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Background: Infections due to extended spectrum β-lactamases (ESBL) producing Escherichia coli and Klebsiella pneumoniae have become an important clinical problem. These organisms are important regarding the infection control by the physicians. Objective: The present study was undertaken to determine the prevalence of ESBLs along with their antimicrobial sensitivity pattern in Escherichia coli and Klebsiella pneumoniae . Methodology: This cross sectional study was conducted in the Department of Microbiology at Sir Salimullah Medical College, Dhaka. Urine samples were collected from patients who were clinically suspected to have UTI. After incubation, plates were checked for presence of suspected pathogens. Organisms were identified to species level by conventional methods. All isolated E. coli and K. pneumoniae were included in the study. The susceptibility to antibiotics was determined by Kirby Bauer method on Muller Hinton agar. Isolates were screened for ESBL production by using disk diffusion of cefotaxime, ceftazidime, ceftriaxone and cefpodoxime placed on inoculated plates containing Muller Hinton agar according to the CLSI recommendations. Phenotypic confirmatory test for ESBL producers was done by combined disc diffusion for all the isolates that were screened positive for the ESBL production following CLSI guidelines. Combined disk diffusion method was also done in this study. Result: A total of 220 non repeated urine samples were cultured of which 132(60%) cases had shown the bacterial growth. Among the 132 samples Escherichia coli had found in 103(78.0%) cases and Klebsiella spp. was found in 14(10.6%) cases. Out of 103 E coli 23(22.3%) cases was found as ESBL strain. On the other hand within 14 Klebsiella species, the ESBL strain was found in 5(35.7%) cases. Both E coli and Klebsiella species were 100% sensitive to imipenem. However, cephamycin was sensitive in 93.7% and 100% in E coli and Klebsiella species respectively. Conclusion: Results indicate that routine ESBL detection should be made imperative and empirical use of third generation cephalosporins must be discouraged. DOI: http://dx.doi.org/10.3329/jssmc.v4i1.11999 J Shaheed Suhrawardy Med Coll, 2012;4(1):22-25

RT-PCR analysis of the genes in the clavulanic acid cluster revealed three transcriptional polycistronic units that comprised the ceaS2-bls2-pah2-cas2, cyp-fd-orf12-orf13 and oppA2-orf16 genes, whereas oat2, car, oppA1, claR, orf14, gcaS and pbpA were expressed as monocistronic transcripts. Quantitative RT-PCR of Streptomyces clavuligerus ATCC 27064 and the mutant S. clavuligerus ccaR::aph showed that, in the mutant, there was a 1000- to 10,000-fold lower transcript level for the ceaS2 to cas2 polycistronic transcript that encoded CeaS2, the first enzyme of the clavulanic acid pathway that commits arginine to clavulanic acid biosynthesis. Smaller decreases in expression were observed in the ccaR mutant for other genes in the cluster. Two-dimensional electrophoresis and MALDI-TOF analysis confirmed the absence in the mutant strain of proteins CeaS2, Bls2, Pah2 and Car that are required for clavulanic acid biosynthesis, and CefF and IPNS that are required for cephamycin biosynthesis. Gel shift electrophoresis using recombinant r-CcaR protein showed that it bound to the ceaS2 and claR promoter regions in the clavulanic acid cluster, and to the lat, cefF, cefD-cmcI and ccaR promoter regions in the cephamycin C gene cluster. Footprinting experiments indicated that triple heptameric conserved sequences were protected by r-CcaR, and allowed identification of heptameric sequences as CcaR binding sites.

AmpC β-lactamases (Bla(AmpC)) are an emerging group of antimicrobial resistance determinants. The lack of an agreed Bla(AmpC) detection method hinders investigation of their epidemiology and understanding of their clinical significance. This study compared the sensitivity and specificity of phenotypic methods of Bla(AmpC) detection in a collection of 246 Enterobacteriaceae with a diverse range of β-lactam resistance profiles. The Bla(AmpC) screening methods evaluated were based on cephamycin, ceftazidime and cefepime susceptibility. These were compared with Bla(AmpC) screening using conventional ESBL detection methods. The confirmatory methods evaluated were biologically based assays, inhibitor-based assays, an AmpC Etest and a rapid chromogenic assay. A multiplex nucleic acid amplification test and the three-dimensional enzyme extraction assay were used as reference methods. Bla(AmpC) activity was present in 74 isolates. The majority of the enzymes were plasmid-encoded and belonged to the CMY, DHA and EBC families. The screening methods had sensitivities between 47 and 99 % and specificities of 45-95 %. The performance of confirmatory tests varied widely, ranging in sensitivity from 19 % to 97 % and in specificity from 88 % to 100 %. Only the Tris-EDTA and MAST ID D68C disc tests had a sensitivity and a specificity above 90 %. Further investigation is needed to establish the most suitable enzyme substrates, inhibitor types, inhibitor concentrations and interpretative cut-offs in order to refine the inhibitor-based methods. A simple disc-based protocol using cefoxitin non-susceptibility as a screening tool, followed by the Tris-EDTA method for confirmation, detects Bla(AmpC) activity with 95 % sensitivity and 98 % specificity.

To increase production of the important pharmaceutical compound clavulanic acid, a beta-lactamase inhibitor, both random mutagenesis approaches and rational engineering of Streptomyces clavuligerus strains have been extensively applied. Here, for the first time, we compared genome-wide gene expression of an industrial S. clavuligerus strain, obtained through iterative mutagenesis, with that of the wild-type strain. Intriguingly, we found that the majority of the changes contributed not to a complex rewiring of primary metabolism but consisted of a simple upregulation of various antibiotic biosynthesis gene clusters. A few additional transcriptional changes in primary metabolism at key points seem to divert metabolic fluxes to the biosynthetic precursors for clavulanic acid. In general, the observed changes largely coincide with genes that have been targeted by rational engineering in recent years, yet the presence of a number of previously unexplored genes clearly demonstrates that functional genomic analysis can provide new leads for strain improvement in biotechnology.

