carbapenems. The best antimicrobial agent was ertapenem with a percentage of 0% of resistance in all bacterial isolates., Competing Interests: The authors declare that they have no conflict of interest.

Cephalosporins are commonly prescribed antibiotics that impair cross-linking of the bacterial cell wall. The Gram-positive opportunistic pathogen, Enterococcus faecalis, is intrinsically resistant to these antibiotics and proliferates substantially during cephalosporin therapy. As a result, the usage of cephalosporins has the potential to lead to life-threatening enterococcal infections. Yet, the molecular mechanisms that drive cephalosporin resistance (CR) are incompletely understood. Previously, we demonstrated that MurAA, an enzyme that catalyzes the first committed step in peptidoglycan (PG) synthesis, is required for CR. However, the mechanism by which MurAA contributes to CR remained unknown. Here, we tested the hypothesis that MurAA drives CR by controlling metabolic flux through the PG synthesis pathway. To do so, we developed and exploited an inducible gene expression system for E. faecalis based on an interspecies chimeric receptor that responds to exogenous nitrate for control of expression from a NisR-regulated promoter (P nisA ). We used this tool to demonstrate synthetic lethality of MurAA with its homolog MurAB, to titrate expression of MurAA, and to conditionally deplete multiple PG synthesis enzymes downstream of MurAA that are predicted to be essential. These genetic manipulations, in addition to pharmacological inhibition of the PG synthesis pathway, all led to reductions in PG synthesis that correlated with reductions in CR. Our findings are consistent with a model in which control of metabolic flux through the PG synthesis pathway is a major driver of CR. IMPORTANCE Enterococci are dangerous opportunistic pathogens with the potential to cause life-threatening infections due in part to their intrinsic resistance to cephalosporin antibiotics. Elucidating the molecular mechanisms that provide this resistance is critical for the development of strategies to both prevent and treat enterococcal infections. Here, we report that the cell wall synthesis enzyme, MurAA, drives cephalosporin resistance at least in part by controlling metabolic flux through the peptidoglycan synthesis pathway. To demonstrate this, we designed and validated an inducible gene expression system based on a chimeric receptor that is functional in multiple lineages of E. faecalis. In doing so, we provided a new tool for inducible gene expression with broad applications beyond our studies, including studies of essential genes.

Cephalosporin C (CPC) production is often accompanied by a typical morphological differentiation of Acremonium chrysogenum, involving the fragmentation of its hyphae into arthrospores. The type I integral plasma membrane protein Axl2 is a central component of the bud site selection system (BSSS), which was identified as the regulatory factor involved in the hyphal septation process and arthrospore formation. Using CRISPR/Cas9 technology and homologous recombination (HR), we inserted an egfp donor DNA sequence into the Acaxl2 locus, causing the generation of the deletion strain Ac-ΔAcaxl2:eGFP from Acremonium chrysogenum FC 3 -5-23, the industrial producer of CPC. The mycelial morphology of the deletion strain Ac-ΔAcaxl2:eGFP was mainly composed of arthrospores with a characteristic diameter of 2-8 µm, which increased from 75% at 48 h to 90% at 72 h post culture and were maintained until the end of the fermentation process. However, the deletion strain showed accelerated production of CPC, and the final titer was 5573 μg/ml, which was nearly three times higher than that of the control strain FC 3 -5-23. The up-regulation of genes related to the biosynthesis gene cluster in Ac-ΔAcaxl2:eGFP, especially the "late" genes, was one reason why its CPC production was higher than that of the original strain. Furthermore, compared with FC 3 -5-23, the more significant increase of genes involved in the BSSS (Acbud3 and Acbud4) in Ac-ΔAcaxl2:eGFP in the late stage of fermentation, may be responsible for this increase in arthrospore formation. Similarily, the transcription of the regulatory factors AcFKH1 and CPCR1 were also markedly increased at this time and may be the factors responsible for the regulation of CPC synthesis. These results indicated that Acaxl2 plays an important role in both arthrospore formation and CPC production, strongly implicating these regulatory factors as having pivotal links between mycelial morphology and secondary metabolite production in high-yielding A. chrysogenum. To the opposite, the axl2 gene knockout of wild strain CGMCC 3.3795 did not significantly influence the CPC production, which reflected the complexity of the secondary metabolic process and the differences in the function of axl2 gene in high- and low-yielding strains., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Penicillins and cephalosporins are the most important class of beta (β) lactam antibiotics, accounting for 65% total antibiotic market. Penicillins are produced by Penicillium rubens (popularly known as P. chrysogenum) were used to synthesize the active pharmaceutical intermediate (API), 6-aminopenicillinic acid (6-APA) employed in semisynthetic antibiotic production. The wild strains produce a negligible amount of penicillin (Pen). High antibiotic titre-producing P. chrysogenum strains are necessitating for industrial Pen production to meet global demand at lower prices. Classical strain improvement (CSI) approaches such as random mutagenesis, medium engineering, and fermentation are the cornerstones for high-titer Pen production. Since, Sir Alexander Fleming Discovery of Pen, great efforts are expanded to develop at a commercial scale antibiotics producing strains. Breakthroughs in genetic engineering, heterologous expression and CRISPR/Cas9 genome editing tools opened a new window for Pen production at a commercial scale to assure health crisis. The current state of knowledge, limitations of CSI and genetic engineering approaches to Pen production are discussed in this review., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp 4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp 4 genes were sequenced and pbp 4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log 10 reduction) at the different concentrations tested. pbp 4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine ( del A) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424 STFK 427 motif I; L475Q, 7 aa upstream of the 482 SDN 484 motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619 KTGT 622 motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp 4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs - which might interfere with the formation of the BPR/PBP4 complex. pbp 4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lazzaro, Cassisi, Stefani and Campanile.)

