Development of an optimized broth enrichment culture medium for the isolation of Clostridium difficile.

Clostridium difficile is a spore forming bacterium and the leading cause of colitis and antibiotic associated diarrhoea in the developed world. Effective recovery of spores, particularly in low numbers, is imperative to obtain accurate prevalence data, due to the low number of spores found within non-clinical samples (<20/ml). Through comparison of C. difficile enrichment media, this study showed the importance of selecting an effective enrichment media. Commonly used broths, such as Cooked Meat broth, promote significantly less growth than other available broths such as Brain Heart Infusion broth, BHI. The optimization of BHI using selective antibiotics, moxalactam and norfloxacin, and sodium taurocholate at a concentration of 0.4%, allowed for high growth rate (0.465 h-1), short lag times (<14 h) and recovery of spores at low concentrations. The optimized broth, designated BHIMN-T, out-performed other commonly used broths so can be recommended for future studies.

Crystal structure of Mox-1, a unique plasmid-mediated class C ß-lactamase with hydrolytic activity towards moxalactam.

Mox-1 is a unique plasmid-mediated class C ß-lactamase that hydrolyzes penicillins, cephalothin, and the expanded-spectrum cephalosporins cefepime and moxalactam. In order to understand the unique substrate profile of this enzyme, we determined the X-ray crystallographic structure of Mox-1 ß-lactamase at a 1.5-Å resolution. The overall structure of Mox-1 ß-lactamase resembles that of other AmpC enzymes, with some notable exceptions. First, comparison with other enzymes whose structures have been solved reveals significant differences in the composition of amino acids that make up the hydrogen-bonding network and the position of structural elements in the substrate-binding cavity. Second, the main-chain electron density is not observed in two regions, one containing amino acid residues 214 to 216 positioned in the Ω loop and the other in the N terminus of the B3 ß-strand corresponding to amino acid residues 303 to 306. The last two observations suggest that there is significant structural flexibility of these regions, a property which may impact the recognition and binding of substrates in Mox-1. These important differences allow us to propose that the binding of moxalactam in Mox-1 is facilitated by the avoidance of steric clashes, indicating that a substrate-induced conformational change underlies the basis of the hydrolytic profile of Mox-1 ß-lactamase.

A potential substrate binding conformation of ß-lactams and insight into the broad spectrum of NDM-1 activity.

New Delhi metallo-ß-lactamase 1 (NDM-1) is a key enzyme that the pathogen Klebsiella pneumonia uses to hydrolyze almost all ß-lactam antibiotics. It is currently unclear why NDM-1 has a broad spectrum of activity. Docking of the representatives of the ß-lactam families into the active site of NDM-1 is reported here. All the ß-lactams naturally fit the NDM-1 pocket, implying that NDM-1 can accommodate the substrates without dramatic conformation changes. The docking reveals two major binding modes of the ß-lactams, which we tentatively name the S (substrate) and I (inhibitor) conformers. In the S conformers of all the ß-lactams, the amide oxygen and the carboxylic group conservatively interact with two zinc ions, while the substitutions on the fused rings show dramatic differences in their conformations and positions. Since the bridging hydroxide ion/water in the S conformer is at the position for the nucleophilic attack, the S conformation may simulate the true binding of a substrate to NDM-1. The I conformer either blocks or displaces the bridging hydroxide ion/water, such as in the case of aztreonam, and is thus inhibitory. The docking also suggests that substitutions on the ß-lactam ring are required for ß-lactams to bind in the S conformation, and therefore, small ß-lactams such as clavulanic acid would be inhibitors of NDM-1. Finally, our docking shows that moxalactam uses its tyrosyl-carboxylic group to compete with the S conformer and would thus be a poor substrate of NDM-1.

Three factors that modulate the activity of class D ß-lactamases and interfere with the post-translational carboxylation of Lys70.

The activity of class D ß-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D ß-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 ß-lactamase.

