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D M Livermore

Publications and source records attributed to D M Livermore.

At least 145 records · Page 8Linked to original sources
PubMed1994

Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to beta-lactam resistance.

Wild-type strains of Pseudomonas aeruginosa are more resistant to various beta-lactam antibiotics as well as other agents than most enteric bacteria. Although resistance to compounds of earlier generations is explained by the synergism between the outer membrane barrier and the inducible beta-lactamase, it was puzzling to see significant levels of resistance to compounds that do not act as inducers or are not hydrolyzed rapidly by the chromosomally encoded enzyme. This intrinsic-resistance phenotype becomes enhanced in those strains with the so-called intrinsic carbenicillin resistance. In the accompanying paper (X.-Z. Li, D. M. Livermore, and H. Nikaido, Antimicrob. Agents Chemother. 38:1732-1741, 1994), we showed that active efflux played a role in the resistance, to various non-beta-lactam agents, of P. aeruginosa strains in general and that the efflux was enhanced in intrinsically carbenicillin-resistant strains. We show in this paper that, in comparison with the drug-hypersusceptible mutant K799/61, less benzylpenicillin was accumulated in wild-type strains of P. aeruginosa and that the accumulation levels were even lower in intrinsically carbenicillin-resistant strains. Deenergization by the addition of a proton conductor increased the accumulation level to that expected for equilibration across the cytoplasmic membrane. In intrinsically carbenicillin-resistant isolates, there was no evidence that either nonspecific or specific permeation rates of beta-lactams across the outer membrane were lowered in comparison with those of the more susceptible isolates. Furthermore, these carbenicillin-resistant isolates were previously shown to have no alteration in the level or the inducibility of beta-lactamase and in the affinity of penicillin-binding proteins. These data together suggest the involvement of an active efflux mechanism also in the resistance to beta-lactams. Hydrophilic beta-lactams with more than one charged group did not cross the cytoplasmic membrane readily. Yet one such compound, ceftriaxone, appeared to be extruded from the cells of more-resistant strains, although with this compound effects of proton conductors could not be shown. We postulate that wild-type strains of P. aeruginosa pump out such hydrophilic beta-lactams either from the periplasm or from the outer leaflet of the lipid bilayer of the cytoplasmic membrane, in a manner analogous to that hypothesized for multidrug resistance protein of human cancer cells (M.M. Gottesman and I. Pastan, Annu. Rev. Biochem. 62:385-427, 1993).

Anti-Bacterial Agents↗
PubMed1993

Determinants of the activity of beta-lactamase inhibitor combinations.

Inhibitor combinations provide one strategy to overcome beta-lactamase-mediated resistance. Their success depends, obviously, on the inhibitor being able to bind and inactivate the beta-lactamase molecules. Clavulanate, sulbactam and tazobactam are irreversible inactivators of many beta-lactamases, forming covalent complexes which resist hydrolysis. 'Suicide' kinetics are seen with some, but not all, enzymes. All three compounds inactivate staphylococcal penicillinase, the chromosomal beta-lactamases of Proteus vulgaris and Bacteroides spp., and the Class IV beta-lactamases present in some klebsiellae. Tazobactam, but not the other compounds, has moderate activity against some Class I (AmpC) chromosomal beta-lactamases, notably that of Morganella morganii, but not that of Enterobacter cloacae. Both clavulanate and tazobactam are strong inhibitors of the widely distributed TEM and SHV plasmid-mediated beta-lactamases; sulbactam is a weaker inhibitor. Other factors, aside from the affinity of the inhibitor for the enzyme, co-determine the success or failure of inhibition. Potentiation is most readily achieved if little enzyme is produced, and if the organism is very permeable to the inhibitor. Thus, resistance to inhibitor combinations is rare in strains of Haemophilus influenzae and Neisseria gonorrhoeae that produce TEM-beta-lactamase, but is commoner in enterobacteria that produce this enzyme, since these are less permeable and sometimes manufacture very large amounts of enzyme. The partner beta-lactam agent is also important. Irrespective of the inhibitor used, piperacillin is easier to protect against TEM beta-lactamases and the M. morganii Class I enzyme than are ampicillin, amoxycillin or ticarcillin. This may relate to the lower affinity of piperacillin for these enzymes, or to its greater affinity for the bacterial penicillin-binding proteins. Finally, pH can affect the degree of inhibition achieved with sulphones for some beta-lactamases, notably TEM-1.

Animals↗
PubMed1993

Multicentre survey of the comparative in-vitro activity of piperacillin/tazobactam against bacteria from hospitalized patients in the British Isles.

