Analysis of bacterial carbapenem antibiotic production genes reveals a novel β-lactam biosynthesis pathway

Carbapenems are β-lactam antibiotics which have an increasing utility in chemotherapy, particularly for nosocomial, multidrug-resistant infections. Strain GS101 of the bacterial phytopathogen, Erwinia carotovora , makes the simple β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have mapped and sequenced the Erwinia genes encoding carbapenem production and have cloned these genes into Escherichia coli where we have reconstituted, for the first time, functional expression of the β-lactam in a heterologous host. The carbapenem synthesis gene products are unrelated to enzymes involved in the synthesis of the so-called sulphur-containing β-lactams, namely penicillins, cephamycins and cephalosporins. However, two of the carbapenem biosynthesis genes, carA and carC , encode proteins which show significant homology with proteins encoded by the Streptomyces clavuligerus gene cluster responsible for the production of the β-lactamase inhibitor, clavulanic acid. These homologies, and some similarities in genetic organization between the clusters, suggest an evolutionary relatedness between some of the genes encoding production of the antibiotic and the β-lactamase inhibitor. Our observations are consistent with the evolution of a second major biosynthetic route to the production of β-lactam-ring-containing antibiotics.     

β-Lactam and aminoglycoside production from streptomycetes

Streptomyces cattleya, S. fradiae and S. griseus produced different amounts of growth when cultured sequentially through sporulation, vegetative and antibiotic production media. Only S. griseus grew well on all three types of medium. Streptomyces cattleya grew poorly on both sporulation and vegetative media. Growth was 1·6 and 8·0 mg/1/h respectively. For all three species, biomass yield in the final antibiotic production medium was dependent on amount of inoculum. Antibiotic yields were obtained only from production media. Under slow growth conditions l -cysteine and l -valine supplementation stimulated S. cattleya β-lactam production, giving 1000 μg/ml β-lactam equivalents compared with 45 μg/ml β-lactam equivalents for no supplementation. For aminoglycosides the agar well diffusion bioassay was more sensitive towards the hydrochloride than the neutral salt. Paper chromatography confirmed the main antibiotic classes. R _F values for replicate samples indicated aminoglycoside homogeneity and β-lactam heterogeneity.     

Methicillin-resistant strains of Staphylococcus aureus; presence of identical additional penicillin-binding protein in all strains examined

Abstract The methicillin-resistant strain of Staphylococcus aureus MR-1 previously reported to possess a penicillin-binding protein 3 (PBP 3) with a decreased affinity for β-lactam antibiotics was re-examined and, in common with other resistant strains, found to contain an additional PBP (PBP 2'). Expression of the additional protein, which has a very low affinity for β-lactams, was not influenced by temperature or osmolarity of the medium in contrast with strains examined previously. It was the only PBP still available to bind radioactive β-lactams and therefore still active enzymically when strain MR-1 was grown in the presence of concentrations of β-lactam antibiotics sufficient to kill sensitive strains of S. aureus . Penicillin-peptides derived by partial proteolysis of PBP 2'-penicillin complexes of MR-1 and 3 other methicillin-resistant strains appeared to be identical and different from the penicillin-peptides derived from PBP 1, PBP 2 and PBP 3, each of which gave rise to a unique series of peptides containing covalently-bound penicillin.     

Cloning and expression in Enterobacteriaceae of the extended-spectrum β-lactamase gene from a Pseudomonas aeruginosa plasmid

Abstract An extended-spectrum β-lactamase, the gene for which is located on plasmid pMS350 in Pseudomonas aeruginosa strains, hydrolyzes carbapenems and other extended-spectrum β-lactam antibiotics. We cloned the pMS350 β-lactamase gene in an Escherichia coli K-12 strain using the vector plasmid pHSG398, and subcloned it into pMS360, a plasmid with a wide host-range. This resulted in the formation of the recombinant plasmid, pMS363, containing a 4.1-kb DNA insert that includes the extended-spectrum β-lactamase gene. Plasmid pMS363 was introduced into the P. aeruginosa PAO strain or into six species of Enterobacteriaceae, and the specific activities of the β-lactamase and MICs of various β-lactam antibiotics were estimated. The cloned gene was capable of expression in these strains and caused resistance to carbapenem, penem and other β-lactam antibiotics, with the exception of aztreonam.     