The β-lactam classes of antibacterials are preeminent in the treatment of bacterial infection due to their unparalleled clinical efficacy and clinical safety. Following the discovery of the penicillins, successive β-lactam drug discovery has added the cephalosporin, penem cephamycin, clavulanate, monobactam, nocardicin, and carbapenem subclasses. The driving force behind much of this era of discovery is the staggering ability of pathogenic bacteria to adapt previous generations of the β-lactam by the acquisition and expression of resistance mechanisms. Although many factors contribute to β-lactam resistance, alterations to the molecular targets of the β-lactams (the penicillin binding proteins) and the use of enzymes (the β-lactamases) capable of the hydrolytic deactivation of the β-lactams are paramount. This review traces the historical development of β-lactam drug discovery, with emphasis on the most recent progress in the medicinal chemistry, biochemistry, and microbiology of the β-lactams leading to the discovery of new generation β-lactam antibacterials effective against the Gram-negative and -positive bacterial pathogens of current medical concern. Keywords: β-lactam; β-lactamase; penicillin; cephalosporin; monobactam

It is approximately 60 years since the discovery of cephalosporin C in Cephalosporium acremonium. Streptomycetes have since been found to produce the structurally related cephamycin C. Studies on the biosynthetic pathways of these two compounds revealed a common pathway including a step governed by deacetoxycephalosporin C synthase which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C. Because of the therapeutic importance of cephalosporins, this enzyme has been extensively studied for its ability to produce these antibiotics. Although, on the basis of earlier studies, its substrate specificity was believed to be extremely narrow, relentless efforts in optimizing the in-vitro enzyme assay conditions showed that it is able to convert a wide range of penicillin substrates differing in their side chains. It is a member of 2-oxoglutarate-dependent dioxygenase protein family, which requires the iron(II) ion as a co-factor and 2-oxoglutarate and molecular oxygen as co-substrates. It has highly conserved HXDX( n ) H and RXS motifs to bind the co-factor and co-substrate, respectively. With advances in technology, the genes encoding this enzyme from various sources have been cloned and heterologously expressed for comparative analyses and mutagenesis studies. A high level of recombinant protein expression has also enabled crystallization of this enzyme for structure determination. This review will summarize some of the earlier biochemical characterization and describe the mechanistic action of this enzyme revealed by recent structural studies. This review will also discuss some of the approaches used to identify the amino acid residues involved in binding the penicillin substrate and to modify its substrate preference for possible industrial application.

Three open reading frames denoted as orf21, orf22, and orf23 were identified from downstream of the currently recognized gene cluster for clavulanic acid biosynthesis in Streptomyces clavuligerus ATCC 27064. The new orfs were annotated after in silico analysis as genes encoding a putative sigma factor, a sensor kinase, and a response regulator. The roles of the individual genes were explored by disruption of the corresponding orfs, and the morphological and antibiotic production phenotypes of the resulting mutants were compared. In orf21 and orf22 mutants, no growth or morphological differences were noted, but modest reduction of cephamycin C (orf21), or both cephamycin C and clavulanic acid production (orf22) compared with wild-type, were observed. In orf23 mutant, cell growth and sporulation was retarded, and clavulanic acid and cephamycin C production were reduced to 40 and 47% of wild-type levels, respectively. Conversely, overexpression of orf23 caused precocious hyperproduction of spores on solid medium, and antibiotic production was increased above the levels seen in plasmid control cultures. Transcriptional analyses were also carried out on orf23 and showed that mutation had little effect on transcription of genes associated with the early stages of cephamycin C or clavulanic acid production but transcription of claR, which regulates the late stages of clavulanic acid production, was reduced in orf23 mutants. These observations suggest that the orf23 product may enable S. clavuligerus to respond to environmental changes by altering cell growth and differentiation. In addition, the effects of ORF23 on growth might indirectly regulate the biosynthesis of secondary metabolites such as clavulanic acid and cephamycin C.

Biosynthesis of cephamycin C in Streptomyces clavuligerus involves the initial conversion of lysine to α-aminoadipic acid. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase carry out this two-step reaction, and genes encoding each of these enzymes are found within the cephamycin C gene cluster. However, while mutation of the lat gene causes complete loss of cephamycin production, pcd mutants still produce cephamycin at 30% to 70% of wild-type levels. Cephamycin production by pcd mutants could be restored to wild-type levels either by supplementation of the growth medium with α-aminoadipic acid or by complementation of the mutation with an intact copy of the pcd gene. Neither heterologous PCR nor Southern analyses showed any evidence for the presence of a second pcd gene. Furthermore, cell extracts from pcd mutants lack detectable PCD activity. Cephamycin production in the absence of detectable PCD activity suggests that S. clavuligerus must have some alternate means of producing the aminoadipyl-cysteinyl-valine needed for cephamycin biosynthesis.

Forty-six cephamycin-resistant Escherichia coli isolates from patients diagnosed with community-acquired urinary tract infection were selected in order to study their resistance mechanism. With the exception of one isolate producing CMY-4, all isolates produced a CMY-2 β-lactamase. Molecular typing showed that the CMY-2-producing isolates were not related. Cephamycin resistance was plasmid encoded and conjugatively transferred. Plasmid digest profiles suggested that the plasmids were different. Thirty-nine of the 45 CMY-2-producing isolates harboured a plasmid containing a specific DNA fragment, IS Ecp1 – bla CMY-2 – blc – sugE , which was identical to those previously published in CMY-2-producing Salmonella enterica serotype Choleraesuis (SCB67) and Salmonella enterica serotype Typhimurium (pNF1358) from Taiwan and the USA, respectively. Among the remaining six isolates, insertion of IS 1294 and IS 1 at different positions was observed in one and five isolates, respectively. The regions surrounding bla CMY-2 of the six isolates were identical to the other 39 isolates as well as to SCB67 and pNF1358. Since the present identical transmissible bla CMY-2 -carrying element was observed in food animal sources both in the USA and Taiwan, its possible transmission to humans, as revealed in this study, is of great concern. Awareness of this mobile resistance element is required to prevent introduction into hospitals and to reduce the spread of this emerging resistance within the community.

To investigate the in vitro activities of cephamycins (cefmetazole and flomoxef) for extended-spectrum β-lactamase (ESBL)- and plasmid-mediated AmpC β-lactamase (pAmpC)-producing Enterobacteriaceae, a total of 574 third-generation cephalosporin-resistant clinical isolates were collected at a Japanese multicenter study. PCR and sequencing identified 394 isolates with only ESBL genes, 63 isolates with only pAmpC genes, and 6 isolates with both ESBL and pAmpC genes. blaCTX-M types predominated 95.5% of the ESBL genes, and blaCMY-2 predominated 91.3% of the pAmpC genes. The MIC50/90 values of cefmetazole and flomoxef were ≤ 1/4 and ≤ 1/≤ 1 μg/mL for isolates with only ESBL genes, respectively, and 16/>16 and 8/16 μg/mL for isolates with only pAmpC genes, respectively. Flomoxef ≥ 4 μg/mL had the best screening performance for the detection of isolates with pAmpC genes. Flomoxef had better in vitro activities against ESBL-producing Enterobacteriaceae and provided a clearer distinction between ESBL and pAmpC-producing Enterobacteriaceae compared to cefmetazole.