Twenty-nine British and Irish hospitals each collected up to 300 bacterial isolates from in-patients. The organisms were identified by an appropriate API system or, for staphylococci, by their Gram and coagulase reactions. Disc susceptibility tests were performed. Isolates that gave zones < or = 25 mm to piperacillin/tazobactam (75 micrograms + 10 micrograms) discs were sent to a central laboratory for re-examination and determination of MIC, together with a sample of the more susceptible organisms. Results were evaluated for 6724 isolates. Over 95% of the isolates of Escherichia coli, klebsiellae, Proteus mirabilis, Pseudomonas aeruginosa, Haemophilus, Moraxella and Bacteriodes, spp. streptococci, pneumococci and Enterococcus faecalis were susceptible to piperacillin/tazobactam (defined as giving a zone > or = 22 mm to a 75 micrograms + 10 micrograms disc), as were 86% of Acinetobacter spp. and 82% of the Citrobacter, Enterobacter, Morganella and Serratia group. Tazobactam particularly extended the activity of piperacillin against E. coli isolates (96% susceptible cf. 61% to piperacillin alone) klebsiellae (95% cf. 70%), P. mirabilis (99% cf. 86%), and Acinetobacter spp. (86% cf. 53%). Occasional (18%) resistance in Enterobacter, Serratia and Citrobacger spp. was probably caused by stable depression of Class I beta-lactamases, which are inhibited poorly by tazobactam. High resistance frequencies (> 25%) were found for Enterococcus faecium and Xanthomonas maltophilia. Tazobactam potentiated piperacillin against beta-lactamase-producing methicillin-susceptible Staphylococcus aureus, but the mode inhibition zone of piperacillin/tazobactam discs was only 26 mm, compared to 38 mm for beta-lactamase-negative isolates. Nevertheless, fewer than 5% of the enzyme producers appeared resistant to 8 + 4 mg/L piperacillin/tazobactam in MIC tests. Similar behaviour was noted for coagulase-negative staphylococci. Amongst the eleven comparator drugs, ceftazidime, gentamicin and ciprofloxacin were as active as piperacillin/tazobactam against most enterobacteria. However, Acinetobacter and Bacteroides spp. and enterococci were resistant to ceftazidime, and Bacteroides spp., enterococci, pneumococci and other streptococci were inherently resistant to ciprofloxacin and gentamicin. Cefuroxime, ampicillin and co-amoxiclav had narrower spectra. Only imipenem showed a consistently wider spectrum and lower frequency of resistance than piperacillin/tazobactam.

Bacteria, Anaerobic↗
PubMed1993

Behaviour of beta-lactamase-positive and -negative Staphylococcus aureus isolates in susceptibility tests with piperacillin/tazobactam and other beta-lactam/beta-lactamase inhibitor combinations.

beta-Lactamase production protects Staphylococcus aureus against piperacillin and amoxycillin. Tazobactam and clavulanate inhibit the enzyme, but beta-lactamase producers remained substantially less susceptible than non-producers to piperacillin/tazobactam and co-amoxiclav in disc tests. These observations reflected the fact the inhibition zones for beta-lactamase non-producers expanded with time, as bacterial growth was killed by the diffusing antibiotic, whereas those for enzyme producers maintained a constant diameter after they first became discernible, indicating equilibrium between diffusion and destruction of the antibiotic. The antibiotic destruction required for this equilibrium depended on biomass formation, which was apparent from the luxuriant growth on beta-lactamase-producers around the inhibition zone edges, and on beta-lactamase induction, which was demonstrable when S. aureus cultures were exposed to discs containing tazobactam alone. beta-Lactamase-related effects on the activity of inhibitor combinations were apparent also in MIC tests, but only when these were performed with large inocula. Thus, the MICs of piperacillin or amoxycillin with tazobactam or clavulanate were only two- to four-fold higher for beta-lactamase producers than non-producers at low inocula (10(4) cfu), but this differential increased to 6- to 16-fold at high inocula (10(6) cfu). Inoculum effects were greater for tazobactam combinations than for clavulanate combinations, irrespective of the partner penicillin. These beta-lactamase-related inhibition zone differentials and inoculum effects for the inhibitor combinations resemble previous observations for first-generation cephalosporins, notably cephazolin and cephaloridine. Their significance for the latter compounds has occasioned long debate.

Anti-Bacterial Agents↗
PubMed1993

Activity of cefepime and other beta-lactam antibiotics against permeability mutants of Escherichia coli and Klebsiella pneumoniae.