Different Penicillin-Binding Protein Profiles in Amoxicillin-Resistant Helicobacter pylori

Background. The β-lactam group of antibiotics kills bacteria by inhibiting the terminal stages of peptidoglycan metabolism. We have recently identified amoxicillin-resistant Helicobacter pylori , none of which expressed β-lactamase. Penicillin-binding proteins (PBPs) represent a group of target enzymes for the β-lactam antibiotic family, and alterations in PBPs have been described in other penicillin-resistant bacteria. The amoxicillin-resistant phenotype characteristically was lost after freezing but could be restored by consecutive transfers into gradient plates.
Materials and Methods. To determine whether amoxicillin resistance in H. pylori was related to alterations in any of the H. pylori PBPs, five H. pylori strains resistant to amoxicillin and three amoxicillin-sensitive strains were tested. PBPs were extracted from bacteria grown to logarithmic phase, labeled in vivo with ³H-benzylpenicillin, and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Four main PBPs were separated from all amoxicillin-sensitive H. pylori strains.
Results. Only three of the four main PBPs were found in the amoxicillin-resistant H. pylori strains. The differentially detectable PBP (PBP D) had an apparent molecular weight of 30 to 32 kD.
Conclusion. These results suggest that PBP D might play a role in the amoxicillin-resistant phenotype of H. pylori strains lacking β-lactamase activity.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Giant linear plasmids of β-lactam antibiotic producing Streptomyces

Abstract A survey of the total cellular DNA from five β-lactam antibiotic-producing Streptomyces spp. by pulsed field gel electrophoresis was conducted to investigate the presence of linear plasmids. Streptomyces clavuligerus NRRL 3585 contained two giant linear plasmids of 120 and 430 kb, in addition to the well-characterized 11.7 kb linear plasmid. Streptomyces griseus NRRL 3851 contained a single giant linear plasmid of 120 kb, and Streptomyces jumonjinensis NRRL 5741 contained two giant linear plasmids (220 and 280 kb), and two smaller linear plasmids. No plasmids were identified in Streptomyces cattleya NRRL 3841 or Streptomyces lipmannii NRRL 3584. Southern hybridization did not reveal any homology shared by these plasmids, and β-lactam antibiotic synthesis gene clusters were located on the chromosome.     

Cloning, sequencing and expression in Escherichia coli of the low-affinity penicillin binding protein of Enterococcus faecalis

Abstract Low-affinity penicillin binding proteins are particular membrane proteins, in several Gram-positive bacteria, which are involved in β-lactam antibiotic resistance. The structural gene for the low-affinity penicillin binding protein 5 (PBP5) of Enterococcus faecalis was cloned and sequenced. From the sequence of the 3378 bp, a 2040 bp coding region was identified. From biochemical analysis it emerges that E. faecalis PBP5 is a type II membrane protein with an uncleaved N-terminal and is composed of 679 amino acids with a molecular weight of 74055. This protein showed 48 and 33% of identity with Enterococcus hirae PBP5 and Staphylococcus aureus PBP2a, both low-affinity PBPs involved in β-lactam resistance. Anti-PBP5 antibodies cross-reacted with a membrane protein present in other species of enterococci, but the entire gene fragment cloned hybridized only with DNAs of E. faecalis strains, thus suggesting that genes coding for low-affinity PBPs of enterococci are not stictly homologous. In this experiment digoxigenin-labelled E. faecalis DNA was used.     