Racemic camphor and isoborneol are readily available as industrial side products, whereas (1R)-camphor is available from natural sources. Optically pure (1S)-camphor, however, is much more difficult to obtain. The synthesis of racemic camphor from α-pinene proceeds via an intermediary racemic isobornyl ester, which is then hydrolyzed and oxidized to give camphor. We reasoned that enantioselective hydrolysis of isobornyl esters would give facile access to optically pure isoborneol and camphor isomers, respectively. While screening of a set of commercial lipases and esterases in the kinetic resolution of racemic monoterpenols did not lead to the identification of any enantioselective enzymes, the cephalosporin Esterase B from Burkholderia gladioli (EstB) and Esterase C (EstC) from Rhodococcus rhodochrous showed outstanding enantioselectivity (E>100) towards the butyryl esters of isoborneol, borneol and fenchol. The enantioselectivity was higher with increasing chain length of the acyl moiety of the substrate. The kinetic resolution of isobornyl butyrate can be easily integrated into the production of camphor from α-pinene and thus allows the facile synthesis of optically pure monoterpenols from a renewable side-product., (© 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

An increase in the number of Salmonella enterica serovar Saintpaul observed in Singapore in 2015-2016 in humans was accompanied by increased resistance to third generation cephalosporins. We aimed to understand the genetic mechanisms contributing to this resistance. Whole genome sequencing using MiSeq was performed on 49 S. Saintpaul isolates collected between 2014-2016. Nanopore sequencing was also performed in an attempt to obtain a full genome of the plasmids. All but one S. Saintpaul isolates sequenced belonged to a single sequence type based on an in silico 7-gene multi-locus sequence typing scheme suggesting a clonal lineage. In total 27/49 were resistant to third generation cephalosporins as confirmed by the broth microdilution method; the resistance was due to the presence of either bla CTX-M-55 (n=23), bla CTX-M-27 (n=1) or bla CMY-2 (n=3) carried on a plasmid. Two isolates were also found to carry the mcr-1 gene on a different plasmid. Our study showed that all S. Saintpaul isolates resistant to third generation cephalosporins carried either bla CTX-M-55 , bla CTX-M-27 or bla CMY-2 on a plasmid. Continuous monitoring of Salmonella serovars is warranted to track the potential spread of these plasmids., (Copyright © 2021 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Aims: Many antibiotics derived from mold metabolites have been found to possess anticarcinogenic properties. We aimed to investigate whether they may elicit anticancer activity, especially against nasopharyngeal carcinoma., Main Methods: The response of nasopharyngeal and other carcinoma cell lines to cephalosporin antibiotics was evaluated in vitro and in vivo. MTT and clonogenic colony formation assays assessed the viability and proliferation of cultured cells. Flow cytometry was used to assess cell cycle parameters and apoptotic markers. Tumor growth was determined using a xenograft model in vivo. Microarray and RT-qPCR expression analyses investigate differential gene expression. Mechanistic assessment of HMOX1 in cefotaxime-mediated ferroptosis was tested with Protoporphyrin IX zinc., Key Findings: Cephalosporin antibiotics showed highly specific and selective anticancer activity on nasopharyngeal carcinoma CNE2 cells both in vitro and vivo with minimal toxicity. Cefotaxime sodium significantly regulated 11 anticancer relevant genes in CNE2 cells in a concentration-dependent manner. Pathway analyses indicate apoptotic and the ErbB-MAPK-p53 signaling pathways are significantly enriched. HMOX1 represents the top one ranked upregulated gene by COS and overlaps with 16 of 42 enriched apoptotic signaling pathways. Inhibition of HMOX1 significantly reduced the anticancer efficacy of cefotaxime in CNE2 cells., Significance: Our discovery is the first to highlight the off-label potential of cephalosporin antibiotics as a specific and selective anticancer drug for nasopharyngeal carcinoma. We mechanistically show that induction of ferroptosis through HMOX1 induction mediates cefotaxime anticancer activity. Our findings provide an alternative treatment for nasopharyngeal carcinoma by showing that existing cephalosporin antibiotics are specific and selective anticancer drugs., (Copyright © 2021. Published by Elsevier Inc.)

