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.     

Detection of Penicillin-binding Proteins in the Endosymbiont of the Trypanosomatid Crithidia deanei

ABSTRACT. Growth by serial transfers of the trypanosomatid Crithidia deanei in culture medium containing 1 mg/ml of the β-lactam antibiotics ampicillin or cephalexin resulted in shape distortion of its endosymbiont. The endosymbiont first appeared as filamentous structures with restricted areas of membrane damage. An increase of electron lucid areas was also observed in the endosymbiont matrix. The continuous treatment with β-lactam antibiotics, resulted in endosymbiont membranes fragmentation; and later on the space previously occupied by the symbiont was identified as an electron lucid area in the host cytoplasm. The putative targets of β-lactam antibiotic were two membrane-bound penicillin-binding proteins (PBPs) detected in the Sarkosyl-soluble fraction of purified symbionts labeled with [³H]-benzylpenicillin. The apparent molecular weight of the proteins were 90 kDa (PBP1) and 45 kDa (PBP2). PBP2 represented 85% of the total PBP content in the membrane fraction of the endosymbionts. Competition experiments using the tested antibiotics and [³H]-benzylpenicillin showed that ampicillin and cephalexin have half saturating concentrations considerably higher than [³H]-benzylpenicillin and indicated that PBP1 is the probable lethal target of the antibiotics tested. These results suggest that a physiologically active PBP is present in the cell envelope of C. deanei endosymbionts and may play important roles in the control of processes such as cell division and shape determination.     

Phylogenetic analysis of the isopenicillin-N-synthetase horizontal gene transfer

A phylogenetic study of the isopenicillin-N-synthetase (IPNS) gene sequence from prokaryotic and lower eukaryotic producers of β-lactam antibiotics by means of a maximum-likelihood approach has been carried out. After performing an extensive search, rather than invoking a global molecular clock, the results obtained are best explained by a model with three rates of evolution. Grouped in decreasing order, these correspond toA. nidulans and then to the rest of the eukaryotes and prokaryotes, respectively. The estimated branching date between prokaryotic and fungal IPNS sequences (852 ±106 MY) strongly supports the hypothesis that the IPNS gene was horizontally transferred from bacterial β-lactam producers to filamentous fungi. Correspondence to: A. Moya     

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.     

NMR spectrometric assay for determining enzymatic hydrolysis of β-lactam antibiotics with bacteria in aqueous solution

Abstract An application of a nuclear magnetic resonance (NMR) spectrometer for the measurement of β-lactamase activity in clinical material containing bacteria is presented. By means of proton (¹H)-NMR, it was easy to measure quantitatively β-lactamase activity in human bacteriuria, without performing any such pretreatment as isolation of bacteria or extraction of crude enzymes and without preparing special reagents for the detection. This is the first report on the application of ¹H-NMR analysis of structural changes for determining hydrolysis of β-lactam antibiotics with β-lactamase-producing bacteria in aqueous solution.     

Detailed studies on Quercus infectoria Olivier (nutgalls) as an alternative treatment for methicillin-resistant Staphylococcus aureus infections

Aims:  To investigate the antimethicillin-resistant Staphylococcus aureus (MRSA) mechanism of Quercus infectoria (nutgalls) extract and its components.
Methods and Results:  Ethanol extract, an ethyl acetate fraction I, gallic acid and tannic acid could inhibit the growth of clinically isolated MRSA strains with minimum inhibitory concentration values between 63 and 250  μ g ml⁻¹. Clumps of partly divided cocci with thickened cell wall were observed by transmission electron microscopy in the cultures of MRSA incubated in the presence of the ethanol extract, the ethyl acetate fraction I and tannic acid. Because cell wall structure of the organism structures seemed to be a possible site for antibacterial mechanisms, their effect with representative β-lactam antibiotics were determined. Synergistic effects with fractional inhibitory concentration index ranged from 0·24 to 0·37 were observed with 76% and 53% of the tested strains for the combination of the ethanol extract with amoxicillin and penicillin G, respectively.
Conclusions:  The appearance of pseudomulticellular bacteria in the treated cells and the synergistic effect of the plant extract with β-lactamase-susceptible penicillins suggest that the extract may interfere with staphylococcal enzymes including autolysins and β-lactamase.
Significance and Impact of the Study:  Our results provide scientific data on the use of the nutgalls, which contain mainly tannin contents up to 70% for the treatment of staphylococcal infections.     

The β-lactam-sensitive d,d-carboxypeptidase activity of Pbp4 controls the l,d and d,d transpeptidation pathways in Corynebacterium jeikeium

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to β-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of β-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d , d and l , d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt_Cjk1, a member of the active site cysteine l , d -transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of β-lactam, the PBPs and the l , d -transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt_Cjk1 and Pbp2C were not inhibited by ampicillin, the participation of the l , d -transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt_Cjk1 for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt_Cjk1 was produced by an ampicillin-sensitive d , d -carboxypeptidase (Pbp4_Cjk). Acquisition and mutational alterations of pbp2C accounted for high-level β-lactam resistance in C. jeikeium .     

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.     

An Electroacoustic Analysis for Determining the Effect of Amoxicillin on Microbial Cells

The probability of determining the effects of amoxicillin, which is one of β-lactam antibiotics, on microbial cells of Escherichia coli by the electroacoustic analysis method was shown for the first time. A piezoelectric resonator with a lateral electric field with a 1-mL liquid container was used as a biological sensor. It has been established that in the presence of amoxicillin the frequency dependence of the real and imaginary parts of the electrical impedance of a resonator loaded with a suspension of sensitive cells differs significantly from those of the resonator with a control of a microbial cell suspension without an antibiotic. When the resonator is loaded with the amoxicillin-resistant cell suspension, these dependencies are virtually the same. These results open prospects for the use of electroacoustic analysis methods to register the effect of β-lactam antibiotics on microbial cells and evaluate their antibacterial activity.     

