OmpA is the principal nonspecific slow porin of Acinetobacter baumannii.

Acinetobacter species show high levels of intrinsic resistance to many antibiotics. The major protein species in the outer membrane of Acinetobacter baumannii does not belong to the high-permeability trimeric porin family, which includes Escherichia coli OmpF/OmpC, and instead is a close homolog of E. coli OmpA and Pseudomonas aeruginosa OprF. We characterized the pore-forming function of this OmpA homolog, OmpA(Ab), by a reconstitution assay. OmpA(Ab) produced very low pore-forming activity, about 70-fold lower than that of OmpF and an activity similar to that of E. coli OmpA and P. aeruginosa OprF. The pore size of the OmpA(Ab) channel was similar to that of OprF, i.e., about 2 nm in diameter. The low permeability of OmpA(Ab) is not due to the inactivation of this protein during purification, because the permeability of the whole A. baumannii outer membrane was also very low. Furthermore, the outer membrane permeability to cephalothin and cephaloridine, measured in intact cells, was about 100-fold lower than that of E. coli K-12. The permeability of cephalothin and cephaloridine in A. baumannii was decreased 2- to 3-fold when the ompA(Ab) gene was deleted. These results show that OmpA(Ab) is the major nonspecific channel in A. baumannii. The low permeability of this porin, together with the presence of constitutive ß-lactamases and multidrug efflux pumps, such as AdeABC and AdeIJK, appears to be essential for the high levels of intrinsic resistance to a number of antibiotics.

Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli.

Multidrug efflux transporters, especially those that belong to the resistance-nodulation-division (RND) family, often show very broad substrate specificity and play a major role both in the intrinsic antibiotic resistance and, with increased levels of expression, in the elevated resistance of Gram-negative bacteria. However, it has not been possible to determine the kinetic behavior of these important pumps so far. This is partly because these pumps form a tripartite complex traversing both the cytoplasmic and outer membranes, with an outer membrane channel and a periplasmic adaptor protein, and it is uncertain if the behavior of an isolated component protein reflects that of the protein in this multiprotein complex. Here we use intact cells of Escherichia coli containing the intact multiprotein complex AcrB-AcrA-TolC, and measure the kinetic constants for various cephalosporins, by assessing the periplasmic concentration of the drug from their rate of hydrolysis by periplasmic beta-lactamase and the rate of efflux as the difference between the influx rate and the hydrolysis rate. Nitrocefin efflux showed a K(m) of about 5 microM with little sign of cooperativity. For other compounds (cephalothin, cefamandole, and cephaloridine) that showed lower affinity to the pump, however, kinetics showed strong positive cooperativity, which is consistent with the rotating catalysis model of this trimeric pump. For the very hydrophilic cefazolin there was little sign of efflux.

The growth rate of Mycobacterium smegmatis depends on sufficient porin-mediated influx of nutrients.

Mycobacteria have a unique outer membrane (OM) that is thicker than any other known biological membrane. Nutrients cross this permeability barrier by diffusion through porins. MspA is the major porin of Mycobacterium smegmatis. In this study we showed that three paralogues of MspA, namely MspB, MspC and MspD are also porins. However, only the mspA and mspC genes were expressed in the wild-type strain. None of the single deletion mutants displayed a significant OM permeability defect except for the mspA mutant. Deletion of the mspA gene caused activation of transcription of mspB and/or mspD in three independent strains by unknown chromosomal mutations. It is concluded that mspB and mspD provide backup porins for M. smegmatis. This also indicated that a minimal porin-mediated OM permeability is essential for survival of M. smegmatis. Electron microscopy in combination with quantitative image analysis of protein gels revealed that the number of pores per cell dropped from 2400 to 800 and 150 for the DeltamspA and DeltamspA DeltamspC mutant (ML10) respectively. The very low number of pores correlated well with the at least 20-fold lower channel activity of detergent extracts of the ML10 strain and its 15- and 75-fold lower permeability to nutrient molecules such as serine and glucose respectively. The amount of Msp porin and the OM permeability of the triple porin mutant lacking mspA, mspC and mspD was not altered. The growth rate of M. smegmatis dropped drastically with its porin-mediated OM permeability in contrast to porin mutants of Escherichia coli. These results show that porin-mediated influx of nutrients is a major determinant of the growth rate of M. smegmatis.