Background: To investigate the trends and correlation between antibacterial consumption and carbapenem resistance in Gram-negative bacteria from 2012 to 2019 in a tertiary-care teaching hospital in southern China., Methods: This retrospective study included data from hospital-wide inpatients collected between January 2012 and December 2019. Data on antibacterial consumption were expressed as defined daily doses (DDDs)/1000 patient-days. Antibacterials were classified according to the Anatomical Therapeutic Chemical (ATC) classification system. The trends in antimicrobial usage and resistance were analyzed by linear regression, while Pearson correlation analysis was used for assessing correlations., Results: An increasing trend in the annual consumption of tetracyclines, β-lactam/β-lactamase inhibitor (BL/BLI) combinations, and carbapenems was observed (P < 0.05). Carbapenem resistance in Acinetobacter baumannii (A. baumannii) significantly increased (P < 0.05) from 18% in 2012 to 60% in 2019. Moreover, significant positive correlations were found between resistance to carbapenems in A. baumannii (P < 0.05) and Escherichia coli (E. coli; P < 0.05) and consumption of carbapenems, while the resistance rate of A. baumannii to carbapenems was positively correlated with cephalosporin/β-lactamase inhibitor (C/BLI) combinations (P < 0.01) and tetracyclines usage (P < 0.05). We also found that use of quinolones was positively correlated with the resistance rate of Burkholderia cepacia (B. cepacia) to carbapenems (P < 0.05), and increasing uses of carbapenems (P < 0.01) and penicillin/β-Lactamase inhibitor (P/BLI) combinations (P < 0.01) were significantly correlated with reduced resistance of Enterobacter cloacae (E. cloacae) to carbapenems., Conclusion: These results revealed significant correlations between consumption of antibiotics and carbapenem resistance rates in Gram-negative bacteria. Implementing proper management strategies and reducing the unreasonable use of antibacterial drugs may be an effective measure to reduce the spread of carbapenem-resistant Gram-negative bacteria (CRGN), which should be confirmed by further studies.

Engineering active sites in inert scaffolds to catalyze chemical transformations with unnatural substrates is still a great challenge for enzyme catalysis. In this research, a p-nitrobenzyl esterase from Bacillus subtilis was identified from the structural database, and a double mutant E115A/E188A was designed to afford catalytic activities toward the hydrolysis of ceftizoxime. A quadruple mutant E115A/E188A/L362S/I270A with enhanced catalytic efficiency was created to catalyze the condensation reaction of ethyl-2-methoxy-amino-2-(2-aminothiazole-4-yl) acetate with 7-amino-3-nor-cephalosporanic acid to produce ceftizoxime in a fully aqueous medium. The catalytic efficiencies of the computationally designed mutants E115A/E188A/L362S/I270A and E115A/Y118 K/E188 V/I270A/L362S can be taken as starting points to further improve their properties towards the practical application in designing more ecology-friendly production of third-generation cephalosporins., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The filamentous fungus Acremonium chrysogenum is the main industrial producer of cephalosporin C (CPC), one of the major precursors for manufacturing of cephalosporin antibiotics. The plasma membrane H+-ATPase (PMA) plays a key role in numerous fungal physiological processes. Previously we observed a decrease of PMA activity in A. chrysogenum overproducing strain RNCM 408D (HY) as compared to the level the wild-type strain A. chrysogenum ATCC 11550. Here we report the relationship between PMA activity and CPC biosynthesis in A. chrysogenum strains. The elevation of PMA activity in HY strain through overexpression of PMA1 from Saccharomyces cerevisiae, under the control of the constitutive gpdA promoter from Aspergillus nidulans, results in a 1.2 to 10-fold decrease in CPC production, shift in beta-lactam intermediates content, and is accompanied by the decrease in cef genes expression in the fermentation process; the characteristic colony morphology on agar media is also changed. The level of PMA activity in A. chrysogenum HY OE::PMA1 strains has been increased by 50-100%, up to the level observed in WT strain, and was interrelated with ATP consumption; the more PMA activity is elevated, the more ATP level is depleted. The reduced PMA activity in A. chrysogenum HY strain may be one of the selected events during classical strain improvement, aimed at elevating the ATP content available for CPC production., Competing Interests: The authors have declared that no competing interests exist.

