Design, Synthesis, and Evaluation of Cephamycin-Based Antisporulation Agents targeting Clostridioides difficile.

With the aim of discovering small molecule inhibitors of the sporulation process in Clostridioides difficile, we prepared a series of C-7 α-(4-substituted-1H-1,2,3-triazol-1-yl)acetamide analogues of cefotetan, a known inhibitor of the C. difficile sporulation-specific protein target CdSpoVD. These analogues were evaluated using both in vitro binding assays with CdSpoVD and antisporulation assays against C. difficile. Further design concepts were aided utilizing the predicted docking scores (DS) using both AlphaFold (AF) models, and a crystal structure of the CdSpoVD protein (PDB 7RCZ). Despite being 1 order of magnitude more potent as a sporulation inhibitor than cefotetan, in vivo studies on compound 6a in a murine-model of C. difficile infection demonstrated comparable spore shedding capabilities as cefotetan. Importantly, compound 6a had no concerning broad spectrum antibacterial activities, toxicity, or hemolytic activity and thus has potential for further drug development.

Proteomic analysis of a hom-disrupted, cephamycin C overproducing Streptomyces clavuligerus.

BACKGROUND: Streptomyces clavuligerus is prolific producer of cephamycin C, a medically important antibiotic. In our former study, cephamycin C titer was 2-fold improved by disrupting homoserine dehydrogenase (hom) gene of aspartate pahway in Streptomyces clavuligerus NRRL 3585. OBJECTIVE: In this article, we aimed to provide a comprehensive understanding at the proteome level on potential complex metabolic changes as a consequence of hom disruption in Streptomyces clavuligerus AK39. METHODS: A comparative proteomics study was carried out between the wild type and its hom disrupted AK39 strain by 2 Dimensional Electrophoresis-Matrix Assisted Laser Desorption and Ionization Time-Of-Flight Mass Spectrometry (2DE MALDI-TOF/MS) and Nanoscale Liquid Chromatography- Tandem Mass Spectrometry (nanoLC-MS/MS) analyses. Clusters of Orthologous Groups (COG) database was used to determine the functional categories of the proteins. The theoretical pI and Mw values of the proteins were calculated using Expasy pI/Mw tool. RESULTS: "Hypothetical/Unknown" and "Secondary Metabolism" were the most prominent categories of the differentially expressed proteins. Upto 8.7-fold increased level of the positive regulator CcaR was a key finding since CcaR was shown to bind to cefF promoter thereby direcly controlling its expression. Consistently, CeaS2, the first enzyme of CA biosynthetic pathway, was 3.3- fold elevated. There were also many underrepresented proteins associated with the biosynthesis of several Non-Ribosomal Peptide Synthases (NRPSs), clavams, hybrid NRPS/Polyketide synthases (PKSs) and tunicamycin. The most conspicuously underrepresented protein of amino acid metabolism was 4-Hydroxyphenylpyruvate dioxygenase (HppD) acting in tyrosine catabolism. The levels of a Two Component System (TCS) response regulator containing a CheY-like receiver domain and an HTH DNA-binding domain as well as DNA-binding protein HU were elevated while a TetR-family transcriptional regulator was underexpressed. CONCLUSION: The results obtained herein will aid in finding out new targets for further improvement of cephamycin C production in Streptomyces clavuligerus.

Extended-Spectrum-Beta-Lactamase- and Plasmid-Encoded Cephamycinase-Producing Enterobacteria in the Broiler Hatchery as a Potential Mode of Pseudo-Vertical Transmission.