Structural insights into the design of inhibitors for the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia.

One mechanism by which bacteria can escape the action of beta-lactam antibiotics is the production of metallo-beta-lactamases. Inhibition of these enzymes should restore the action of these widely used antibiotics. The tetrameric enzyme L1 from Stenotrophomonas maltophilia was used as a model system to determine a series of high-resolution crystal structures of apo, mono and bi-metal substituted proteins as well as protein-inhibitor complexes. Unexpectedly, although the apo structure revealed only few significant structural differences from the holo structure, some inhibitors were shown to induce amino acid side-chain rotations in the tightly packed active site. Moreover, one inhibitor employs a new binding mode in order to interact with the di-zinc center. This structural information could prove essential in the process of elucidation of the mode of interaction between a putative lead compound and metallo-beta-lactamases, one of the main steps in structure-based drug design.

A theoretical study on the substrate deacylation mechanism of class C beta-lactamase.

The whole reaction of the deacylation of class C beta-lactamase was investigated by performing quantum chemical calculations under physiological conditions. In this study, the X-ray crystallographic structure of the inhibitor moxalactam-bound class C beta-lactamase (Patera et al. J. Am. Chem. Soc. 2000, 122, 10504-10512.) was utilized and moxalactam was changed into the substrate cefaclor. A model for quantum chemical calculations was constructed using an energy-minimized structure of the substrate-bound enzyme obtained by molecular mechanics calculation, in which the enzyme was soaked in thousands of TIP3P water molecules. It was found that the deacylation reaction consisted of two elementary processes. The first process was formation of a tetrahedral intermediate, which was initiated by the activation of catalytic water by Tyr150, and the second process was detachment of the hydroxylated substrate from the enzyme, which associated with proton transfer from the side chain of Lys67 to Ser64O(gamma). The first process is a rate-determining process, and the activation energy was estimated to be 30.47 kcal/mol from density functional theory calculations considering electron correlation (B3LYP/6-31G**). The side chain of Tyr150 was initially in a deprotonated state and was stably present in the active site of the acyl-enzyme complex, being held by Lys67 and Lys315 cooperatively.

Noncovalent interaction energies in covalent complexes: TEM-1 beta-lactamase and beta-lactams.

The class A beta-lactamase TEM-1 is a key bacterial resistance enzyme against beta-lactam antibiotics, but little is known about the energetic bases for complementarity between TEM-1 and its inhibitors. Most inhibitors form a covalent adduct with the catalytic Ser70, making the measurement of equilibrium constants, and hence interaction energies, technically difficult. This study evaluates noncovalent interactions within covalent complexes by examining the differential stability of TEM-1 and its inhibitor adducts. The thermal denaturation of TEM-1 follows a two-state, reversible model with a melting temperature (T(m)) of 51.6C and a van't Hoff enthalpy of unfolding (DeltaH(VH)) of 146.2 kcal/mol at pH 7.0. The stability of the enzyme changes on forming an inhibitor adduct. As expected, some inhibitors stabilize TEM-1; transition-state analogues increase the T(m) by up to 3.7C (1.7 kcal/mol). Surprisingly, all beta-lactam covalent acyl--enzyme complexes tested destabilize TEM-1 significantly relative to the apo-enzyme. For instance, the clinically used inhibitor clavulanic acid and the beta-lactamase-resistant beta-lactams moxalactam and imipenem destabilize TEM-1 by over 2.6C (1.2 kcal/mol) in their covalent adducts. Based on the structure of the TEM-1/imipenem complex (Maveyraud et al., J Am Chem Soc 1998;120:9748--52), destabilization by moxalactam and imipenem is thought to be caused by a steric clash between the side-chain of Asn132 and the 6(7)-alpha group of these beta-lactams. To test this hypothesis, the mutant enzyme N132A was made. In contrast with wild-type, the covalent complexes between N132A and both imipenem and moxalactam stabilize the enzyme, consistent with the hypothesis. To investigate the structural bases of this dramatic change in stability, the structure of N132A/imipenem was determined by X-ray crystallography. In the complex with N132A, imipenem adopts a very different conformation from that observed in the wild-type complex, and the putative destabilizing interaction with residue 132 is relieved. Studies of several enzymes suggest that beta-lactams, and covalent inhibitors in general, can have either net favorable or net unfavorable noncovalent interaction energies within the covalent complex. In the case of TEM-1, such unfavorable interactions convert substrate analogues into very effective inhibitors.