The conjugation of organometallic groups to current β-lactam antibiotics is a field of increasing study due to the ability of certain organometallic groups to enhance the antibiotic potency of these drugs. Herein, we report the antibacterial properties of two metallocenyl (ferrocenyl and ruthenocenyl) 7-aminocephalosporanic acid (7-ACA) antibiotic conjugates. Continuing a trend we found in our previous studies, the ruthenocenyl conjugate showed greater antibacterial activity than its ferrocenyl counterpart. Compared with the previously published 7-aminodesacetoxycephalosporanic acid (7-ADCA) conjugates, the 3-acetyloxymethyl group significantly improved the compounds' activity. Furthermore, the Rc-7-ACA compound was more active against clinical Staphylococcus aureus isolates than the ampicillin reference. Noticeably, neither of the two new compounds showed an undesirable toxic effect in HeLa and L929 cells at the concentrations at which they displayed strong antibacterial effects. The antibacterial activity of the two metallocenyl 7-ACA derivatives was further confirmed by scanning electron microscopy (SEM). SEM micrographs showed that bacteria treated with metallocenyl 7-ACA derivatives feature cell wall damage and morphology changes. Using a CTX-M-14 β-lactamase competition assay based on nitrocefin hydrolysis, we showed that the Rc-7-ACA bound more favorably to CTX-M-14 than its ferrocenyl counterpart, again confirming the superiority of the ruthenocenyl moiety over the ferrocenyl one in interacting with proteins. We also report a 1.47 Å resolution crystal structure of Rc-7-ACA in complex with the CTX-M-14 E166A mutant, an enzyme sharing a similar active site configuration with penicillin-binding proteins, the molecular target of β-lactam antibiotics. These results strengthen the case for the antibacterial utility of the Rc and Fc groups., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels into chemically complex natural products is particularly important for techniques such as NMR, IR, mass spectrometry, imaging, and kinetic isotope effects. Toward this goal, we developed a biosynthetic strategy for the site-specific incorporation of 13 C labels into the canonical β-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin C. This was achieved through sulfur-replacement with 1- 13 C-l-cysteine, resulting in high isotope incorporations and milligram-scale yields. Using 13 C NMR and isotope-edited IR difference spectroscopy, we illustrate how these molecules can be used to interrogate interactions with their protein targets, e.g., TEM-1 β-lactamase. This method provides a feasible route to isotopically labeled penicillin and cephalosporin precursors for future biophysical studies.

Ceftobiprole is an advanced-generation broad-spectrum cephalosporin antibiotic with potent and rapid bactericidal activity against Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus , as well as susceptible Gram-negative pathogens, including Pseudomonas sp. pathogens. In the case of Pseudomonas aeruginosa , ceftobiprole acts by inhibiting P. aeruginosa penicillin-binding protein 3 (PBP3). Structural studies were pursued to elucidate the molecular details of this PBP inhibition. The crystal structure of the His-tagged PBP3-ceftobiprole complex revealed a covalent bond between the ligand and the catalytic residue S294. Ceftobiprole binding leads to large active site changes near binding sites for the pyrrolidinone and pyrrolidine rings. The S528 to L536 region adopts a conformation previously not observed in PBP3, including partial unwinding of the α11 helix. These molecular insights can lead to a deeper understanding of β-lactam-PBP interactions that result in major changes in protein structure, as well as suggesting how to fine-tune current inhibitors and to develop novel inhibitors of this PBP., (Copyright © 2020 American Society for Microbiology.)