Central steps in the biosynthetic pathways of some of the most commonly used antibiotics, the cephalosporins, are catalysed by 2-oxoglutarate (2OG)-dependent oxygenases. Deacetoxycephalosporin C synthase (DAOCS) catalyses the 2OG-dependent oxidative expansion of the five-membered thiazolidine ring of the penicillin nucleus into the six-membered dihydrothiazine ring of the cephalosporin nucleus. DAOCS uses dioxygen to create a reactive iron–oxygen intermediate from ferrous ion to drive the reaction. In prokaryotic cephalosporin producers, the cephalosporin product, DAOC, is hydroxylated at the 3′-position to form deacetylcephalosporin C (DAC) as catalysed by a second 2OG-dependent enzyme, DAC synthase (DACS). In eukaryotic cephalosporin producers, the reaction is catalysed by a bifunctional enzyme, DAOC/DACS, that catalyses both the ring expansion and the 3′-hydroxylation reactions. The prokaryotic and eukaryotic enzymes are closely related to DAOCS by sequence, suggesting these enzymes may have evolved by gene duplication. Cephamycin C-producing microorganisms use two enzymes, encoded by the genes cmcI/J, to convert cephalosporins to their 7α-methoxy derivatives that are less vulnerable to β-lactam hydrolysing enzymes. The methoxylation reaction is dependent on Fe(ii), 2OG and S-adenosylmethionine, suggesting the involvement of another 2OG-dependent oxygenase. Herein, structural and mechanistic features are summarized for these 2OG enzymes that utilize this common and flexible mode of dioxygen activation.

In 2013, an outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 occurred in a nursery school in Japan. The outbreak affected 12 school children and five members of their families. All 17 isolates obtained from these individuals were found to be clonal, as determined by pulsed-field gel electrophoresis analysis and multilocus variable number tandem repeat analysis. The antimicrobial susceptibility profiles of the isolates to 20 drugs were examined, with three isolates showing resistance to the extended-spectrum cephalosporins (ESC) and cephamycin, including cefotaxime, ceftazidime, and cefminox. The resistant isolates carried the blaCMY-2 AmpC β-lactamase gene. It is proposed that the ESC-resistant EHEC O157:H7 isolates might have acquired the resistance plasmid encoding the blaCMY-2 gene during human to human infection in the nursery school.

The transcriptional and translational control of the biosynthesis of the beta-lactamase inhibitor clavulanic acid is a subject of great scientific and industrial interest. To study the role of the ribosomal protein L11 on control of clavulanic acid gene transcription, the DNA region aspC-tRNA(trp)-secE-rplK-rplA-rplJ-rplL of Streptomyces clavuligerus was cloned and characterized. An S. clavuligerus rplK(DeltaPALG) mutant, with an internal 12 nucleotides in-frame deletion in the rplK gene, encoding the L11 (RplK) ribosomal protein lacking amino acids (29)PALG(32), was constructed by gene replacement. This deletion alters the L11 N-terminal domain that interacts with the RelA and class I releasing factors-mediated translational termination. The mutant grew well, showed threefold higher resistance to thiostrepton, did not form spores and lacked diffusible brown pigments, as compared with the wild-type strain. The wild-type phenotype was recovered by complementation with the native rplK gene. S. clavuligerus rplK(DeltaPALG) produced reduced levels of clavulanic acid (15-26% as compared with the wild type) and cephamycin C (40-50%) in cultures grown in defined SA and complex TSB media. The decreased yields resulted from an impaired transcription of the regulatory genes ccaR and claR and the cefD and ceaS2 genes for cephamycin and clavulanic acid biosynthesis respectively. Expression of ceaS2 encoding carboxyethylarginine synthase (CEAS), the precursor-committing enzyme for clavulanic acid biosynthesis, was particularly affected in this mutant. In the wild-type strain polyphosphorylated nucleotides peaked at 36-48 h of growth in SA cultures whereas expression of the cephamycin and clavulanic acid genes occurred 12-24 h earlier than the increase in ppGpp indicating that there is no strict correlation between the peak of ppGpp and the onset of transcription of the clavulanic acid and cephamycin C biosynthesis. The drastic effect of the rplK(DeltaPALG) mutation on the onset of expression of the ceaS2 and the regulatory ccaR and claR genes and the lack of correlation with ppGpp levels suggest that the onset of transcription of these genes is modulated by the conformational alteration of the N-terminal region of L11 probably by interaction with the nascent peptide releasing factors and with RelA.

Many secondary metabolites (e.g. antibiotics and mycotoxins) are toxic to the microorganisms that produce them. The clusters of genes that are responsible for the biosynthesis of secondary metabolites frequently contain genes for resistance to these toxic metabolites, such as different types of multiple drug resistance systems, to avoid suicide of the producer strains. Recently there has been research into the efflux systems of secondary metabolites in bacteria and in filamentous fungi, such as the large number of ATP-binding cassette transporters found in antibiotic-producing Streptomyces species and that are involved in penicillin secretion in Penicillium chrysogenum. A different group of efflux systems, the major facilitator superfamily exporters, occur very frequently in a variety of bacteria that produce pigments or antibiotics (e.g. the cephamycin and thienamycin producers) and in filamentous fungi that produce mycotoxins. Such efflux systems include the CefT exporters that mediate cephalosporin secretion in Acremonium chrysogenum. The evolutionary origin of these efflux systems and their relationship with current resistance determinants in pathogenic bacteria has been analyzed. Genetic improvement of the secretion systems of secondary metabolites in the producer strain has important industrial applications.

On the basis of molecular dynamics simulations, we investigate the dynamic properties of the carbapenem- and cephamycin-resistant dinuclear zinc metallo-beta-lactamase from Bacteroides fragilis and its complex with a biphenyl tetrazole inhibitor, 2-butyl-6-hydroxy-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-quinazolin-4-one 1 (L-159061). The results obtained with the newly developed force field parameters for the coordination environment of the catalytic zinc ions show that the active site gorge comprising major and minor loops gets deeper and narrower upon binding of the inhibitor, which supports the previous experimental implication that the structural flexibility of the loop structures plays a significant role in enzymatic action. In the presence of the inhibitor, the Trp32 side chain at the apex of the major loop covers the entrance of active site channel, thereby contributing to the stabilization of the enzyme-inhibitor complex. In addition to a direct coordination of the inhibitor tetrazole ring to the second zinc ion in the active site, the hydrogen bonding of Lys167 to the inhibitor carbonyl group and hydrophobic interactions between the inhibitor and side chains of loop residues prove to be significant binding forces of the enzyme-inhibitor complex.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.