Antimicrobial resistance through extended-spectrum beta-lactamases (ESBLs) and transferable (plasmid-encoded) cephamycinases (pAmpCs) represents an increasing problem in human and veterinary medicine. The presence of ESBL-/pAmpC-producing commensal enterobacteria in farm animals, such as broiler chickens, is considered one possible source of food contamination and could therefore also be relevant for human colonization. Studies on transmission routes along the broiler production chain showed that 1-day-old hatchlings are already affected. In this study, ESBL-/pAmpC-positive broiler parent flocks and their corresponding eggs, as well as various environmental and air samples from the hatchery, were analyzed. The eggs were investigated concerning ESBL-/pAmpC-producing enterobacteria on the outer eggshell surface (before/after disinfection), the inner eggshell surface, and the egg content. Isolates were analyzed concerning their species, their phylogroup in the case of Escherichia coli strains, the respective resistance genes, and the phenotypical antibiotic resistance. Of the tested eggs, 0.9% (n = 560) were contaminated on their outer shell surface. Further analyses using pulsed-field gel electrophoresis showed a relationship of these strains to those isolated from the corresponding parent flocks, which demonstrates a pseudo-vertical transfer of ESBL-/pAmpC-producing enterobacteria into the hatchery. Resistant enterobacteria were also found in environmental samples from the hatchery, such as dust or surfaces which could pose as a possible contamination source for the hatchlings. All 1-day-old chicks tested negative directly after hatching. The results show a possible entry of ESBL-/pAmpC-producing enterobacteria from the parent flocks into the hatchery; however, the impact of the hatchery on colonization of the hatchlings seems to be low. IMPORTANCE: ESBL-/pAmpC-producing enterobacteria occur frequently in broiler-fattening farms. Recent studies investigated the prevalence and possible transmission route of these bacteria in the broiler production chain. It seemed very likely that the hatcheries play an important role in transmission and/or contamination events. There are only few data on transmission investigations from a grandparent or parent flock to their offspring. However, reliable data on direct or indirect vertical transmission events in the hatchery are not available. Therefore, we conducted our study and intensively investigated the broiler hatching eggs from ESBL-/pAmpC-positive broiler parent flocks as well as the hatchlings and the environment of the hatchery.

Heterologous expression of Streptomyces clavuligerus ATCC 27064 cephamycin C gene cluster.

The Streptomyces clavuligerus cephamycin C gene cluster has been subcloned in a SuperCos-derived cosmid and introduced in Streptomyces flavogriseus ATCC 33331, Streptomyces coelicolor M1146 and Streptomyces albus J1074. The exconjugant strains were supplemented with an additional copy of the S. clavuligerus cephamycin regulatory activator gene, ccaRC, expressed from the constitutive Pfur promoter. Only S. flavogriseus-derived exconjugants produced a compound active against Escherichia coli ESS22-31 that was characterized by HPLC-MS as cephamycin C. The presence of an additional ccaR copy resulted in about 40-fold increase in cephamycin C production. Optimal heterologous cephamycin C production was in the order of 9% in relation to that of S. clavuligerus ATCC 27064. RT-qPCR studies indicated that ccaRC expression in S. flavogriseus::[SCos-CF] was 7% of that in S. clavuligerus and increased to 47% when supplemented with a copy of Pfur-ccaR. The effect on cephamycin biosynthesis gene expression was thus improved but not in an uniform manner for every gene. In heterologous strains, integration of the cephamycin cluster results in a ccaR-independent increased resistance to penicillin, cephalosporin and cefoxitin, what corresponds well to the strong expression of the pcbR and pbpA genes in S. flavogriseus-derived strains.

Transcriptomic analysis of Streptomyces clavuligerus ΔccaR::tsr: effects of the cephamycin C-clavulanic acid cluster regulator CcaR on global regulation.