Deacetyl-7-aminocephalosporanic acid (D-7-ACA) is required for producing of many semisynthetic β-lactam antibiotics; therefore, enzymes capable of converting 7-aminocephalosporanic acid (7-ACA) to D-7-ACA present a valuable resource to the pharmaceutical industry. In the present study, a putative acetylesterase, EstZY, was identified and characterized from a thermophilic bacterium Alicyclobacillus tengchongensis. Sequence alignment showed that EstZY was an acetylesterase which belonged to carbohydrate esterase family 7 (CE7), with substrate preference for short-chain acyl esters p-NPC 2 to p-NPC 8 . Maximum enzyme activity was recorded at pH 9.0 and 50 °C, where K m and V max were calculated as 1.9 ± 0.23 mM and 258 ± 18.5 μM min - 1 , respectively. The residues Ser185, Asp274, and His303 were identified as the putative catalytic triad by homology modelling, site-directed mutagenesis and molecular docking. Moreover, EstZY can remove the acetyl group at C3' position of 7-ACA to form D-7-ACA; this is the first report of a 7-ACA deacetylase from CE7 family in A. tengchongensis and may represent a new enzyme with industrial values., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Infections caused by Mycobacterium abscessus are increasing in prevalence in cystic fibrosis patients. This opportunistic pathogen's intrinsic resistance to most antibiotics has perpetuated an urgent demand for new, more effective therapeutic interventions. Here we report a prospective advance in the treatment of M. abscessus infection; increasing the susceptibility of the organism to amoxicillin, by repurposing the β-lactamase inhibitor, relebactam, in combination with the front line M. abscessus drug imipenem. We establish by multiple in vitro methods that this combination works synergistically to inhibit M. abscessus. We also show the direct competitive inhibition of the M. abscessus β-lactamase, Bla Mab , using a novel assay, which is validated kinetically using the nitrocefin reporter assay and in silico binding studies. Furthermore, we reverse the susceptibility by overexpressing Bla Mab in M. abscessus, demonstrating relebactam-Bla Mab target engagement. Finally, we highlight the in vitro efficacy of this combination against a panel of M. abscessus clinical isolates, revealing the therapeutic potential of the amoxicillin-imipenem-relebactam combination.

In present work, the degradation of antibiotic and inactivation of antibiotic resistance genes (ARGs) in cephalosporin C fermentation (CEPF) residues were performed using ionizing radiation, ozonation and thermal treatment. The results showed that the three treatment methods could degrade cephalosporin C effectively, with the removal efficiency of 85.5% for radiation at dose of 100 kGy, 79.9% for ozonation at dosage of 5.2 g O 3 /L, and 71.9% and 87.3% for thermal treatment at 60 °C and 90 °C for 4 h. The cephalosporin resistance gene tolC was detected in the raw CEPF residues, and its abundance was decrease 74.2% by radiation, 64.6% by ozonation and 26.9%-37.1% by thermal treatment respectively. The presence of protein, glucose and acetate in the CEPF residues had inhibitive influence on the degradation of cephalosporin C by ionizing radiation, and the effect was more significant when the antibiotic concentration was lower. The total content of COD, polysaccharides and protein changed slightly after radiation and thermal treatment, while they were decreased greatly by ozonation. The primary techno-economic analysis showed that the operational cost of ionizing radiation by electron beam at 50 kGy ($5.2/m 3 ) was comparable to thermal treatment ($4.3-7.9/m 3 ), which was more economical than ozonation ($14.6/m 3 )., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cephalosporin C acylase (CCA) is the key enzyme in the production of 7-aminocephalosporanic acid (7-ACA) via a one-step enzymatic process. To improve the soluble expression level of CCA in recombinant Escherichia coli at elevated temperatures, a library of T7 promoter mutants was created by site-saturation mutagenesis, and a series of mutated promoters were subsequently screened. Green fluorescent protein (GFP) was fused to the C-terminus of CCA to facilitate library screening, and the expression of the CCA and GFP fusion proteins was investigated under the control of the T7 promoter. Twenty-four mutants were selected by detecting the fluorescence intensity of colonies on agar plates to form a library with different expression levels. The enzyme activities of the mutants were positively correlated with their fluorescence intensities. The highest enzyme activity among these mutant promoters was 1.3-fold higher than the enzyme activity resulting from the wild-type promoter when the cells were cultured at 32 °C for 16 h. In addition, the transcription and expression levels of several typical promoters were discussed, and the effects of GFP fusion on the enzyme activity of CCA were investigated.