Calorimetric analysis of cephalosporins using an immobilized TEM-1 beta-lactamase on Ni2+ chelating sepharose fast flow.

Two beta-lactamases, penicillinase type I from Bacillus cereus and TEM-1 beta-lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of beta-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 beta-lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone.

MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis.

MspA is an extremely stable, oligomeric porin from Mycobacterium smegmatis that forms water-filled channels in vitro. Immunogold electron microscopy and an enzyme-linked immunosorbent assay demonstrated that MspA is localized in the cell wall. An mspA deletion mutant did not synthesize detectable amounts of mspA mRNA, as revealed by amplification using mspA-specific primers and reverse-transcribed RNA. Detergent extracts of the DeltamspA mutant exhibited a significantly lower porin activity in lipid bilayer experiments and contained about fourfold less porin than extracts of wild-type M. smegmatis. The chromosome of M. smegmatis encodes three proteins very similar to MspA. Sequence analysis of the purified porin revealed that mspB or mspC or both genes are expressed in the DeltamspA mutant. The properties of this porin, such as single channel conductance, extreme stability against denaturation, molecular mass and composition of 20 kDa subunits, are identical to those of MspA. Deletion of mspA reduced the cell wall permeability towards cephaloridine and glucose nine- and fourfold respectively. These results show that MspA is the main general diffusion pathway for hydrophilic molecules in M. smegmatis and was only partially replaced by fewer porins in the cell wall of the DeltamspA mutant [corrected] This is the first experimental evidence that porins are the major determinants of the exceptionally low permeability of mycobacteria to hydrophilic molecules.

Nucleotide sequence and characterization of a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-10) isolated in Spain.

A cefotaxime-resistant, ceftazidime-susceptible Escherichia coli isolate was obtained from a patient with sepsis in 1997, from which a beta-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate = 100), whereas ceftazidime hydrolysis was not detected. The nucleotide sequence revealed a bla gene related to that coding for CTX-M-3. Despite 21 nucleotide substitutions, only 2 determined amino acid changes (Ala27Val and Arg38Gln). The amino acid sequence identity between this enzyme, designated CTX-M-10, and the chromosomal beta-lactamase of Kluyvera ascorbata was 81%.

The interaction and transport of beta-lactam antibiotics with the cloned rat renal organic anion transporter 1.

In the present study, we investigated the interactions between antibiotics, especially beta-lactam antibiotics, and rat renal organic anion transporter 1 (OAT1). [(14)C]p-Aminohippurate (PAH) uptake via OAT1 expressed in Xenopus laevis oocytes was inhibited by all of the penicillins and cephalosporins tested. Penicillin G, carbenicillin, cephaloridine, cephalothin, cefazolin, and cephalexin inhibited [(14)C]PAH uptake via OAT1 in a competitive manner (K(i) = 0.29-2.33 mM). Cinoxacin, a quinolone gyrase inhibitor, also inhibited PAH uptake via OAT1. Other antibiotics, such as gentamicin, streptomycin, and vancomycin, which do not contain anionic moieties, did not interact with OAT1. [(3)H]Penicillin G and [(14)C]cephaloridine were demonstrated to be transported via OAT1. Using the cells that stably expressed OAT1, we analyzed the cytotoxicity of several beta-lactam antibiotics. Cells expressing OAT1 showed higher susceptibility to cephaloridine (a potentially nephrotoxic beta-lactam antibiotic) toxicity than did control cells. The present study suggests that OAT1 is the major organic anion transporter in the kidney that is responsible for the renal secretion of antibiotics, especially that of beta-lactam antibiotics. Furthermore, the culture cell system expressing OAT1 was revealed to be useful for the prediction of the nephrotoxicity of beta-lactam antibiotics.