Involvement of O8-antigen in altering β-lactam antibiotic susceptibilities in Escherichia coli

In spite of being dispensable, O-antigens are believed to facilitate various cellular processes and alter antibiotic sensitivities. Escherichia coli K-12 (CS109) strains are lacking in O-antigens and are reported to be sensitive to antibiotics. To our surprise, E. coli 2443 (expressing O8-antigen) manifested two- to fourfold higher sensitivities toward penicillin and its derivatives than strain CS109. However, sensitivities toward other structurally unrelated antibiotics remained unchanged. To understand the rationale behind such observations, we replaced the rfb locus of strain 2443 with that of E. coli K-12. The β-lactam sensitivities of 2443 cells with replaced rfb locus appeared to be identical to those for CS109. Therefore, it is quite reasonable to hypothesize the possible involvement of O8-antigen in β-lactam sensitization.     

A microtitre plate-based assay for the screening of β-lactams

A.R. QUESADA, A. CAÑEDO, M.A. MORENO AND J.L. FERNÁNDEZ-PUENTES. 1996. A simple, rapid, sensitive and automatizable method for the detection and quantification of bacterial cell wall inhibitors has been developed. The procedure is characterized by the use of a micro-organism hypersensitive to β-lactam antibiotics that contains an inducible cytosolic β-galactosidase; this enzyme is released when the micro-organism cell wall is disrupted by the antibiotic action, and then measured by the use of a chromogenic substrate. The present method allows the detection of β-lactam traces in other non-β-lactam antibiotics, and has been successfully applied in the detection of small amounts of β-lactams in biological fluids such as milk and Actinomycetes fermentation broths. The easy automatization of this method makes it specially suitable for the screening of new antibiotics of natural origin.     

Combinations of Clavulanic Acid and other β-lactam Antibiotics against Strains of Pseudomonas aeruginosa, Serratia marcescens and other Bacteria

Combinations of clavulanic acid, a new β-lactamase inhibitor, with five cephalosporins and one cephamycin were tested against cell-free β-lactamases obtained from Serratia marcescens, Pseudomonas aeruginosa and an Enterobacter strain, 265A. Cefotaxime was the most resistant antibiotic and cephalothin the most sensitive antibiotic to β-lactamases. Low concentrations of clavulanic acid gave some protection against the Serratia and Pseudomonas enzymes. The most active source of β-lactamase was the 265A strain, against which only cefotaxime was highly resistant. Clavulanic acid had only a slight inhibitory effect on this enzyme, which was confirmed by an agar method, and potentiated slightly the activity of cephalothin and cefoxitin against two β-lactamase producing strains of Staphylococcus aureus. Lysis by cephalothin of one strain of S. marcescens was potentiated in the presence of clavulanic acid.     

Clavulanic acid biosynthesis and genetic manipulation for its overproduction

Clavulanic acid, a β-lactamase inhibitor, is used together with β-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other β-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.     

Preparation and application of microarrays for the detection of antibiotic resistance genes in samples isolated from Changchun,China

The emergence of antibiotic-resistant bacteria, especially tetracycline- and β-lactam-resistant bacteria, poses a great threat to human health. The purpose of this study was to develop and apply a suitable gene microarray for the detection of antibiotic resistance genes. We isolated 463 strains of bacteria from a hospital, a veterinary station, an animal nursery, and living environment of Changchun, China. After screening, it was found that 93.9% of these bacteria were resistant to tetracycline, 74.9% to ampicillin, 55.6% to deoxycycline, and 41.7% to ciprofloxacin. For amplification of antibiotic genes, we designed 28 pairs of primers. In addition, 28 hybridization probes for these genes were developed. The DNA microarray analysis was performed at 42°C for 5 h. We were successful in detecting 12 resistance genes by microarray analysis. After detection, we also evaluated the sensitivity of the microarray analysis. The LDL (Lowest Detection Level) of the microarray was 1 × 10⁶ copies/ml of template DNA. It is believed that such microarray-based determination of tetracycline and β-lactam resistance genes can have a potential application in clinical studies in the future.     