A series of substituted di-indolglyoxylamido-spermine analogues were prepared and evaluated for intrinsic antimicrobial properties and the ability to enhance antibiotic action. As a compound class, intrinsic activity was typically observed towards Gram-positive bacteria and the fungus Cryptococcus neoformans, with notable exceptions being the 5-bromo- and 6-chloro-indole analogues which also exhibited modest activity (MIC 34-50 μM) towards the Gram-negative bacteria Escherichia coli and Klebsiella pneumoniae. Several analogues enhanced the activity of doxycycline towards the Gram-negative bacteria Pseudomonas aeruginosa, E. coli, K. pneumoniae and Acinetobacter baumannii. Of particular note was the identification of five antibiotic enhancing analogues (5-Br, 7-F, 5-Me, 7-Me, 7-OMe) which also exhibited low to no cytotoxicity and red blood cell haemolytic properties. The mechanisms of action of the 5-Br and 7-F analogues were attributed to the ability to disrupt the integrity of, and depolarize, bacterial membranes., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Enzyme immobilization is widely used for large-scale industrial applications. However, the weak absorption through physical methods limits the recovery ability. Here, affinity-binding immobilization of enzymes was explored using a silica-specific affinity peptide (SAP) as a fusion tag to intensify the binding force between the enzyme and mesoporous silica (MPS) carrier. D-amino acid oxidase (DAAO) of Rhodosporidium toruloides was used as a model enzyme. The optimal screened SAP (LPHWHPHSHLQP) was selected from a M13 phage display peptide library and fused to the C-terminal of DAAO to obtain fused DAAOs with one, two and three SAP tags, respectively. The activity of DAAO-SAP-MPS was superior comparing with DAAO-2SAP-MPS and DAAO-3SAP-MPS; meanwhile DAAO-SAP-MPS shows 36% higher activity than that of DAAO-MPS. Fusion with one SAP improved the thermal stability with a 10% activity increase for immobilized DAAO-SAP-MPS compared to that of DAAO-MPS at 50 °C for 3 h. Moreover, the activity recovery of immobilized DAAO-SAP-MPS was 25% higher in operation stability assessment after six-batch conversions of cephalosporin to glutaryl-7-amino cephalosporanic acid than that of DAAO-MPS.

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others.

In a stirred tank reactor, during catalysis with immobilized cephalosporin C acylase (CCA), the microenvironmental pH dropped to 7.2 in a nonbuffered system (with the pH maintained at 8.5 by adding alkali) due to the existence of diffusional resistance. Moreover, the immobilized CCA only catalyzed five batch reactions, suggesting that the sharp pH gradient impaired the enzyme stability. To buffer the protons produced in the hydrolysis of cephalosporin C by CCA, phosphate and bicarbonate buffers were introduced. When CCA was catalyzed with 0.1 M ammonium bicarbonate buffer, no obvious gradient between the bulk solution and intraparticle pH was detected, and the catalysis of 15 batch reactions was achieved. Accordingly, with 0.2 M ammonium bicarbonate buffer in a packed bed reactor, the immobilized CCA exhibited continuous catalysis with high conversion rates (≥95%) for 21 days. Reactions with ammonium bicarbonate buffer showed significant increases in the stability and catalytic efficiency of the immobilized CCA in different reactors compared to those in nonbuffered systems., (© 2019 American Institute of Chemical Engineers.)

Ceftobiprole, a novel last generation parenteral cephalosporin, has an extended spectrum of activity, notably against methicillin-resistant Staphylococcus aureus (MRSA), ampicillin-susceptible enterococci, penicillin-resistant pneumococci, Enterobacterales and susceptible Pseudomonas aeruginosa. It exerts an inhibitory action on essential peptidoglycan transpeptidases, interfering with cell wall synthesis. The inhibitory action of ceftobiprole through binding to abnormal PBPs like PBP2a in methicillin-resistant staphylococci and PBP2b and PBP2x in the case of β-lactam-resistant pneumococci, ultimately leads to rapid bacterial cell death. In the case of Enterobacterales, ceftobiprole retains activity against narrow spectrum β-lactamases but is hydrolysed by their extended-spectrum counterparts, overexpressed Amp C, and carbapenemases. It is also affected by certain efflux pumps from P. aeruginosa. For anaerobic bacteria, ceftobiprole is active against Gram-positive Clostridioides difficile and Peptococcus spp. and Gram-negative Fusobacterium nucleatum but not against Bacteroides group or other anaerobic Gram-negatives. In in vitro studies, a low propensity to select for resistant subpopulations has been demonstrated. Currently, ceftobiprole is approved for the treatment of community-acquired pneumonia and hospital-acquired pneumonia with the exception of ventilator-associated pneumonia. Ceftobiprole's place in therapy appears to lie mainly in its combined activity against Gram-positive organisms, such as S. aureus and S. pneumoniae alongside that against Gram-negative organisms such as P. aeruginosa.