AmpC β-lactamase, altered porins, or both are usually responsible for cefoxitin resistance in Escherichia coli. We examined the relative importance of each. We studied 18 strains of clinical isolates with reduced cefoxitin susceptibility and 10 initially-susceptible strains passaged through cefoxitin-gradient plates. Of 18 wild-resistant strains, 9 had identical promoter mutations (including creation of a consensus 17-bp spacer) and related pulsed-field gel electrophoresis patterns; the other 9 strains were unrelated. Nine strains had attenuator mutations; two strains did not express OmpC or OmpF. After serial passage, 8 of 10 strains developed cefoxitin resistance, none developed promoter or attenuator mutations, 6 lost both the OmpC and OmpF porin proteins, and 1 showed decreased production of both. One strain had neither porin alteration or increased AmpC production. Porin mutants may occur more commonly and be less fit and less inclined to spread or cause disease than strains with increased β-lactamase expression.

The genetic organization of the region upstream of the car gene of the clavulanic acid biosynthetic gene cluster of Streptomyces clavuligerus has been determined. Sequence analysis of a 12·1 kb region revealed the presence of 10 ORFs whose putative functions, according to database searches, are discussed. Three co-transcriptional units are proposed: ORF10–11, ORF12–13 and ORF15–16–17–18. Potential transcriptional terminators were identified downstream of ORF11 (fd) and ORF15. Targeted disruption of ORF10 (cyp) gave rise to transformants unable to produce clavulanic acid, but with a considerably higher production of cephamycin C. Transformants inactivated at ORF14 had a remarkably lower production of clavulanic acid and similar production of cephamycin C. Significant improvements of clavulanic acid production, associated with a drop in cephamycin C biosynthesis, were obtained with transformants of S. clavuligerus harbouring multiple copies of plasmids carrying different constructions from the ORF10–14 region. This information can be used to guide strain improvement programs, blending random mutagenesis and molecular cloning, to optimize the yield of clavulanic acid.

In the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149 H240 , and TEM-149 H164-H240 versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149 H240 , and TEM-149 H164-H240 β-lactamases, and the K i (inhibition constant), K (dissociation constant of the Henri-Michaelis complex), k +2 and k +3 (first-order acylation and deacylation constants, respectively), and k +2 / K values were calculated.

Many early studies on penicillin and cephamycin biosynthesis were carried out in Streptomyces clavuligerus, but because of the commercial importance of clavulanic acid, most recent studies have focused on that compound. In particular, genetic engineering to generate improved clavulanic acid producer strains has been a main interest. S. clavuligerus grows and produces secondary metabolites optimally at 28 to 30°C, but it is unusually sensitive to elevated temperature and will not grow above 31 to 32°C. Liquid media employed by research groups studying the production of the various types of β-lactam metabolites produced by S. clavuligerus vary widely. Numerous bioprocess-type studies examining growth medium optimization as a means of improving production of clavulanic acid have appeared in recent years, typically reinforcing the association of soy-based growth medium constituents with high productivity. In S. clavuligerus, biosynthesis begins with the amino acids L-lysine, L-cysteine, and L-valine and proceeds through the synthesis of penicillin and cephalosporin intermediates to give the final cephamycin product. While improvements in productivity can be achieved by increasing production of pathway enzymes, another approach to achieving this is to identify genes encoding critical regulatory proteins and increase their expression levels. More global approaches to improvement of antibiotic production could also be coupled with strategies aimed at the pathway-specific regulators. Effects of greater magnitude are seen when pathway-specific regulators are targeted for overproduction, and combination approaches targeting both global and specific regulators along with individual pathway enzymes for overproduction, together with elimination of competing pathways, will likely yield even greater results.

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.

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.

cysteine is a precursor of the penicillin, cephalosporin and cephamycin families of β-lactam antibiotics. Cystathionine-γ-lyase (encoded by the mecB gene), an enzyme that splits cystathionine releasing cysteine, is required for high-level cephalosporin production in methionine-supplemented medium. By amplification of the mecB gene in Acremonium chrysogenum C10, several transformants were obtained that produced 10-40% higher levels of cephalosporin. All selected transformants contained at least two or three copies of the mecB gene as shown by Southern hybridization with a probe internal to mecB. Two of these transformants, A. chrysogenum T27 and A. chrysogenum T58, showed 4- to 10-fold higher cystathionine-γ-lyase activity than the control strain. Northern hybridizations indicated that the levels of the two mecB transcripts of 1.7 and 1.5 kb were greatly increased in transformants T27 and T58. Fermentor studies using controlled conditions confirmed that transformant T27 was a cephalosporin overproducer, reaching titers of nearly 2000 μg/ml of cephalosporin in Shen-defined medium that correlated with two- to fourfold higher cystathionine-γ-lyase levels than in the control strain. Transformant T58 containing five- to sixfold higher levels of cystathionine-γ-lyase in fermentor cultures showed a reduced growth rate and a slow cephalosporin accumulation rate. In conclusion, moderately increased levels of cystathionine-γ-lyase stimulated cephalosporin production but very high levels of this enzyme were deleterious for growth and cephalosporin biosynthesis. Journal of Industrial Microbiology & Biotechnology (2001) 27, 252–258.

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, Askα of 421 aa and M r 44108, and Askβ 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 Ser301 to Tyr, or Gly345 to Asp mutations of the ‘A. lactamdurans’ ask gene.

Two protein bands with strong esterase activity are present in broths of Nocardia lactamidurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed Km values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C.