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

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

Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation.

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.

A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae.

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum beta-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Glu104-->Lys, Met182-->Thr, and Gly238-->Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated blaTEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (Ki, 0.16 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM). Allelic replacement analysis revealed that the combination of Lys104, Thr182, and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.

DNA shuffling of a family of genes from diverse species accelerates directed evolution.

DNA shuffling is a powerful process for directed evolution, which generates diversity by recombination, combining useful mutations from individual genes. Libraries of chimaeric genes can be generated by random fragmentation of a pool of related genes, followed by reassembly of the fragments in a self-priming polymerase reaction. Template switching causes crossovers in areas of sequence homology. Our previous studies used single genes and random point mutations as the source of diversity. An alternative source of diversity is naturally occurring homologous genes, which provide 'functional diversity'. To evaluate whether natural diversity could accelerate the evolution process, we compared the efficiency of obtaining moxalactamase activity from four cephalosporinase genes evolved separately with that from a mixed pool of the four genes. A single cycle of shuffling yielded eightfold improvements from the four separately evolved genes, versus a 270- to 540-fold improvement from the four genes shuffled together, a 50-fold increase per cycle of shuffling. The best clone contained eight segments from three of the four genes as well as 33 amino-acid point mutations. Molecular breeding by shuffling can efficiently mix sequences from different species, unlike traditional breeding techniques. The power of family shuffling may arise from sparse sampling of a larger portion of sequence space.

Improvement of the rectal bioavailability of latamoxef sodium by adjuvants following administration of a suppository.

The absorption of an antibiotic, latamoxef sodium (LMOX), following the rectal administration of a suppository containing adjuvants was investigated. A lipophilic base (Witepsol H15) was used. The rectal absorption of LMOX following the administration of a suppository without adjuvants was very low. Diclofenac sodium (DF) was used as an absorption promoter; it enhances rectal membrane permeability. The blood level of LMOX following the addition of DF(10 mg) to the base was increased only about 1.3-fold compared with that achieved with LMOX alone (difference not significant); even with a higher dose of DF, the absorption of LMOX was not sufficient. The release rate of LMOX from the base was slow. When Tween 80, a non-ionic surfactant, was added to improve the release rate of LMOX, the rate was sufficiently increased. The rectal absorption of LMOX on the addition of both Tween 80 and DF was markedly increased compared to that achieved with LMOX alone or with DF. These results indicate that the rectal absorption of LMOX after administration by a suppository was sufficiently improved by enhancing both the release rate from the base and the membrane permeability of the rectum. Lymphatic uptake and blood levels of LMOX were also investigated after the rectal administration of the LMOX preparation containing both Tween 80 and DF; the lymphatic uptake of LMOX was significantly enhanced compared with the LMOX preparation in which only DF was used as an adjuvant. The mechanism whereby adjuvants lead to the absorption of a non-absorbable drug, and the subsequent drug transportation routes through the membrane are discussed.

[Protein binding of various cephems in healthy subjects and patients with chronic renal failure].