The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase.

The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.

Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.

Gyrase mutations in laboratory-selected, fluoroquinolone-resistant mutants of Mycobacterium tuberculosis H37Ra.

To characterize mechanisms of resistance to fluoroquinolones by Mycobacterium tuberculosis, mutants of strain H37Ra were selected in vitro with ofloxacin. Their quinolone resistance-determining regions for gyrA and gyrB were amplified and sequenced to identify mutations in gyrase A or B. Three types of mutants were obtained: (i) one mutant (TKp1) had no mutations in gyrA or gyrB; (ii) mutants that had single missense mutations in gyrA, and (iii) mutants that had two missense mutations resulting in either two altered gyrase A residues or an altered residue in both gyrases A and B. The TKp1 mutant had slightly reduced levels of uptake of [14C]norfloxacin, which was associated with two- to fourfold increases in the MICs of ofloxacin, ciprofloxacin, and sparfloxacin. Gyrase mutations caused a much greater increase in the MICs of fluoroquinolones. For mutants with single gyrA mutations, the increases in the MICs were 4- to 16-fold, and for mutants with double gyrase mutations, the MICs were increased 32-fold or more compared with those for the parent. A gyrA mutation in TKp1 secondary mutants was associated with 32- to 128-fold increases in the MICs of ofloxacin and ciprofloxacin compared with the MICs for H37Ra and an eight-fold increase in the MIC of sparfloxacin. Sparfloxacin was the most active fluoroquinolone tested. No sparfloxacin-resistant single-step mutants were selected at concentrations of > 2.5 micrograms/ml, and high-level resistance (i.e., MIC, > and = 5 micrograms/ml) was associated with two gyrase mutations. Mutations in gyrB and possibly altered levels of intracellular accumulation of drug are two additional mechanisms that may be used by M. tuberculosis in the development of fluoroquinolone resistance. Because sparfloxacin is more active in vitro and selection of resistance appears to be less likely to occur, it may have important advantage over ofloxacin or ciprofloxacin for the treatment of tuberculosis.

Polyamines decrease Escherichia coli outer membrane permeability.

The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels. We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis. In both cases, inhibition was observed. Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation. The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine. Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder. These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins. These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.

Penicillin-binding protein 5 as an inhibitory target of cefozopran in Enterococcus faecalis.

The concentration of cefozopran which inhibits binding of [14C]benzylpenicillin to penicillin-binding protein (PBP) 5 of Enterococcus faecalis TN2OO5 by 50% was 11 mg/L, and its MIC was 12.5 mg/L. Ceftazidime and cefmenoxime, which were inactive at 100 mg/L, showed no affinity for PBP 5 at this concentration. Ampicillin, benzylpenicillin and imipenem showed higher affinity for PBPs 3/4 and PBP 5 than cefozopran, and their MICs were lower than that of cefozopran. No correlation between MICs of the test compounds and the affinity for PBP 1, 2 or 6 was found. These results suggest that cefozopran exhibits antimicrobial activity against E. faecalis TN2OO5 by binding to PBP 5.

An engineered Staphylococcus aureus PC1 beta-lactamase that hydrolyses third-generation cephalosporins.

The beta-lactamase from Staphylococcus aureus PC1 has been cloned into an Escherichia coli vector for site-directed mutagenesis and high-level protein expression. A mutant enzyme has been produced in which Ala238 is replaced by a serine, and Ile239 is deleted (A238S:I239del). The engineered enzyme hydrolyses third-generation cephalosporins substantially more rapidly than the parental enzyme does, while hydrolysis of benzylpenicillin is slower with the mutant than with the wild-type and native enzymes. The mutant beta-lactamase has been crystallized and the structure determined and refined at 2.8 A resolution. The disposition of the beta-strand which forms the side of the active site is altered in comparison with the native S. aureus beta-lactamase structure, widening the active site cleft and providing space to accommodate the bulky side-chains of the third-generation cephalosporins.