Secondary metabolism in simulated microgravity: β-lactam production by Streptomyces clavuligerus

Rotating bioreactors designed at NASA’s Johnson Space Center were used to simulate a microgravity environment in which to study secondary metabolism. The system examined was β-lactam antibiotic production by Streptomyces clavuligerus. Both growth and β-lactam production occurred in simulated microgravity. Stimulatory effects of phosphate and L-lysine, previously detected in normal gravity, also occurred in simulated microgravity. The degree of β-lactam antibiotic production was markedly inhibited by simulated microgravity. Received 17 May 1996/ Accepted in revised form 27 August 1996     

Evaluation of the antibiotic biosynthetic potential of the genus Amycolatopsis and description of Amycolatopsis circi sp. nov., Amycolatopsis equina sp. nov. and Amycolatopsis hippodromi sp. nov

Aims: To describe three new Amycolatopsis strains and assess the antibiotic biosynthetic potential of the genus. Methods and Results: Three strains, designated S1·3^T, S3·6^T and SE(8)3^T, belonging to the genus Amycolatopsis were isolated and found to cluster together by 16S rRNA and gyrB gene‐based phylogenetic analysis. Genetic distance values, based on the gyrB gene, were calculated between the strains and their closest relatives and were all above the threshold value of 0·02 that has been proposed to distinguish Amycolatopsis type strains. DNA–DNA hybridization experiments against related type strains confirmed that strain S3·6^T represents a unique genomic species. Strain S3·6^T was also found to be distinct from strains S1·3^T and SE(8)3^T, the latter two of which were also shown to be distinct from each other. Antibiotic biosynthetic genes were identified from multiple Amycolatopsis strains, and their presence was found to be phylogenetically associated. Conclusions: The data presented in this study indicate that strains S1·3^T, SE(8)3^T and S3·6^T belong to three novel species, for which the names Amycolatopsis circi sp. nov. (= DSM 45561^T = NRRL B‐24841^T), Amycolatopsis equina sp. nov. (= DSM 45563^T = NRRL B‐24842^T) and Amycolatopsis hippodromi sp. nov. (= DSM 45562^T = NRRL B‐24843^T) are proposed. Significance and Impact of the Study: Three new species of Amycolatopsis are described, and the knowledge of the antibiotic biosynthetic potential of the genus has been extended.     

A Note on the Anti-Pseudomonas Activity of Cephalosporins with Chlorhexidine

Thirty clinical isolates of Pseudomonas aeruginosa were tested for susceptibility to cephaloridine, cefuroxime, cefotaxime and cefoxitin with and without chlorhexidine in agar dilution 'chequerboard' titrations and antibiotic disc tests. A synergistic response (σFIC = < 0.7) in combined titration with chlorhexidine was seen in 50% of the strains with cefuroxime and cefoxitin and 13% with cefotaxime. Cephaloridine MICs mostly exceeded the tested range but a definite reduction in combination was shown with 33% of strains. Susceptibility to cephaloridine, cefuroxime and cefoxitin was not enhanced to clinically useful levels and the effect was seen only in chlorhexidine concentrations close to the MIC. Disc tests failed to show the antibacterial activity of the combinations. It is suggested that the level of the principal lesion induced by chlorhexidine in the inner membrane might be too deep to affect the penetration of β-lactam antibiotics to their site of action.     

Compensatory evolution of pbp mutations restores the fitness cost imposed by β-lactam resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.     

Conversion of phenylacetyl-cysteinyl-valine in vitro into penicillin G by isopenicillin N synthase of Streptomyces lactamdurans

Abstract Purified isopenicillin N synthase of Streptomyces lactamdurans JC1843 converted phenylacetyl-cysteinyl-valine, a structural analogue of the natural substrate (α-amino-adipyl-cysteinyl-valine), into a penicillinase-sensitive β-lactam antibiotic. The antibiotic was identified as penicillin G by HPLC. Cyclization of phenylacetyl cysteinylvaline into penicillin G required dithiothreitol (DTT), ascorbic acid and Fe²⁺. The affinity of the enzyme for phenylacetyl cysteinylvaline was about 20 times lower than the affinity for α-aminoadipyl-cysteinyl-valine.     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.