Permeability mutants were obtained, under selection pressure with cefoxitin, from five strains each of Escherichia coli and Klebsiella pneumoniae. Most mutants showed alterations to their outer membrane protein profiles. In general, the porins OmpC and F were lost from E. coli mutants, and one or both components of a 40 kD doublet were lost from K. pneumoniae mutants. Additional minor changes to outer membrane proteins, of unknown significance, were detected in many of the mutants. MICs of cefepime, ceftazidime and cefpirome for the porin-deficient mutants were up to 128-fold above those for their parent strains, but remained < or = 4 mg/L, whereas those of cefoxitin, cefuroxime, and penicillins were raised above accepted breakpoints. The detection of reduced susceptibility to third- and fourth-generation cephalosporins is in contrast to the findings of other groups. Most (9/10) mutants with high-level cefoxitin resistance were reasonably stable during sequential batch culture in rich media, but susceptible variants tended to overgrow under nutrient depletion in continuous culture. These variants were not true revertants, having failed to regain the major porins: rather they appeared to have undergone compensatory mutations. Their instability probably explains why porin-deficient enterobacteria have not become a significant clinical problem, despite the ease with which they can be selected in vitro.

Anti-Bacterial Agents↗
PubMed1993

OXA-11, an extended-spectrum variant of OXA-10 (PSE-2) beta-lactamase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa ABD, which was isolated in October 1991 from blood cultures of a burn patient in Turkey, was resistant to cephalosporins, particularly ceftazidime (MIC, 512 micrograms/ml), penicillins, aztreonam, and meropenem, but not to imipenem. Cephalosporin and penicillin resistance transferred to P. aeruginosa PU21 and was associated with a beta-lactamase with a pI of 6.4 encoded by a 100-MDa plasmid designated pMLH52. Like extended-spectrum TEM and SHV beta-lactamases, this enzyme hydrolyzed penicillins and newer cephalosporins but did not hydrolyze cefoxitin or carbapenems. However, it differed from TEM and SHV derivatives in being a potent oxacillinase, and its encoding gene did not hybridize with probes to TEM and SHV genes. To characterize the enzyme, libraries of total DNA were cloned into plasmid pUC19 and were transformed into Escherichia coli DH5 alpha. Recombinant plasmids that gave ceftazidime resistance all contained a 3.65-kb BamHI fragment. Deletions from this fragment allowed the beta-lactamase gene to be located on a 1.4-kb section of DNA, which contained an open reading frame of 798 bases. This encoded a protein that was deduced to differ from PSE-2 beta-lactamase only in having serine instead of asparagine at position 143 and aspartate instead of glycine at position 157. It is concluded that the resistance of isolate ABD dependent on an extended-spectrum variant of the PSE-2 enzyme. The ability of this enzyme to cause ceftazidime resistance dependent primarily on a low Km for the compound; Vmax remained low. It is proposed that PSE-2 should be transferred to the OXA group as OXA-10 and that the new enzyme be designated OXA-11.

Amino Acid Sequence↗
PubMed1992

Carbapenemases.

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Aspergillus↗
PubMed1992

Survey of the prevalence of beta-lactamases amongst 1000 gram-negative bacilli isolated consecutively at the Royal London Hospital.

beta-Lactamase expression was examined in 1000 consecutive Gram-negative bacilli isolated from urine, wound swab, sputum or blood specimens received at the Microbiology Laboratory of the Royal London Hospital. This survey, performed between January and April, 1991, followed a similar study undertaken in early 1982. The distribution of species was similar in the two surveys, except that the proportion of Pseudomonas aeruginosa isolates had increased from 11% in 1982 to 17.5% in the present study. This increase was balanced by a decreased proportion of enterobacteria. Amongst plasmid-mediated beta-lactamases, TEM-1 (especially), TEM-2, SHV-1 and OXA types continued to predominate in enterobacteria. Their frequency in Escherichia coli was unchanged (46% in 1991 compared with 43% in 1982), but had increased from 5 to 22% amongst Proteus mirabilis isolates. An apparent decrease in their frequency amongst Enterobacter cloacae isolates, from 48% in 1982 to 17% in 1991, probably reflected changes to strain prevalence rather than enzyme prevalence. Plasmid type beta-lactamases were present in fewer than 2% of P. aeruginosa isolates in both surveys. In the present study, chromosomal beta-lactamase derepression (constitutive hyperproduction) was detected in 10/76 isolates of E. cloacae, Enterobacter aerogenes, Citrobacter freundii, Serratia spp. and Morganella morganii, and in 2/170 P. aeruginosa isolates. These proportions were increased, compared with those seen the 1982 survey, though the significance was borderline (P approximately 0.05; chi 2 test). Extended-spectrum plasmid mediated beta-lactamases, unknown in 1982, were found in 11/70 Klebsiellae pneumoniae isolates in the present study. Ten of these organisms, representing at least five distinct strains, produced TEM-10 enzyme, encoded by a plasmid of c. 90 kb; the remaining organism had an unidentified SHV-derived enzyme.