Streptomyces clavuligerus ATCC 27064 and S. clavuligerus ΔccaR::tsr cultures were grown in asparagine-starch medium, and samples were taken in the exponential and stationary growth phases. Transcriptomic analysis showed that the expression of 186 genes was altered in the ccaR-deleted mutant. These genes belong to the cephamycin C gene cluster, clavulanic acid gene cluster, clavams, holomycin, differentiation, carbon, nitrogen, amino acids or phosphate metabolism and energy production. All the clavulanic acid biosynthesis genes showed Mc values in the order of -4.23. The blip gene-encoding a ß-lactamase inhibitory protein was also controlled by the cephamycin C-clavulanic acid cluster regulator (Mc -2.54). The expression of the cephamycin C biosynthesis genes was greatly reduced in the mutant (Mc values up to -7.1), while the genes involved in putative ß-lactam resistance were less affected (Mc average -0.88). Genes for holomycin biosynthesis were upregulated. In addition, the lack of clavulanic acid and cephamycin production negatively affected the expression of genes for the clavulanic acid precursor arginine and of miscellaneous genes involved in nitrogen metabolism (amtB, glnB, glnA3, glnA2, glnA1). The transcriptomic results were validated by quantative reverse transcription polymerase chain reaction and luciferase assay of luxAB-coupled promoters. Transcriptomic analysis of the homologous genes of S. coelicolor validated the results obtained for S. clavuligerus primary metabolism genes.

Kinetic study on cephamycin C degradation.

Cephamycin C (CepC) is a ß-lactam antibiotic that belongs to the cephalosporin class of drugs. This compound stands out from other cephalosporins for its greater resistance to ß-lactamases, which are enzymes produced by pathogenic microorganisms that present a major mechanism of bacterial resistance to ß-lactam antibiotics. Cephamycin C is produced by the bacterium Streptomyces clavuligerus. Knowledge about the stability of the compound under different values of pH is important for the development of the process of production, extraction, and purification aimed at obtaining higher yields. Therefore, the stability of cephamycin C under different pH levels (2.2, 6.0, 7.0, 7.6, and 8.7) at 20 °C was evaluated in this study. Ultrafiltered broth from batch fermentations of S. clavuligerus was used in the trials. The results indicated that cephamycin C is a more stable compound than other ß-lactam compounds such as penicillin and clavulanic acid. A higher degradation rate was observed at very acidic or basic pH levels, while this rate was lower at quasi-neutral pH levels. After 100 h of trial, the initial CepC showed 46 % degradation at pH 2.2, 71 % degradation at pH 8.7, and varied from 15 to 20 % at quasi-neutral pH levels.

Complete genome sequence of Streptomyces cattleya NRRL 8057, a producer of antibiotics and fluorometabolites.

Streptomyces cattleya, a producer of the antibiotics thienamycin and cephamycin C, is one of the rare bacteria known to synthesize fluorinated metabolites. The genome consists of two linear replicons. The genes involved in fluorine metabolism and in the biosynthesis of the antibiotic thienamycin were mapped on both replicons.

Chapter 16. Enzymology of beta-lactam compounds with cephem structure produced by actinomycete.

Cephamycins are beta-lactam antibiotics with a cephem structure produced by actinomycetes. They are synthesized by a pathway similar to that of cephalosporin C in filamentous fungi but the actinomycetes pathway contains additional enzymes for the formation of the alpha-aminoadipic acid (AAA) precursor and for the final steps specific to cephemycins. Most of the biochemical and genetic studies on cephemycins have been made on cephemycin C biosynthesis in the producer strains Streptomyces clavuligerus ATCC27064 and Amycolatopsis lactamdurans NRRL3802. Genes encoding cephamycin C biosynthetic enzymes are clustered in both actinomycetes. Ten enzymatic steps are involved in the formation of cephamycin C. The precursor alpha-AAA is formed by the sequential action of lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase. Steps common to cephalosporin C biosynthesis include the formation of the tripeptide L-delta-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV) by ACV synthetase, the cyclization of ACV to form isopenicillin N (IPN) by IPN synthase, the epimerization of IPN to penicillin N by isopenicillin N epimerase, the ring expansion of penicillin N to a six member cephem ring by deacetoxycephalosporin C synthase (DAOCS) and the hydroxylation at C-3' by deacetylcephalosporin C hydroxylase. However, in actinomycetes, the epimerization step is different from that in cephalosporin-producing fungi, and the expansion of the ring and its hydroxylation are performed by separate enzymes. Specific steps in cephamycin biosynthesis include the carbamoylation at C-3' by cephem carbamoyl transferase and the introduction of a methoxyl group at C-7 by the joint action of a C-7 cephem-hydroxylase and a methyltransferase. All the enzymes of the pathway have been purified almost to homogeneity and the DAOC synthase and 7-hydroxycephem-methyltransferase (CmcI) of S. clavuligerus have been crystallized giving insights into the mode of action of these enzymes. The cefE gene of S. clavuligerus, encoding DAOCS, has been extensively used to expand the penicillin ring in filamentous fungi in vivo using DNA recombinant technology.