Lack of additive effect between mechanisms of resistance to carbapenems and other beta-lactam agents in Pseudomonas aeruginosa.

Eighty-nine clinical isolates resistant (n = 61) or susceptible (n = 28) to imipenem and exhibiting the main patterns of susceptibility to other beta-lactam agents (wild type pattern, penicillinase pattern, constitutive cephalosporinase pattern) were studied in order to investigate (i) the mechanism of resistance involved and (ii) whether resistance to carbapenems affects the level of resistance to other beta-lactam agents and, conversely, if resistance to other beta-lactam agents affects the level of resistance to carbapenems. For this purpose, the presence of OprD protein in the cell wall was detected by Western blot and beta-lactamase activity by spectrophotometric assay and isoelectric focusing. OprD expression was not detectable in the imipenem-resistant (MIC > or = 16 micrograms/ml) strains. It was decreased in half the strains for which MICs of imipenem were 2 to 8 micrograms/ml and was close to a normal level in the most susceptible strains (MIC < or = 1 microgram/ml), thus demonstrating a direct correlation between the level of susceptibility to imipenem and the level of OprD expression. No imipenemase activity was detected in imipenem-resistant strains. Synergy between imipenem or meropenem and BRL 42715 was observed for all of the strains, demonstrating the role of cephalosporinase in carbapenem resistance. Within each pattern of susceptibility, the mean MICs of beta-lactam agents other than carbapenems were similar, whether the strains were susceptible or resistant to imipenem. Conversely, the mean MICs of imipenem or meropenem for either the imipenem-resistant or the imipenem-susceptible strains were similar, regardless of the susceptibility of these strains to the other beta-lactam agents. Thus, when several mechanisms of resistance to beta-lactam agents are present in the same strain of Pseudomonas aeruginosa, there is no additive effect between these mechanisms.

Beta-lactamase activity in mycobacteria other than M. tuberculosis.

Beta-lactamase activity was studied in 142 non-tuberculosis mycobacterial strains. The distribution according to species was as follows: four M. avium, 14 M. chelonae, 10 M. fortuitum, 59 M. gordonae, 55 M. kansasii. The spectrum of hydrolysis of the beta-lactamases was screened using an acidimetric method and the characterization was performed by analytical isoelectric focusing.

Acid pH decreases OmpF and OmpC channel size in vivo.

To be effective against gram-negative organisms, beta-lactam antibiotics must be able to penetrate the outer membrane. For Escherichia coli, these compounds generally cross this barrier through non-specific channels in porins OmpF and OmpC. In vitro studies have shown that increased pH induces a switch in the structure of OmpF and OmpC from a small channel conformation to a set of larger-sized channel conformations. In this study, the permeability of two cephalosporins into cells producing either OmpC or OmpF was examined at various pHs. The results suggest that the pH-induced switch in channel size observed in vitro also occurs in vivo.

Imipenem resistance in Enterobacter aerogenes is associated with derepression of chromosomal cephalosporinases and impaired permeability.

Enterobacter aerogenes mutants with high-level resistance to imipenem were studied. They were derived from strains characterized by stable over-production of a class-I beta-lactamase. This enzyme (pI = 8.2) exhibited high affinity toward imipenem and hydrolysed the drug slowly. Imipenem-resistant mutants lacked a major 43-kDa outer membrane protein and displayed decreased permeability to cephaloridine. Introduction of a plasmid coding for the regulatory ampD gene abolished beta-lactamase production and rendered the mutants susceptible to imipenem.

Regulation of ompF porin expression by salicylate in Escherichia coli.