Anti-Bacterial Agents↗
PubMed1992

Effects of CO2 and pH on inhibition of TEM-1 and other beta-lactamases by penicillanic acid sulfones.

Incubation in 5% CO2 reduced the inhibition zones of piperacillin-tazobactam (75/10 micrograms) disks for Escherichia coli strains with TEM-1, TEM-2, and SHV-1 beta-lactamases. Similarly, MICs of piperacillin-tazobactam and other penicillin-sulfone combinations for TEM producers were up to 500-fold higher at pH 6.5 than at pH 8.0. This effect was greatest for organisms with high levels of enzyme activity. CO2 and mild acidity did not affect the susceptibility of beta-lactamase-negative strains to penicillin-sulfone combinations, and the effects of these conditions were variable for organisms with beta-lactamases other than TEM-1, TEM-2, and SHV-1. These last observations discounted acid-mediated inactivation of piperacillin or tazobactam. MICs of amoxicillin or piperacillin alone or with clavulanate for TEM and SHV producers were affected only less than or equal to 16-fold by 5% CO2 or acidity, indicating that the greater effects seen with the penicillin-sulfone combinations depended on the behavior of the sulfones and not on that of the penicillins. This pH effect was studied in detail for TEM-1 enzyme. Inhibition of this enzyme by sulfones but not clavulanate varied grossly with pH, with 50% inhibitory concentrations of tazobactam and sulbactam up to 300-fold higher at pH 6.5 than at 8.0. By contrast, the hydrolytic activity of TEM-1 enzyme for substrates and its level of production varied threefold or less between pH 6.5 and pH 8.0. Increased inhibition at pH 8.0 reflected sequestration of the enzyme into a secondary noncovalent complex rather than increased irreversible inactivation.

Carbon Dioxide↗
PubMed1992

Interplay of impermeability and chromosomal beta-lactamase activity in imipenem-resistant Pseudomonas aeruginosa.

Mutational loss of the D2 porin causes imipenem resistance in Pseudomonas aeruginosa. It was found that this mechanism could function only when the chromosomal beta-lactamase was expressed. Mutants lacking both the beta-lactamase and the D2 porin were almost as susceptible as those that lacked the beta-lactamase but retained the porin. Thus, imipenem resistance reflected an interplay of the enzyme and impermeability, not either factor alone. These findings suggest that the activity of a carbapenem more beta-lactamase stable than imipenem should be less affected by the porin loss. Meropenem approached this behavior.

Bacterial Outer Membrane Proteins↗
PubMed1991

Activity of clavulanate combinations against TEM-1 beta-lactamase-producing Escherichia coli isolates obtained in 1982 and 1989.

Recent reports of decreased susceptibility to amoxycillin/clavulanate and ticarcillin/clavulanate combinations amongst Escherichia coli isolates have been attributed to an increased frequency of hyperproduction of TEM-1 beta-lactamase. To test this claim we compared the activities of clavulanate combinations against consecutive E. coli isolates producing TEM-1 beta-lactamase that were obtained from clinical material at The London Hospital during 1982 (n = 50) and 1989 (n = 46). Enzyme production was quantified and related to the level of clavulanate required to potentiate amoxycillin and ticarcillin. The levels of TEM-1 enzyme production varied 150-fold amongst the isolates, partly because of variation in the gene copy number. A clear correlation existed between enzyme quantity and levels of resistance to the clavulanate combinations. However, there was no significant difference (P greater than 0.05, Mann-Whitney U test) between the isolates obtained in 1982 and 1989 in terms of the clavulanate concentrations required to potentiate the penicillins, or in the distribution of beta-lactamase activities present.

Amoxicillin↗
PubMed1991

Susceptibility of Escherichia coli isolates with TEM-1 beta-lactamase to combinations of BRL42715, tazobactam or clavulanate with piperacillin or amoxycillin .