Summary The cephamycin producer strain ‘ Nocardia lactamdurans ’ contains four ribosomal RNA ( rrn ) operons. One of them ( rrn D) was cloned from a DNA library in the bifunctional cosmid pJAR4. A 2229 bp region of rrn D has been sequenced. The ‘ N. lactamdurans ’ rrn D operon maintains the canonical order 5′-16S-23S-5S-3′. Four of the consensus Gurtler-Stanisch sequences were found in the 16S rRNA gene and a fifth one in the sequenced 5′ region of the 23S rRNA gene. The and Shine-Dalgarno sequence of ‘ N. lactamdurans ’ (located in the 3′-end of the 16S rRNA gene) was found to be 5′-CCUCCUUUCU-3′ and is identical to that of Corynebacterium lactofermentum and Mycobacterium tuberculosis . A phylogenetic analysis of ‘ N. lactamdurans ’ by the neighbor-joining method using the entire 16S rRNA nucleotide sequence revealed that this actinomycete is closely related to Amycolatopsis orientalis subsp orientalis, Amycolatopsis coloradensis, Amycolatopsis alba, Amycolatopsis sulphurea and other Amycolatopsis sp. but only distantly related to species of the genus Nocardia . The cephamycin producer ‘ N. lactamdurans ’ NRRL 3802 should be, therefore, classified as Amycolatopsis lactamdurans . The deduced secondary structure of the 16S rRNA is very similar to that of A. colorandensis and A. alba but different from those of species of the Nocardia genus supporting the incorporation of ‘ N. lactamdurans ’ into the genus Amycolatopsis .

In vitro synthesis of cefminox by cell-free extracts of Streptomyces clavuligerus was investigated using alpha-ketoglutarate, L-ascorbic acid, FeSO(4), S-adenosyl-L-methionine, and 7alpha-demethoxycefminox as the substrates. The formation of cefminox was detected both by a biological assay with Proteus vulgaris GN 76/C-1 and by high performance liquid chromatography. Although the conversion rate of 7alpha-demethoxycefminox to cefminox was observed to be quite low, it still demonstrated the potential for an enzymatic process to replace the chemical steps which are currently in use for the production of cefminox.

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans , in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the α-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbAB promoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans . Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

The discovery and development of the β-lactam antibiotics are among the most powerful and successful achievements of modern science and technology. Since Fleming's accidental discovery of the penicillin-producing mold, seventy years of steady progress has followed, and today the β-lactam group of compounds are the most successful example of natural product application and chemotherapy. Following on the heels of penicillin production by Penicillium chrysogenum came the discoveries of cephalosporin formation by Cephalosporium acremonium, cephamycin, clavam and carbapenem production by actinomycetes, and monocyclic β-lactam production by actinomycetes and unicellular bacteria. Each one of these groups has yielded medically-useful products and has contributed to the reduction of pain and suffering of people throughout the world. Research on the microbiology, biochemistry, genetics and chemistry of these compounds have continued up to the present with major contributions being made by both individual and collaborative groups from industry and academia. The discovery of penicillin not only led to the era of the wonder drugs but provided the most important antibiotics available to medicine. Continued efforts have resulted in the improvement of these compounds with respect to potency, breadth of spectrum, activity against resistant pathogens, stability and pharmacokinetic properties. On the research front, major advances are being made on structural and regulatory biosynthetic genes and metabolic engineering of the pathways involved. New semisynthetic compounds especially those designed to combat resistance development are being examined in the clinic, and unusual non-antibiotic activities of these compounds are being pursued. Although seventy years of age, the β-lactams are not yet ready for retirement.

Cephamycin C is produced in a nine steps pathway by the actinomycetes S. clavuligerus and N. lactamdurans. The genes encoding the biosynthesis enzymes are clustered in both microorganisms as well as in the cephabacin producer Lysobacter lactamgenus, a Gram negative bacterium. The clusters of genes include genes encoding enzymes common to the biosynthesis of penicillin and cephalosporin C by the eukaryotic producers Penicillium chrysogenum and Cephalosporiun acremonium and genes for steps specific for the formation of the precursor α-aminoadipic acid as well as for the enzymes involved in the late modification of the cephalosporin intermediates of the pathway. Present are also genes for proteins involved in the export and/or resistance to cephamycin C. In S. clavuligerus a gene encoding a regulatory protein controlling the formation of cephamycin C and clavulanic acid is also present in the cluster.

A proper description of the biosynthesis of fungal β-lactam antibiotics requires detailed knowledge of the cell biology of the producing organisms. This involves a delineation of the compartmentalization of the biosynthetic pathways, and of the consequential transport steps across the cell-boundary plasma membrane and across organellar membranes. Of the enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum and Aspergillus nidulans, δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS) probably have a cytosolic location. Acyl-coenzyme A:isopenicillin N acyltransferase (IAT) is located in microbodies. Of the two enzymes that may be involved in activation of the side chain, acetyl-coenzyme A synthetase (ACS) is located in the cytosol, and phenylacetyl-coenzyme A ligase (PCL) is probably located in the microbody. All enzymes of the cephalosporin biosynthesis pathway in Cephalosporium acremonium probably have a cytosolic location. The vacuole may play an ancillary role in the supply of precursor amino acids, and in the storage of intermediates. The distribution of precursors, intermediates, end- and side-products, the transport of nutrients, precursors, intermediates and products across the plasma membrane, and the transport of small solutes across organellar membranes, is discussed. The relevance of compartmentalization is considered against the background of recent biotechnological innovations of fungal β-lactam biosynthesis pathways.

Nutritional requirements of a mutant strain of Streptomyces clavuligerus for Cephamycin C production were derived by testing various indigenous substrates and their concentrations in production medium. Upon initial screening, six components: tapioca starch (TS), corn steep liquor (CSL), sunflower cake (SFC), K 2 HPO 4 , MgSO 4 .7H 2 O and MnSO 4 were identified as supporting high production of Cephamycin C. Statistical analysis (ANOVA) of media components indicated that addition of oil cake SFC to the medium enhanced production significantly. SFC controlled foam during fermentation and served as a pH regulator.

Batch cultures of Nocardia lactamdurans MA4213 in stirred tank fermentors (400 rpm) became oxygen limited after 45 h of incubation. When the stirrer speed was increased at 36 h from 400 to 600 rpm there was a 5-fold increase in the dissolved oxygen (DO) in the broth what resulted in a 2-fold stimulation of cephamycin biosynthesis. Immunoblotting studies using antibodies against the α-aminoadipyl-cysteinyl-valine synthetase and the protein P7 (cephem-7-hydroxylase) component of the methoxylation system showed that cultures with increased DO concentrations maintained high levels of these proteins for an extended period of time. Under these conditions there was an enhanced conversion of cephamycin intermediates into cephamycin C. Allophane and alumina exerted a strong stimulatory effect on cephamycin biosynthesis that correlated with high DO levels in allophane- or alumina-supplemented cultures at low agitation speeds. Allophane or alumina supplementation of N. lactamdurans cultures resulted in a delayed decay of the cephem-7-hydroxylase protein in the cells favoring conversion of the cephamycin intermediates into cephamycin C.