This study was employed to investigate whether the serum protein binding of various cephems [cefpiramide (CPM), cefalotin (CET), latamoxef (LMOX)] differ among healthy subjects and patients with chronic renal failure (CRF) by means of in vitro equilibrium dialysis. The protein binding capacities of cephems in patients with CRF (hemodialysis, continuous ambulatory peritoneal dialysis, non-dialysis) decreased significantly compared to those in healthy subjects. The binding capacities correlated directly with total protein, albumin concentration and correlated inversely with blood urea nitrogen and serum creatinine concentration. In the study of protein binding during and after hemodialysis, the binding capacities of CPM and LMOX decreased immediately after dialysis and then increased with the time. However, the binding capacities of CET increased immediately after dialysis and then decreased. The binding capacities of CPM and LMOX correlated inversely with non-esterified fatty acids (NEFA) and those of CET correlated directly with NEFA. In the study of protein binding in pooled sera from healthy subjects with or without palmitic acid (PA), the binding capacities of CPM and LMOX decreased by increasing the concentration of PA, while those of CET increased by increasing PA up to 3 mM. The changes in binding capacity of cephems during and after hemodialysis have been possibly caused by increase of NEFA due to activation of lipase in use of heparin as an anticoagulant. In conclusion, changes in protein binding capacity of cephems in sera from CRF, which should be taken into consideration to avoid possible side effects.

Comparison of N-methylthiotetrazole dispositions in healthy volunteers following single intravenous doses of moxalactam, cefoperazone, and cefotetan.

The N-methylthiotetrazole side chain (NMTT) that is present on several cephalosporins has been implicated in the development of antibiotic-associated hypoprothrombinemia. A randomized three-way crossover trial was conducted to compare the release of the NMTT side chain from three NMTT-containing antibiotics. Single 2-g doses of moxalactam, cefoperazone, and cefotetan were given, followed by serial blood and urine sampling. The concentrations of the parent compound and the NMTT side chain in plasma, urine, and the reconstituted antibiotic solution were determined by high-pressure liquid chromatography. Peak NMTT concentrations ranged from 0.42 to 16.50 micrograms/ml and were significantly higher after moxalactam administration than after cefoperazone or cefotetan administration (P less than 0.01). The NMTT trough concentrations (12.5 h) ranged from nondetectable to 2.47 micrograms/ml and tended to be greater following cefoperazone administration. The amounts of NMTT administered (e.g., the amount in the reconstituted antibiotic solution) were 25.8 +/- 1.4, 15.2 +/- 0.9, and 22.1 +/- 3.0 mg following moxalactam, cefoperazone, and cefotetan administration, respectively (P less than 0.01). In contrast, urinary recoveries of NMTT were 57.4 +/- 26.2, 73.6 +/- 44.3, and 29.7 +/- 22.9 mg following moxalactam, cefoperazone, and cefotetan, respectively. The amount of NMTT formed in vivo and excreted unchanged, as assessed by subtracting in vitro NMTT formation from NMTT urinary recovery, was significantly higher after cefoperazone than after moxalactam or cefotetan administration (P less than 0.05). The discrepancy between in vitro NMTT production (moxalactam > cefotetan > cefoperazone) and the amount of NMTT formed in vivo and excreted unchanged (cefoperazone > moxalactam > cefotetan) demonstrated that the in vivo production of NMTT is dependent on the disposition of the parent cephalosporin.

A survey of the kinetic parameters of class C beta-lactamases. Cephalosporins and other beta-lactam compounds.

Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates of six class C beta-lactamases. Nitrocefin, cephaloridine, cefazolin, cephalothin and cephalexin were good substrates, with kcat. values ranging from 27 to 5000 s-1. Cefuroxime, cefotaxime and cefoxitin exhibited low kcat. values (0.010-1.7 s-1) and low Km values, which suggested a rate-limiting deacylation. Imipenem and aztreonam were even poorer substrates (kcat. 2 x 10(-4)-3 x 10(-2) s-1) and, in the presence of a reporter substrate, behaved as transient inactivators. With moxalactam, biphasic kinetics were observed, indicating a possible rearrangement of the acyl-enzyme.

Disposition of moxalactam and N-methyltetrazolethiol in rats and monkeys.