Cloning of a beta-lactam resistance determinant of Enterobacter cloacae affecting outer membrane proteins of Enterobacteriaceae

Abstract In this paper we describe the cloning of a restriction fragment of Enterobacter cloacae chromosomal DNA that causes β-lactam resistance in both Escherichia coli HB101 and the parental strain E. cloacae 2249-1.
The increase in minimum inhibitory concentration (MIC) of the β-lactam antibiotics studied was not the result of enhanced β-lactamase production, but of a decrease in the concentration of the pore proteins OmpF and OmpC in E. coli and of a 37-kDa membrane protein in E. cloacae . The results obtained thus far indicate that we have cloned a gene encoding a 20 kDa polypeptide that is involved in the regulation of outer membrane protein synthesis.     

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-d-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.     

A sensitive enzymic assay for benzylpenicillin

A method has been developed for the determination of sodium benzylpenicillin concentrations in the range 3·3–33 μg/ml. 6-Aminopenicillanic acid is released from the benzylpenicillin by the action of the enzyme penicillin acylase and is estimated from its reaction with fluorescamine at pH 4.7-Aminocephalosporanic acid shows a similar trend to 6-aminopenicillanic acid in its reaction at pH 4. The open β-lactam ring form of each compound shows little fluorescence with fluorescamine at pH but shows strong fluorescence in the pH range 7–9. 6-Aminopenicillanic acid and its open β-lactam ring form give different fluorescent responses to increasing volumes of a solution of the fluorigenic agent at pH 7·8. This effect can be used to estimate concentrations in a mixture of the two components providing other amino material is absent.     

Cell Envelopes of Gram negative Bacteria: Composition, Response to Chelating Agents and Susceptibility of Whole Cells to Antibacterial Agents

The effects of ethylenediamine tetraacetic acid (EDTA) and related chelating agents on the sensitivity of isolated cell envelopes of some β-lactamase +ve and -ve strains of Gram negative bacteria have been investigated. Envelopes from Pseudomonas aeruginosa (especially strain NCTC 1999) contained the greatest amounts of Mg²⁺ and were the most sensitive to these agents in terms of (i) lysis, (ii) release of cations, (iii) release of readily extractable lipid. Cyclohexane—1,2, -diamine-tetraacetic acid was the most effective chelator, followed by EDTA and N -hydroxy-ethylethylenediamine triacetic acid, with nitriloacetic acid and iminodiacetic acid having little effect. A lysozyme–Tris–EDTA system also caused lysis of P. aeruginosa envelopes. The sensitivity of whole cells of the various strains to some β-lactam antibiotics and other antibacterial agents has been carried out and the basis of sensitivity or resistance in relation to drug destruction and the above envelope composition discussed.     

The increased sensitivity of rifamycin-resistant mutants of Methylobacterium AM1 to a variety of antimicrobial agents

Rifamycin-resistant strains of Methylobacterium AM1 are more sensitive to β-lactam antibiotics, chloramphenicol, tetracycline, spectinomycin, puromycin, nalidixic acid, benzalkonium chloride and p -fluorophenylalanine than are rifamycin-sensitive strains. This suggests that mutation to rifamycin resistance is associated with a general increase in membrane permeability and may have a bearing on the use of rifamycin-resistant mutants in genetic and biochemical studies of this organism.     

Supersensitivity to β-Lactam Antibiotics in Escherichia coli Caused by D-Alanine Carboxypeptidase IA Mutation

Escherichia coli cells acquired supersensitivity to various β-lactam antibiotics by dacA mutation, a defect in D-alanine carboxypeptidase IA activity. The mutant cells were rather less sensitive to mecillinam than the dacA⁺ cells. This mutation did not result in either thermosensitivity of cell growth or appreciable increase of the generation times in usual rich media, but the resulting appearance of supersensitivity to β-lactam antibiotics suggests that the cell wall or envelope of this mutant is somewhat abnormal and thus that D-alanine carboxypeptidase IA is involved in cell wall or envelope synthesis.     

Susceptibility of Gram-negative bacteria to β-lactam antibiotics and rapid characterization of β-lactamase activity

Various species of Gram-negative bacteria were tested for susceptibility to β-lactam antibiotics and for production of β-lactamase. The rapid cephalosporin 87/312 visual test was more sensitive than either the acidometric or the iodometric test; the iodometric test was least sensitive. Characteristic β-lactamase hydrolysis product patterns were obtained by scanning mixtures of β-lactamase-producing bacteria and cephalosporin substrates. β-Lactamase could not be detected on bacterial cells by fluorescent antibody techniques. The presence of β-lactamase can be correlated with minimal inhibitory concentrations of β-lactam antibiotics only in certain Gram-negative bacilli.     

Selective agents for listeria can inhibit their growth

The minimum inhibitory concentrations of four antibiotics used in listeria selective agars were determined at 30° and 37°C. Differences of susceptibility to β-lactam antibiotics and fosfomycin were observed in Listeria monocytogenes, L. seeligeri and L. ivanovii. Incubation at 30°C is recommended for listeria selective agars containing ceftazidime, cefotetan, latamoxef and fosfomycin.     

Critical Role of a Ferritin-Like Protein in the Control of Listeria monocytogenes Cell Envelope Structure and Stability under β-lactam Pressure

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenes Δ fri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure.     

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.