Delta-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into alpha-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 microM) and NAD+ (apparent Km 115 microM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6 amino-transferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but no in actinomycetes that do no produce beta-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to alpha-aminoadipic acid.

The ß-lactam antibiotics continue providing health to the world population by virtue of industrial production and discoveries of new molecules with useful activities. Sales of these remarkable compounds, including penicillins, cephalosporins, cefoxitin, monobactams, clavulanic acid, and carbapenems, have reached over $20 billion dollars per year. Strain improvement of the penicillin-producing strains of Penicillium chrysogenum has been truly remarkable, with present strains producing about 100,000 times more penicillin than the original Penicillium notatum of Sir Alexander Fleming. The traditional strain improvement programs based on random mutation and screening in combination with recombinant DNA techniques allowed an impressive ß-lactam yield enhancement at industrial scale. A remarkable amount of information has been gathered on the biosynthetic enzymes involved, the pathways of biosynthesis of ß-lactams as well as their regulation, and the genomics and proteomics of the producing organisms. The rational metabolic engineering of ß-lactam-producing microorganisms has resulted in productivity increments and the design of biosynthetic pathways giving rise to new antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms (GMOs). Modern aspects of the processes are discussed in the present review including genetics, molecular biology, metabolic engineering, genomics, and proteomics. © 2014 Springer-Verlag Berlin Heidelberg. All rights are reserved.

The effect of the CcaR regulatory protein on expression of the cephamycin C gene cluster is studied. Quantitative reverse transcription PCR (qRT-PCR) expression analysis of the cephamycin biosynthesis genes in the ccaR-disrupted strain, S. clavuligerus ccaR::aph, revealed that in the absence of CcaR, the lat and cmcI genes expression was reduced 2,200-and 1,087-fold compared with the wild type. Expression of pcbAB-pcbC-cefD-cefE-cmcJ-cmcH and blp was 225- to 359-fold lower, while expression of pcbR-pbpA-bla and orf10 was only slightly affected if at all, indicating that resistance and regulatory genes are not under CcaR control as opposed to pathway biosynthetic genes. In the intergenic cmcH-ccaR region, a small messenger RNA (mRNA) overlaps with the cmcH transcription terminator. Deletion of 688 bp of the intergenic region results in a strain, S. clavuligerus Delta RI, still able to produce cephamycin C and clavulanic acid but at levels 30-40 % lower than the parental strain. Therefore, specific sequences in the intergenic region upstream of ccaR enhance the expression of ccaR but are not essential for its expression. Strains containing an additional ccaR gene integrated in the chromosome, S. clavuligerus pSET-PC, or multiple copies of ccaR expressed from the PglpF promoter, S. clavuligerus pAK23, were constructed. Fermentations of the pAK23 strain resulted in a 6.1-fold increase in specific cephamycin C production relative to the wild type. In the same experiments, qRT-PCR analysis of the cephamycin biosynthesis genes showed a 5.1-fold increase in ccaR expression and similar increases in expression of lat and cmcI, while expression of other cluster genes were increased in the order of 2- to 3-fold.

Clavulanic acid (CA) is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus and has been successfully used in combination with β-lactam antibiotics (for example, Augmentin) to treat infections caused by β-lactamase-producing pathogens. Since the discovery of CA in the late 1970s, significant information has accumulated on its biosynthesis, and regarding molecular mechanisms involved in the regulation of its production. Notably, the genes directing CA biosynthesis are clustered along with the genes responsible for the biosynthesis of the β-lactam antibiotic, cephamycin C, and co-regulated, which makes this organism unique in that the production of an antibiotic and production of a small molecule to protect the antibiotic from its enzymatic degradation are controlled by shared mechanisms. Traditionally, the industrial strain improvement programs have relied significantly on random mutagenesis and selection approach. However, the recent availability of the genome sequence of S. clavuligerus along with the capability to build metabolic models, and ability to engineer the organism by directed approaches, has created exciting opportunities to improve strain productivity more efficiently. This review will include focus mainly on the gene organization of the CA biosynthetic genes, regulatory mechanisms that affect its production, and will include perspectives on improving strain productivity.

Background Long Term Care Facilities (LTCFs) play a key role in guaranteeing care to patients in developed countries. Many patients, mostly elderly, access LTCFs at some time in their lives, and their healthcare pathways often require them to move back and forth between hospital and outpatient settings. These patterns bring about new challenges regarding infection control, especially healthcare associated infections. Methods A point prevalence study was conducted in 23 Italian LTCFs, to identify colonization in patients with urinary catheter (>24 hours). Species identification, susceptibility tests and extended spectrum beta lactamase (ESBL) production screenings were performed using Vitek 2 System. Enterobacteria identified by Vitek 2 System as ESBL-producers or suspected AmpC hyperproducers on the basis of cephamycin resistance, were sent to a research laboratory where they underwent a double-disk synergy test. Finally, ESBL-producers were screened for bla resistance genes by PCR assay. Results 211 patients with catheter were screened, 185 out of 211 patients showed positive samples for the presence of Enterobacteriaceae, 114 of these 185 patients were colonized by extended spectrum cephalosporins resistant microorganisms. We identified a total of 257 Gram negative pathogens, of which 51.8% (133/257) were extended spectrum cephalosporins resistant. 7 out of 133 cephamycin not susceptible strains proved to be AmpC-type beta-lactamases and 125/133 ESBL-producers; 1 was not further characterized. 43 out of 257 (16.7%) E. coli, 37/257 (14.4%) P. mirabilis, 20/257 (7.8%), P. stuartii, 14/257 (5.4%) M. morganii, 7/257 (2.7%), K. pneumoniae, 4/257 (1.6%) C. koseri proved to be overall ESBL-producers by double-disk synergy test. Third and fourth generation cephalosporin resistant P. mirabilis, P. stuartii and M. morganii strains mainly harboured a blaTEM gene (95.9%), while 89.1% of E. coli were positive for the blaCTX-M determinant by PCR and sequencing. Patients with decubitus had a higher risk of colonization by at least one resistant isolate (p