Multichannel liquid chromatography-tandem mass spectrometry cocktail method for comprehensive substrate characterization of multidrug resistance-associated protein 4 transporter.

PURPOSE: To develop a comprehensive substrate-screening method for the ATP-binding cassette (ABC) transporter, and identify new substrates for multidrug resistance-associated protein 4 (MRP4/ABCC4). METHODS: Human MRP4-expressing membrane vesicles were incubated with a mixture of 50 compounds, including methotrexate, a known MRP4 substrate. The amounts transported were simultaneously determined by liquid chromatography-tandem mass spectrometry. RESULTS: From 49 compounds, 12 were identified as substrate candidates for MRP4 in the first screening. The second screening was performed involving the uptake of mixture using single quadrupole multichannel mode, and the third screening was performed involving the uptake of individual compounds using multiple reaction monitoring multichannel mode. As a result, eight substrate candidates were additionally identified. Subsequently, in the fourth step, osmotic pressure-dependent transport was demonstrated for 18 compounds (cefmetazole, piperacillin, rebamipide, tetracycline, ampicillin, benzylpenicillin, bumetanide, cephalosporin C, enalapril, pipemidic acid, furosemide, ceftazidime, pravastatin, hydrochlorothiazide, sulbactam, baclofen, bezafibrate and alacepril) among the 20 substrate candidates, thereby confirming them as MRP4 substrates. By contrast, the uptakes of meloxicam and nateglinide did not depend on osmolarity, indicating that these compounds were not substrates, but bound to MRP4. CONCLUSIONS: The new comprehensive substrate-screening method for ABC transporters allowed the identification of 18 new substrates for MRP4.

Connecting primary and secondary metabolism: AreB, an IclR-like protein, binds the ARE(ccaR) sequence of S. clavuligerus and modulates leucine biosynthesis and cephamycin C and clavulanic acid production.

A protein binding to the autoregulatory element (ARE) upstream of the regulatory ccaR gene of Streptomyces clavuligerus was isolated previously by DNA affinity binding. The areB gene, encoding this protein, is located upstream and in opposite orientation to the leuCD operon of S. clavuligerus; it encodes a 239-amino-acid protein of the IclR family with a helix-turn-helix motif at the N-terminal region. An areB-deleted mutant, S. clavuligerusDeltaareB, has been constructed by gene replacement. This strain requires leucine for optimal growth in defined media. Expression of the leuCD operon is retarded in S. clavuligerusDeltaareB, because AreB binds the areB-leuCD intergenic region acting as a positive modulator. Clavulanic acid and cephamycin C production are improved in the DeltaareB mutant although no drastic difference in ccaR expression was observed. Pure recombinant AreB protein does not bind the ARE(ccaR) sequence (as shown by EMSA) unless filtered extracts from S. clavuligerus ATCC 27064-containing molecules of Mr lower than 10 kDa are added to the binding reaction. Restoration of binding to the ARE(ccaR) sequence is not observed when filtered extracts are obtained from the DeltaareB mutant, suggesting that biosynthesis of the small-molecular-weight effector is also controlled by AreB.

Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A beta-lactamase, GES-4, possessing a single G170S substitution in the omega-loop.