The disposition of moxalactam (MOX) and N-methyltetrazolethiol (NMTT) in rats and monkeys after intravenous injection was investigated, focusing on the in vivo liberation of NMTT, by using [NMTT-14C]MOX and [14C]NMTT. After [NMTT-14C]MOX injection, MOX levels in plasma quickly became high in both rats and monkeys and then declined, with half-lives at the beta phase of 18.8 and 67.1 min, respectively. The levels of NMTT liberated from MOX were much lower than those of MOX, but the apparent elimination was significantly slow. The levels of MOX and NMTT in rat liver were almost comparable but lower than those in plasma. With [14C]NMTT administration, the level of NMTT in plasma declined, with half-lives at the beta phase of 21.5 min in rats and 54.0 min in monkeys. After [NMTT-14C]MOX injection, most of the radioactivity was excreted in urine as MOX, with 11% of the dose in rats and 8% of the dose in monkeys eliminated as NMTT until 24 h. Total biliary excretion was 26% of the injected radioactivity in rats, and most of it was due to MOX. In one monkey, the total biliary excretion was only 0.2% of the injected radioactivity. With [14C]NMTT administration, most radioactivity was excreted in the urine as unchanged NMTT in both animals. Oral administration in rats showed that part of the biliary-excreted MOX was degraded to NMTT in the intestine and then absorbed. Repeated administration of [NMTT-14C]MOX to rats did not change the levels of MOX and NMTT in plasma or liver nor did it change the excretion profiles. Thus, accumulation of MOX and NMTT did not occur.

Single-dose antibiotic prophylaxis for biliary surgery. Cefazolin vs moxalactam.

Cefazolin was compared with moxalactam for single-dose prophylaxis against infection in a double-blind, prospective, randomized trial of 90 patients undergoing cholecystectomy. Risk factors for infection were present in 65 (72%) of the 90 patients and were evenly distributed. Antibiotic levels in plasma, bile, and tissue measured when the cystic duct was divided were similar for both drugs. Age greater than 65 years but not recent cholecystitis or type of antibiotic was predictive of recovery of bacteria from bile cultures. Wound infections occurred in two patients receiving cefazolin and one patient receiving moxalactam for an overall infection rate of 3%. No toxic reactions to antibiotics, including bleeding disorders, were observed. In conclusion, no significant difference in prophylactic efficacy was detected in this comparison of a first-generation with a third-generation cephalosporin. Because of its lower cost and narrower antimicrobial spectrum, however, cefazolin should remain the agent of choice.

'Covalent trapping' and latamoxef resistance in beta-lactamase-derepressed Pseudomonas aeruginosa.

Three Pseudomonas aeruginosa strains which constitutively produced chromosomal (Id, or Sabath and Abraham) beta-lactamase in large amounts were resistant to latamoxef (moxalactam) MICs, 128-256 mg/l). Their beta-lactamase-basal mutants, which produced 1200-18,000-fold less enzyme, were latamoxef-sensitive (MICs, 4-16 mg/l), suggesting that the enzyme caused the resistance of the parent organisms. Latamoxef was a feeble substrate of the enzyme (kcat less than 0.5/min) but reacted to form a stable complex that lacked catalytic activity against benzylpenicillin. The complex was isolated by gel filtration and was shown to be stable to isoelectric focusing, suggesting a covalent link between the enzyme and latamoxef. During incubation the complex underwent a slow breakdown, regenerating active enzyme. This breakdown obeyed first-order kinetics, and the half-life of the inactivated form was 19 +/- 1 min at 37 degrees C. Binding of antibiotic molecules in this complex may contribute to the latamoxef-resistance observed in the beta-lactamase-derepressed strains. This 'covalent trapping' should be distinguished from the 'non-covalent trapping' proposed elsewhere as a general mechanism of beta-lactamase-mediated resistance to reversibly-bound weak-substrate beta-lactams.