Cephamycin C-producing microorganisms contain a two-protein enzyme system that converts cephalosporins to 7-methoxycephalosporins. Interaction between the two component proteins P7 (Mr 27,000) and P8 (Mr 32,000) has been studied by immunoaffinity chromatography using anti-P7 and anti-P8 antibodies, cross-linking with glutaraldehyde, and fluorescence spectroscopy analysis. Co-renaturation of the P7 and P8 polypeptides resulted in the formation of a protein complex with a molecular mass of 59 kDa, which corresponds to a heterodimer of P7 and P8. Glutaraldehyde cross-linking of the polypeptides after assembly of the protein complex showed the presence of a single heterodimer form that reacted with antibodies against P7 and P8. Each separate protein did not associate with itself into multimers. The P7·P8 complex co-purified by immunoaffinity chromatography from extracts of Nocardia lactamdurans and Streptomyces clavuligerus, suggesting that both proteins are present as an aggregate in vivo. Fluorescence spectroscopy studies of 5-methylaminonaphthalene-1-sulfonyl-P7 in response to increasing concentrations of P8 showed a blue shift in the fluorophore emission, indicating a conformational change of P7 in response to the interaction of P8 with an apparent dissociation constant of 47 μM. NADH showed affinity for the P7 component. The P7·P8 complex interacted strongly with the substrates S-adenosylmethionine and cephalosporin C, differently from that occurring with the separate P7 or P8 components, resulting in a strong blue shift in the fluorescence emission spectra of the complex.

Summary: Addition of allophane, a phosphate-trapping agent, to two different strains of Nocardia lactamdurans exerted a large stimulatory effect on cephamycin biosynthesis. The biosynthesis of cephamycin is inhibited by inorganic phosphate at concentrations above 5 mM and allophane reversed this inhibitory effect. Allophane-supplemented cultures showed increased activities and/or an extended life in the cell of four cephamycin biosynthetic enzymes: isopenicillin N synthase, the two-protein-component 7α-cephem methoxylase (7α-cephem hydroxylase and 7-hydroxycephem methyltransferase) and 3′-hydroxymethylcephem O-carbamoyltransferase. However, the first enzyme of the pathway, lysine 6-aminotransferase, was not stimulated by allophane. Allophane-supplemented cultures showed increased protein levels of (i) α-aminoadipyl-cysteinyl-valine synthetase (the condensing multienzyme that forms the tripeptide intermediate), and (ii) the two proteins involved in the 7α-cephem methoxylase, as shown by immunoblotting with antibodies against each of these proteins. Phosphate repressed the de novo synthesis of these proteins but did not increase their degradation. These results indicated that allophane stimulates expression of the cluster of genes extending from the pcbAB gene (encoding α-aminoadipyl-cysteinyl-valine synthetase) to cmcl-cmcJ (encoding the two-protein methoxylase) and cmcH (encoding the O-carbamoyltransferase).

The ornithine carbamoyltransferases (OTCases) from the β-lactam-producing actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans have been purified to near-homogeneity by Δ-N-phosphonoacetylornithine-Sepharose 4B affinity chromatography. The S. clavuligerus and N. lactamdurans OTCase monomers had a molecular mass of 37 kDa. The native OTCases of S. clavuligerus, N. lactamdurans and Streptomyces coelicolor had molecular masses of 248, 251 and 247 kDa respectively, which correspond to a hexameric structure. The apparent Km values for ornithine and carbamoylphosphate of the S. clavuligerus enzyme were respectively 2.3 and 6.0 mM at pH 8.0. The enzyme showed a reverse activity on citrulline and used lysine and putrescine as substrates. The hexameric complex showed coupled arginase–OTCase activities and was able to convert arginine into citrulline in a carbamoylphosphate-dependent manner. The requirement for carbamoylphosphate might prevent the arginase–OTCase complex from carrying out a futile cycle of arginine biosynthesis and degradation.

A Streptomyces clavuligerus gene (designated pcbR) which is located immediately downstream from the gene encoding isopenicillin N synthase in the cephamycin gene cluster was characterized. Nucleotide sequence analysis and database searching of PcbR identified a significant similarity between PcbR and proteins belonging to the family of high-molecular-weight group B penicillin-binding proteins (PBPs). Eight of nine boxes (motifs) conserved within this family of proteins are present in the PcbR protein sequence in the same order and with approximately the same spacing between them. When a mutant disrupted in pcbR was constructed by gene replacement, the resulting pcbR mutant exhibited a significant decrease in its resistance to benzylpenicillin and cephalosporins, indicating that pcbR is involved in beta-lactam resistance in this organism. Western blot (immunoblot) analysis of S. clavuligerus cell membranes using PcbR-specific antibodies suggested that PcbR is a membrane protein. PcbR was also present in cell membranes when expressed in Escherichia coli and was able to bind radioactive penicillin in a PBP assay, suggesting that PcbR is a PBP. When genomic DNAs from several actinomycetes were probed with pcbR, hybridization was observed to some but not all beta-lactam-producing actinomycetes.

Streptomyces clavuligerus produced cephamycin C under all nutrient limitations investigated in batch culture, whilst clavulanic acid was produced under phosphate and carbon limitations. Guanosine 5′-diphosphate 3′-diphosphate (ppGpp) was produced at very low levels in all fermentations, prior to the detection of cephamycin C in the fermentation broth. Guanosine 5′-diphosphate 3′-monophosphate (ppGp) was observed at high levels in all fermentations; it appeared following a downturn in nutrient levels and immediately prior to detection of isopenicillin N synthase (IPNS). ppGp was detected following nutritional shiftdown by amino acid depletion and it was not produced via degradation of ppGpp. The results point to a possible role for ppGp in the regulation of cephamycin production in S. clavuligerus.

The bla gene of the cephamycin cluster of Nocardia lactamdurans has been subeloned in the shuttle plasmids pULVK2 and pULVK2A and amplified in N. lactamdurans LC411. The transformants showed two- to threefold higher beta-lactamase activity. Formation of beta-lactamase preceded the onset of cephamycin biosynthesis. The beta-lactamase of N. lactamdurans inactivated penicillins and, to a lesser extent, cephalosporin C but did not hydrolyse cephamycin C. This beta-lactamase was highly sensitive to clavulanic acid (50% inhibition was observed at 0.48 microgram/ml clavulanic acid). The N. lactamdurans bla gene was disrupted in vivo by inertion of the kanamycin-resistance gene. Three bla-disrupted mutants, BD4, BD8 and BD12, were selected that lacked beta-lactamase activity. Overexpresion of the bla gene resulted in N. lactamdurans transformants that were resistant to penicillin whereas mutants in which the bla gene was disrupted were super-sensitive to this antibiotic. The three N. lactamdurans mutants with the bla gene disrupted showed a significant increase of cephamycin biosynthesis in solid medium, whereas transformants with the amplified bla gene produced reduced levels of cephamycin. The cephamycin-overproducing Merck strain N. lactamdurans MA4213 showed no detectable levels of beta-lactamase activity. The beta-lactamase plays a negative role in cephamycin biosynthesis in solid medium, but not in liquid medium.