The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type beta-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new beta-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 microg/ml) and beta-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 +/- 1.7 microM). The GES-4 enzyme had a single G170S substitution in the Omega-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of beta-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla(GES-4) genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla(GES-4) genes found in strain KG502 were almost identical to that of bla(GES-3) in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla(GES-4) gene found in strain KG502 might well emerge from a point mutation in the bla(GES-3) gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.

A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase.

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).

Inactivation of Aeromonas hydrophila metallo-beta-lactamase by cephamycins and moxalactam.

Incubation of moxalactam and cefoxitin with the Aeromonas hydrophila metallo-beta-lactamase CphA leads to enzyme-catalyzed hydrolysis of both compounds and to irreversible inactivation of the enzyme by the reaction products. As shown by electrospray mass spectrometry, the inactivation of CphA by cefoxitin and moxalactam is accompanied by the formation of stable adducts with mass increases of 445 and 111 Da, respectively. The single thiol group of the inactivated enzyme is no longer titrable, and dithiothreitol treatment of the complexes partially restores the catalytic activity. The mechanism of inactivation by moxalactam was studied in detail. Hydrolysis of moxalactam is followed by elimination of the 3' leaving group (5-mercapto-1-methyltetrazole), which forms a disulfide bond with the cysteine residue of CphA located in the active site. Interestingly, this reaction is catalyzed by cacodylate.

Structure of the ask-asd operon and formation of aspartokinase subunits in the cephamycin producer 'Amycolatopsis lactamdurans'.

The first two genes of the lysine pathway are closely linked forming a transcriptional operon in the cephamycin producer 'Amycolatopsis lactamdurans'. The asd gene, encoding the enzyme aspartic semialdehyde dehydrogenase, has been cloned by complementation of Escherichia coli asd mutants. It encodes a protein of 355 aa with a deduced M(r) of 37109. The ask gene encoding the aspartokinase (Ask) is located upstream of the asd gene as shown by determination of Ask activity conferred to E. coli transformants. asd and ask are separated by 2 nt and are transcribed in a bicistronic 2.6 kb mRNA. As occurs in corynebacteria, the presence of a ribosome-binding site within the ask sequence suggests that this ORF encodes two overlapping proteins, Askalpha of 421 aa and M(r) 44108, and Askbeta of 172 aa and M(r) 18145. The formation of both subunits of Ask from a single gene (ask) was confirmed by using antibodies against the C-terminal end of Ask which is identical in both subunits. Ask activity of 'A. lactamdurans' is regulated by the concerted action of lysine plus threonine and this inhibition is abolished in E. coli transformants containing Ser(301) to Tyr, or Gly(345) to Asp mutations of the 'A. lactamdurans' ask gene.

Analysis of temporal and spatial expression of the CcaR regulatory element in the cephamycin C biosynthetic pathway using green fluorescent protein.

The DNA-binding capability of a key secondary metabolite regulatory element (CcaR) in the Streptomyces clavuligerus cephamycin C pathway was investigated by gel mobility retardation and DNase I footprinting analysis. These results revealed that CcaR specifically binds to the promoter region of the lysine-epsilon-aminotransferase gene (lat). Green fluorescent protein (GFP) was subsequently used as a reporter to analyse in vivo expression of CcaR. The corresponding isogenic strain containing ccaR:gfp in the chromosome produced cephamycin C at levels similar to those of wild-type S. clavuligerus. Confocal laser scanning microscopy revealed that expression of CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. The highly fluorescent seed culture mycelia quickly lost fluorescence upon inoculation into fresh culture medium. The characteristic green colour reappeared in a small portion of mycelia during mid-exponential growth phase. As the culture aged, the population expressing CcaR expanded, and the expression level increased. This was followed by a reduction in the CcaR-expressing population towards the end of the culture period. During peak expression, CcaR was distributed uniformly in mycelia, but became localized distal to the chromosome when the culture entered stationary phase. In solid phase analysis, abundant CcaR expression was evident in the substrate mycelia, but was completely absent in aerial hyphae. These results show regulatory linkage between ccaR and lat, whose expression profile showed a similar spatial decoupling between morphogenesis and antibiotic production. In addition, visualizing CcaR within S. clavuligerus mycelia demonstrates a distinct pattern of localization over the course of physiological differentiation.