The expression of ompF, the gene encoding a major outer membrane protein of Escherichia coli, is regulated by various environmental factors. The mechanism by which salicylate (SAL) drastically reduces ompF expression was studied here by means of lacZ fusions to ompF, ompC, and micF, by sodium dodecyl sulfate-gel electrophoresis of outer membrane proteins, and by measurements of outer membrane permeability. Growth of E. coli in LB broth containing SAL strongly reduced ompF-specific translation of an ompF-lacZ fusion. The extent of this reduction varied with the SAL concentration from 64% at 0.5 mM to 95% at 2 mM and greater than 99% at 10 mM. ompF-lacZ transcription was not affected by SAL, whereas ompC-lacZ transcription was elevated by 70%. Since the micF transcript is antisense to a portion of the ompF transcript and is capable of decreasing the translation of ompF, the effect of SAL on micF transcription was measured in a micF-lacZ fusion strain. SAL-grown cells contained three- to fourfold more micF transcript during the logarithmic phase of growth than did the control cultures. However, micF was not absolutely required for the response to SAL. In micF-deleted strains, the effects of SAL on ompF translation, on OmpF in the outer membrane, and on outer membrane permeability were diminished but still evident. The effect of SAL on ompF expression was independent of the osmolarity of the medium and was epistatic to certain ompB regulatory mutations: the high levels of ompF expression found in envZ3 and ompR472 strains were greatly reduced by growth in SAL. Unexpectedly, the OmpC- phenotypes of these mutants were suppressed by SAL. Thus, growth in SAL severely decreases the translation of ompF while enhancing the transcription of micF and ompC. In this respect, SAL-grown cells resemble certain marA and tolC mutants that have high levels of micF and ompC transcripts and low levels of OmpF.

One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli.

Site-directed mutagenesis was performed with the phosphate starvation-inducible outer membrane porin PhoE of Escherichia coli K-12 to study the molecular basis of its anion selectivity. Lysines 18, 29, 64, and 125 were replaced by glutamic acids, and the properties of the mutant porins were investigated in in vivo and in vitro experiments. Lipid bilayer experiments showed that all these mutations had no influence on the pore structure because PhoE and the mutants had the same single channel conductance in KCl solution. Selectivity measurements revealed that the mutations changed the ionic selectivity of PhoE, but the change was dependent on the location of the lysine. Replacement of Lys18 and Lys29 by glutamic acid had a relatively small influence. The effect of the Lys64 substitution was somewhat larger, and the effect of the replacement of Lys125 resulted in the most drastic change in selectivity and in the loss of the interaction of PhoE with polyphosphate, whereas the replacement of the other lysines had no effect on the polyphosphate interaction behavior. The results are consistent with the assumption that the charge spot in PhoE consists of only 1 lysine per monomer, located in position 125 of the primary sequence and probably close to the pore interior.

Evaluation of cefotaxime alone and in combination with desacetylcefotaxime against strains of Staphylococcus aureus that produce variants of staphylococcal beta-lactamase.

We evaluated cefotaxime (CTX) alone and in combination with its metabolite, desacetylcefotaxime (dCTX) against strains of Staphylococcus aureus that produce the four recognized variants of staphylococcal beta-lactamase and a beta-lactamase-producing isolate characterized by the expression of borderline resistance to methicillin. Although macrodilution MICs revealed that dCTX was less active than CTX against these strains (geometric means of 16 micrograms/ml and 4 micrograms/ml, respectively), the addition of clinically achievable concentrations of dCTX to CTX resulted in a reduction in the observed CTX MICs. This effect was similar to although less pronounced than that obtained by combining clavulanic acid with cefazolin. The increased antistaphylococcal activity noted by MIC determinations was confirmed with kill-kinetic studies. Determination of the relative rates of hydrolysis of selected cephalosporins showed that neither CTX nor dCTX were appreciably hydrolyzed by the variant staphylococcal enzymes. Evaluation of the effect of CTX and dCTX upon the staphylococcal beta-lactamases demonstrated that neither agent inhibited the destruction of a 100 microM solution of nitrocefin, although the reduction of CTX and cefazolin MICs by low concentrations of dCTX suggests that the dCTX metabolite may act as a competitive inhibitor of beta-lactamase. These observations may explain the previously demonstrated clinical efficacy of CTX used alone for the treatment of serious infections caused by S. aureus.