The phenotype of Klebsiella pneumoniae HEL-1 indicates a plasmidic cephamycinase gene (blaCMY-2). Its sequence shows one open reading frame coding for a protein of 381 amino acids. CMY-2 is classified as class C beta-lactamase that is closely related to the plasmidic enzymes BIL-1 and LAT-1 and the chromosomal AmpC of Citrobacter freundii. The blaCMY-2 gene possibly was translocated onto a plasmid of C. freundii which spread to K. pneumoniae.

Sequencing of ORF10 (gene cmcH) of the Nocardia lactamdurans cephamycin gene cluster proved that it encodes a protein with a deduced molecular mass of 57,149 Da. This protein showed significant similarity to the putative O-carbamoyltransferases (O-Cases) encoded by the nodU genes of Rhizobium fredii and Bradyrhizobium japonicum, involved in the synthesis of nodulation factors. The carbamoyl-phosphate (CP)-binding amino-acid sequence of human OTCase is conserved in the cmcH product. A similar cmcH (80% identify in a 160-nt fragment) in the cephamycin (CmC) cluster of cmc genes of Streptomyces clavuligerus was partially sequenced. The cmcH gene is closely linked to and in the same orientation as cefF in both organisms. Both cmcH were subcloned in pIJ702 and expressed in Streptomyces lividans. Extracts of transformants could carbamoylate decarbamoylcefuroxime. A similar cmcH was found by Southern hybridization in Streptomyces cattleya, but not in Streptomyces griseus or Streptomyces lipmanii which produce non-carbamoylated CmC.

The genes pcbAB, pcbC and penDE encoding enzymes involved in the biosynthesis of penicillin have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are clustered in chromosome I (10.4 Mb) of P. chrysogenum, but they are located in chromosome II of Penicillium notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. Expression studies have shown that each gene is expressed as a single transcript from separate promoters. Enzyme regulation studies and gene expression analysis have provided useful information to understand the control of gene expression leading to overexpression of the genes involved in penicillin biosynthesis. Cephalosporin genes have been studied in Cephalosporium acremonium and also in cephalosporin-producing bacteria. In C. acremonium the genes involved in cephalosporin biosynthesis are separated in at least two clusters. Cluster I (pcbAB-pcbC) encodes the first two enzymes of the cephalosporin pathway which are very similar to those involved in penicillin biosynthesis. Cluster II (cefEF-cefG), encodes the last three enzymatic activities of the cephalosporin pathway. It is unknown, at this time, if the cefD gene encoding isopenicillin epimerase is linked to any of the two clusters. In cephamycin producing bacteria the genes encoding the entire biosynthetic pathway are located in a single cluster extending for about 30 kb in Nocardia lactamdurans, and in Streptomyces clavuligerus. The cephamycin clusters of N. lactamdurans and S. clavuligerus include a gene lat which encodes lysine-6-aminotransferase an enzyme involved in formation of the precursor alpha-aminoadipic acid. The N. lactamdurans cephamycin cluster includes, in addition, a beta-lactamase (bla) gene, a penicillin binding protein (pbp), and a transmembrane protein gene (cmcT) that is probably involved in secretion of the cephamycin. Little is known however about the mechanism of control of gene expression in the different beta-lactam producers. The availability of most of the structural genes provides a good basis for further studies on gene expression. This knowledge should lead in the next decade to a rational design of strain improvement procedures. The origin and evolution of beta-lactam genes is intriguing since their nucleotide sequences are extremely conserved despite their restricted distribution in the microbial world.

The class A carbapenemase KPC-6 produces resistance to a broad range of β-lactam antibiotics. This enzyme hydrolyzes penicillins, the monobactam aztreonam, and carbapenems with similar catalytic efficiencies, ranging from 10 5 to 10 6 M −1 s −1 . The catalytic efficiencies of KPC-6 against cephems vary to a greater extent, ranging from 10 3 M −1 s −1 for the cephamycin cefoxitin and the extended-spectrum cephalosporin ceftazidime to 10 5 to 10 6 M −1 s −1 for the narrow-spectrum and some of the extended-spectrum cephalosporins.

We investigated the efficiency of the cephamycin cefoxitin as an alternative to carbapenems for the treatment of urinary tract infections (UTIs) due to Escherichia coli producing CTX-M-type extended-spectrum β-lactamases. The susceptible, UTI-inducing E. coli CFT073-RR strain and its transconjugant CFT073-RR Tc (p bla CTX-M-15 ), harboring a bla CTX-M-15 carrying-plasmid, were used for all experiments. MICs of cefoxitin (FOX), ceftriaxone (CRO), imipenem (IMP), and ertapenem (ETP) for CFT073-RR and CFT073-RR Tc (p bla CTX-M-15 ) were 4 and 4, 0.125 and 512, 0.5 and 0.5, and 0.016 and 0.032 μg/ml, respectively. Bactericidal activity was similarly achieved in vitro against the two strains after 3 h of exposure to concentrations of FOX, IMI, and ETP that were 2 times the MIC, whereas CRO was not bactericidal against CFT073-RR Tc (p bla CTX-M-15 ). The frequencies of spontaneous mutants of the 2 strains were not higher for FOX than for IMP or ETP. In the murine model of UTIs, mice infected for 5 days were treated over 24 h. Therapeutic regimens in mice (200 mg/kg of body weight every 3 h or 4 h for FOX, 70 mg/kg every 6 h for CRO, 100 mg/kg every 2 h for IMP, and 100 mg/kg every 4 h for ETP) were chosen in order to reproduce the percentage of time that free-drug concentrations above the MIC are obtained in humans with standard regimens. All antibiotic regimens produced a significant reduction in bacterial counts (greater than 2 log 10 CFU) in kidneys and bladders for both strains ( P < 0.001) without selecting resistant mutants in vivo , but the reduction obtained with CRO against CFT073-RR Tc (p bla CTX-M-15 ) in kidneys was significantly lower than that obtained with FOX. In conclusion, FOX appears to be an effective therapeutic alternative to carbapenems for the treatment of UTIs due to CTX-M-producing E. coli .