Ceftobiprole shows many similar pharmacokinetic properties to other cephalosporins, except for not being orally bioactive, and that it is administered by IV infusion as the prodrug ceftobiprole medocaril, which is subsequently hydrolyzed in the blood into the active molecule. Distribution focus in extracellular fluid and active antibiotic concentration has been proven in different corporal tissues using dosing regimen of 500 mg intravenous infusion over 2 h every 8 h. Ceftobiprole is eliminated exclusively into the urine, thus the reason why dose adjustment is required for patients with moderate or severe renal impairment, or increased creatinine clearance. However, there is no need for dose adjustments related with other comorbidities and patients' conditions such as age, body weight. Although considering distribution features, molecular weight and dose fraction, increase dosing regimen might be necessary in patients using renal replacement therapy. The half-life of ceftobiprole is more than 3 h, allowing to easily reach optimal PK/PD parameters with the infusion time of 2 h, using the usual dosing regimen.

Objectives: To obtain a new acetyl esterase from Paenibacillus sp. XW-6-66 and apply the enzyme to 7-aminocephalosporanic acid (7-ACA) deacetylation., Results: The acetyl esterase AesZY was identified from Paenibacillus sp. XW-6-66, and its enzymatic properties were investigated. With the putative catalytic triad Ser114-Asp203-His235, AesZY belongs to the Acetyl esterase (Aes) family which is included in the α/β hydrolase superfamily and contains the consensus Gly-X-Ser-X-Gly motif. The maximum activity of AesZY was detected at pH 8.0 and 40 °C. AesZY was stable at different pH values ranging from 5.0 to 12.0, and was tolerant to several metal ions. Furthermore, the deacetylation activity of AesZY toward 7-ACA was approximately 7.5 U/mg, and the K cat /K m value was 2.04 s -1  mM -1 ., Conclusions: Our results demonstrate the characterization of a new acetyl esterase belonging to the Aes family with potential biotechnological applications.

Ceftobiprole is a fifth-generation cephalosporin with potent antimicrobial activity against Gram positive and Gram-negative bacteria. It has been approved in major European countries for the treatment of community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP), excluding ventilator-associated pneumonia (VAP). Ceftobiprole is currently in a phase 3 clinical program for registration in the U.S. In 2015, it was designated as an infectious disease product qualified for the treatment of lung and skin infections by the FDA. The efficacy of ceftobiprole in pneumonia has been demonstrated in two-phase III clinical trials conducted in patients with CAP and HAP. The recommended dose in the adult with pneumonia is 500 mg every 8 h infused in 2 h; in case of renal failure, the regimen of administration must be adjusted according to the patient's renal function. It is not necessary to adjust the dose according to gender, age, body weight or liver failure. In case of hyperfiltration, an extension to 4 h infusion of the 500mg TID is required.

Semisynthetic cephalosporins are widely used antibiotics currently produced by different chemical steps under harsh conditions, which results in a considerable amount of toxic waste. Biocatalytic synthesis by the cephalosporin acylase from Pseudomonas sp. strain N176 is a promising alternative. Despite intensive engineering of the enzyme, the catalytic activity is still too low for a commercially viable process. To identify the bottlenecks which limit the success of protein engineering efforts, a series of MD simulations was performed to study for two acylase variants (WT, M6) the access of the substrate cephalosporin C from the bulk to the active site and the stability of the enzyme-substrate complex. In both variants, cephalosporin C was binding to a non-productive substrate binding site (E86α, S369β, S460β) at the entrance to the binding pocket, preventing substrate access. A second non-productive binding site (G372β, W376β, L457β) was identified within the binding pocket, which competes with the active site for substrate binding. Noteworthy, substrate binding to the protein surface followed a Langmuir model resulting in binding constants K = 7.4 and 9.2 mM for WT and M6, respectively, which were similar to the experimentally determined Michaelis constants K M  = 11.0 and 8.1 mM, respectively.

D-Amino acid oxidase (DAAO) is a valuable flavoenzyme capable of being used in various practical applications, such as in determining D-amino acids and producing a material for semisynthetic cephalosporins, requiring higher thermal stability, higher catalytic activity, and broad substrate specificity. In this study, we isolated the thermophilic fungus Rasamsonia emersonii strain YA, which can grow on several D-amino acids as the sole nitrogen source, from a compost and characterized DAAO (ReDAAO) of the fungus. ReDAAO expressed in Escherichia coli exhibited significant oxidase activity against various neutral and basic D-amino acids, in particular hydrophobic D-amino acids. In addition, the enzyme also significantly acted on cephalosporin C, a starting material for semisynthetic antibiotics, and D-Glu, a general substrate for D-aspartate oxidase but not for DAAO, showing its unique and practically useful substrate specificity. The apparent k cat and K m values of the enzyme toward good substrates were comparable to those of higher catalytic fungal DAAOs, and the thermal stability (T 50 value of ~60 °C) was comparable to that of a thermophilic bacterial DAAO and significantly higher than that of other eukaryotic DAAOs. These results highlight the great potential of ReDAAO for use in practical applications.