Production of TEM-1 beta-lactamase is the commonest cause of acquired resistance to amoxycillin and piperacillin in Escherichia coli, now occurring in c. 50% of isolates. Consecutive E. coli isolates producing TEM-1 beta-lactamase were collected at The London Hospital in 1982 (n = 50) and 1989 (n = 46). Enzyme quantities varied 150-fold amongst the isolates. Randomly-selected isolates from both years (n = 36; nine per quartile of the beta-lactamase activity distribution) were tested for susceptibility to combinations of amoxycillin or piperacillin with clavulanate or tazobactam or with BRL42715, a novel penem. The inhibitor concentrations needed to potentiate the penicillins related to the amount of beta-lactamase produced. BRL42715, at 1 mg/l, rendered all the isolates, including TEM-1 hyperproducers, susceptible to the recommended BSAC breakpoints of 8 mg amoxycillin/1 and 16 mg piperacillin/l. At 2 mg/l, BRL42715 almost always reduced amoxycillin and piperacillin MICs to the levels (1-2 mg/l) expected for E. coli isolates that lack TEM-1 enzyme. Tazobactam, at 1-2 mg/l, reduced piperacillin MICs to 1-2 mg/l for strains in the lower half of the beta-lactamase distribution, but greater than 8 mg tazobactam/l was required to reduce piperacillin MICs to 16 mg/l for one-third of the top quartile isolates. Clavulanate was a stronger potentiator of piperacillin than was tazobactam. On the other hand, amoxycillin was a more difficult substrate to potentiate than piperacillin, and isolates with enzyme levels in the top half of the distribution generally required greater than or equal to 8 mg clavulanate/l to reduce amoxycillin MICs to less than or equal to 8 mg/l.

Amoxicillin↗
PubMed1991

Effect of media composition on the susceptibility of Xanthomonas maltophilia to beta-lactam antibiotics.

The susceptibility of Xanthomonas maltophilia strains to beta-lactams was shown to depend on the concentrations at which individual media were prepared. MIC and disc susceptibility tests were performed on solidified Mueller-Hinton and Iso-Sensitest media prepared at 0.1, 0.3, 1, and 3 x the concentrations recommended by the manufacturers. Nine of ten X. maltophilia strains tested showed increasing sensitivity to meropenem, cefotaxime, cefoperazone, piperacillin and latamoxef as the nutrient concentrations of the two media were increased. The opposite pattern was found with one strain (NCTC 10257) on Mueller-Hinton agar. This strain behaved inconsistently on Iso-Sensitest agar. Previous studies have shown that medium-dependent susceptibility in X. maltophilia is not related to beta-lactamase expression. The present study demonstrated that 22 and 25 kDa outer membrane proteins were induced on media with higher nutrient concentrations. The possible relationship of these proteins to sensitivity is considered.

Anti-Bacterial Agents↗
PubMed1991

Susceptibility to beta-lactam antibiotics of mutant strains of Xanthomonas maltophilia with high- and low-level constitutive expression of L1 and L2 beta-lactamases.

Xanthomonas maltophilia produces two inducible beta-lactamases, L1 and L2, and resists the antimicrobial activity of beta-lactam antibiotics, including carbapenems. L1 is a zinc-metaloenzyme with carbapenemase activity; L2 is an unusual cephalosporinase. Mutant strains with high- and low-level constitutive expression of these enzymes were derived from three reference strains of X. maltophilia. With a single exception, the mutant strains had altered expression of both enzymes, indicating that these beta-lactamases share regulatory components. The exception was a mutant strain that had low-level constitutive (basal) expression of L1 enzyme but remained inducible for L2. A parent strain with low-level beta-lactamase inducibility was more susceptible to penicillins, cephalosporins and carbapenems than were those in which higher levels of enzyme activity were inducible. Mutations that caused high-level constitutive beta-lactamase expression increased resistance to penicillins and newer cephalosporins. beta-Lactamase basal mutant strains, including the one that remained inducible for L2 enzyme, were more susceptible than inducible strains to these drugs. Organisms with inducible or high-level constitutive beta-lactamase expression were equally resistant to meropenem and imipenem but basal mutant strains, including the one that remained inducible for L2 enzyme, were more susceptible to meropenem than imipenem. Minimal inhibitory concentrations of meropenem, penicillins and cephalosporins, but not imipenem, were greater on Mueller Hinton agar than on IsoSensitest or Diagnostic Sensitivity Test agars. This behaviour was independent of beta-lactamase inducibility, and may reflect permeability differences between cells grown on different media.

Anti-Bacterial Agents↗