Applications of gene replacement technology to Streptomyces clavuligerus strain development for clavulanic acid production.

Cephamycin C production was blocked in wild-type cultures of the clavulanic acid-producing organism Streptomyces clavuligerus by targeted disruption of the gene (lat) encoding lysine epsilon-aminotransferase. Specific production of clavulanic acid increased in the lat mutants derived from the wild-type strain by 2- to 2.5-fold. Similar beneficial effects on clavulanic acid production were noted in previous studies when gene disruption was used to block the production of the non-clavulanic acid clavams produced by S. clavuligerus. Therefore, mutations in lat and in cvm1, a gene involved in clavam production, were introduced into a high-titer industrial strain of S. clavuligerus to create a double mutant with defects in production of both cephamycin C and clavams. Production of both cephamycin C and non-clavulanic acid clavams was eliminated in the double mutant, and clavulanic acid titers increased about 10% relative to those of the parental strain. This represents the first report of the successful use of genetic engineering to eliminate undesirable metabolic pathways in an industrial strain used for the production of an antibiotic important in human medicine.

Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization.

Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.

[Analysis of secretion into the saliva of cefoxitin (Mefoxin), imipenem (Tienam) and meropenem (Meronem)]. / Cefoxitin (Mefoxin), imipenem (Tienam) és meropenem (Meronem) nyálba történö kiválasztódásának vizsgálata.

The salivary excretion of cefoxitin (Mefoxin), a second-generation beta-lactam antibiotic of the cefalosporin group, which shows enhanced anti-anaerobic effect was investigated in oral surgery patients. In animal experiments the saliva levels of imipenem (Tienam) and meropenem (Meronem), which also belong to the betalactam carbapenem group were studied. The antibiotics were administered parenterally in single therapeutic doses, then blood samples were taken first after half an hour then hourly, and mixed saliva was collected for 6 hours. Cefoxitin was found to reach top level in the 1st hour, then this level decreased rapidly, and in the 4th hour it was no longer measurable. Out of the carbapenems imipenem showed highest level in the 2nd hour and in the 4th hour its concentration in the saliva was minimal. Meropenem reached a higher level in the saliva (1,5-2 times higher than the serum level) in the 2nd hour after administration, which persisted even in the 6th hour. The experimental results justify the administration of these antibiotics in dentistry and oral surgery.

The pcd gene encoding piperideine-6-carboxylate dehydrogenase involved in biosynthesis of alpha-aminoadipic acid is located in the cephamycin cluster of Streptomyces clavuligerus.

Three open reading frames (ORFs) have been located downstream of cefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74, 094).

A novel class C beta-lactamase (FOX-2) in Escherichia coli conferring resistance to cephamycins.

An Escherichia coli strain resistant to a broad spectrum of beta-lactams, including cephamycins, was isolated from a patient suffering from urinary tract infection. A resistance plasmid (pMVP-7) was transferred from the clinical isolate to an Escherichia coli recipient. Both strains produce a cefoxitin-hydrolyzing beta-lactamase focusing at pI 6.7. The phenotype was similar to that of a Klebsiella pneumoniae strain producing cephamycinase FOX-1, so primers were selected from the FOX-1 sequence to amplify the bla gene of the transconjugant. The PCR product obtained was sequenced. The percentage of identity of the deduced amino acid sequence with sequences of other AmpC-type beta-lactamases was 96.9% with FOX-1, 74.9% with CMY-1, and 67.7% with MOX-1. This new plasmid-mediated enzyme is most closely related to FOX-1 (11 amino acid exchanges). We therefore propose the designation FOX-2.