Cephalosporins are of particular importance in human medicine and should be reserved for second-line curative treatment in the veterinary field to avoid any emerging antimicrobial resistance. Due to misuse of ceftiofur in the poultry sector in France, it is now recommended to completely stop using cephalosporins in this sector. Methods currently used for the control of veterinary practices are mostly based on liquid chromatography coupled to mass spectrometry in a targeted mode, including parent compounds and any major metabolites. The aim of the present study was to evaluate the relevance of untargeted metabolomic approaches to highlight a possible exposure of laying hens to cephalosporins using a predictive model including selected treatment biomarkers. An experimentation carried out on living animals involved the administration of cefquinome and ceftiofur. Three biological matrices-droppings, eggs and liver-were investigated. Metabolites were extracted and analysed by liquid chromatography coupled to high resolution mass spectrometry in a full scan mode. Metabolites impacted by the treatment were selected by using univariate and multivariate statistical analyses. Predictive models built from the potential biomarkers selected in the "droppings" matrix were validated and able to classify "treated" and "control" hens. PLS-DA and logistic regression models were compared and both models gave satisfactory results in terms of prediction. Results were of less interest for other matrices in which only biomarkers of exposure to cefquinome were detected., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The drug/proton antiporter AcrB, part of the major efflux pump AcrABZ-TolC in Escherichia coli, is characterized by its impressive ability to transport chemically diverse compounds, conferring a multidrug resistance phenotype. However, the molecular features differentiating between good and poor substrates of the pump have yet to be identified. In this work, we combined molecular docking with molecular dynamics simulations to study the interactions between AcrB and two representative cephalosporins, cefepime and ceftazidime (a good and poor substrate of AcrB, respectively). Our analysis revealed different binding preferences of the two compounds toward the subsites of the large deep binding pocket of AcrB. Cefepime, although less hydrophobic than ceftazidime, showed a higher affinity than ceftazidime for the so-called hydrophobic trap, a region known for binding inhibitors and substrates. This supports the hypothesis that surface complementarity between the molecule and AcrB, more than the intrinsic hydrophobicity of the antibiotic, is a feature required for the interaction within this region. Oppositely, the preference of ceftazidime for binding outside the hydrophobic trap might not be optimal for triggering allosteric conformational changes needed to the transporter to accomplish its function. Altogether, our findings could provide valuable information for the design of new antibiotics less susceptible to the efflux mechanism.

Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A β-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined., (Copyright © 2019 Brouwer et al.)

Expression of β-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to β-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a β-lactamase-cleavable motif. The prodrug is only bactericidal after activation by β-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse β-lactamases; bactericidal activity was not observed in strains without β-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target β-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce β-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.

Background: Cefovecin has a long duration of antibiotic activity in cats and dogs, somewhat attributable to its high plasma protein binding., Aims: To determine the cefovecin binding to plasma proteins in vitro in selected Australian marsupials and to quantify the change in cetovecin concentration over time following subcutaneous injection in koalas., Methods and Results: Various cefovecin concentrations were incubated with plasma and quantified using HPLC. The median (range) bound percentages when 10 μg/mL of cefovecin was incubated with plasma were 11.1 (4.1-20.4) in the plasma of the Tasmanian devil, 12.7 (5.8-17.3) in the koala, 18.9 (14.6-38.0) in the eastern grey kangaroo, 16.9 (15.7-30.2) in the common brush-tailed possum, 37.6 (25.3-42.3) in the eastern ring-tailed possum and 36.4 (35.0-38.3) in the red kangaroo, suggesting that cefovecin may have a shorter duration of action in these species than in cats and dogs. Cefovecin binding to plasma proteins in thawed, frozen equine plasma was also undertaken for assay quality control and the median (range) plasma protein binding (at 10 μg/mL) was 95.6% (94.9-96.6%). Cefovecin was also administered to six koalas at 8 mg/kg subcutaneously and serial blood samples were collected at 3, 6, 24, 48, 72, 96 h thereafter. Cefovecin plasma concentrations were not quantifiable in four koalas and in the other two, the mean plasma concentration at t = 3 h was 1.04 ± 0.01 μg/mL., Conclusion: Because of the limited pharmacokinetic data generated, no further pharmacokinetic analysis was performed; however, a single injected bolus of cefovecin is likely to have a short duration of action in koalas (hours, rather than days)., (© 2019 Australian Veterinary Association.)