Alterations In Outer Membrane Proteins Research Articles

Prevalence of a transferable SHV-5 type β-lactamase in clinical isolates of Klebsiella pneumoniae and Escherichia coli in Greece

Analysis with a double-disc synergy test (DDST) of clinical isolates of Klebsiella pneumoniae and Escherichia coli during the period October 1988-September 1989 revealed that 24% of the former and 4% of the latter, mainly isolated from urine, possessed an extended-spectrum beta-lactamase. During this period no DDST was positive for isolates of other enterobacterial species. Transfer of ceftazidime resistance was demonstrated from six K. pneumoniae and two E. coli isolates resistant to third generation cephalosporins and aztreonam. Apart from one strain of K. pneumoniae, these strains harboured self-transferable multiresistance plasmids (c. 91 kb) with closely related EcoRI, HindIII, AvaII and PstI restriction patterns. These plasmids encoded an extended-spectrum beta-lactamase that conferred an unusually high level of resistance to ceftazidime and aztreonam (MIC greater than or equal to 64 mg/l). This enzyme had a pI of 8.2 and a substrate profile similar to that of the SHV-5 enzyme isolated initially in Chile, and later in France (CAZ-4). The remaining K. pneumoniae isolate harboured a transmissible multiresistance plasmid (c. 182 kb) that encoded the widely distributed SHV-2 enzyme. The resistance to cefoxitin that was observed in some of these strains was associated with outer membrane protein alterations.

Enhanced resistance to cefotaxime and imipenem associated with outer membrane protein alterations in Enterobacter aerogenes

Mutants exhibiting enhanced resistance to cefotaxime and imipenem were selected by plating a strain of Enterobacter aerogenes, which already produced chromosomal beta-lactamase constitutively, on to varying concentrations of different beta-lactam antibiotics. Frequencies of mutation varied from 10(-5) to 10(-8), depending upon the particular antibiotic and concentration used for selection. Only minor variations in beta-lactamase specific activities were observed and these could not be directly correlated with changes in resistance when compared with the original strain. In the majority of mutants, the selection of an enhanced level of resistance to cefotaxime was associated with a significant increase in resistance to imipenem, but no increase in resistance to the non-beta-lactam antibiotics tested was observed. Examination of outer membrane protein profiles revealed a number of complex changes in the mutants when directly compared to the original strain. In one mutant imipenem/cefotaxime resistance was directly associated with almost total loss of a 42K protein which was non-covalently associated with peptidoglycan and therefore possibly a porin protein.

Meropenem: activity against resistant gram-negative bacteria and interactions with beta-lactamases.

The activity of meropenem, a new carbapenem, was determined against 82 Gram-negative bacteria in agar dilution tests. Many of these isolates were resistant to one or more beta-lactam antibiotics and the mechanisms responsible for the resistance had been characterized. The production of beta-lactamases had little influence on susceptibility to either meropenem or imipenem except in tests with Aeromonas hydrophila and Pseudomonas (Xanthomonas) maltophilia. These species produced metalloenzymes capable of hydrolyzing the carbapenems, and strains expressing high levels of these enzymes were resistant to both meropenem and imipenem. Clinical isolates of P. aeruginosa that had developed resistance to imipenem during therapy with the drug were two- to 32-fold less susceptible to meropenem than the corresponding pretreatment isolates. Alterations in outer membrane proteins were associated with this change in susceptibility to the carbapenems. Meropenem was a less potent inducer of Class I beta-lactamases than imipenem but was still a better inducer than ceftazidime or piperacillin. Overall, meropenem showed excellent activity against bacteria producing a variety of beta-lactamases, but cross-resistance between meropenem and imipenem due to enzymatic and non-enzymatic mechanisms did occur.

Outer membrane alterations in multiresistant mutants of Pseudomonas aeruginosa selected by ciprofloxacin

Spontaneous mutants of Pseudomonas aeruginosa selected by ciprofloxacin were studied for outer membrane alterations. Acquisition of ciprofloxacin resistance was at least partially related to defects in lipopolysaccharide synthesis. When ciprofloxacin resistance was combined with resistance to beta-lactams and aminoglycosides, several alterations in outer membrane proteins were noted.

Isolation by streptonigrin enrichment and characterization of a transferrin-specific iron uptake mutant of Neisseria meningitidis

The bactericidal action of the antibiotic streptonigrin is enhanced by large intracellular iron pools. Using this observation, we have utilized a simple enrichment protocol to aid in the isolation of iron uptake mutants of N. meningitidis, based on the relative resistance of iron-starved meningococci to streptonigrin. One such mutant, FAM29, was impaired in its use of transferrin-bound iron; transferrin is the principal iron-binding protein in human plasma. FAM29 retained wild-type ability to utilize iron bound to lactoferrin, heme, or ferric citrate. FAM29 did not produce two iron-repressible outer membrane proteins, of 85000 and 95000 daltons, made by the parent strain. However, genetic transformation experiments indicated that the outer membrane protein alterations were not necessary for the transferrin-deficient phenotype.

Selective ceftazidime resistance in Escherichia coli: association with changes in outer membrane protein.

A strain of Escherichia coli (MG/32) was recovered from the blood of a patient who had received ceftazidime for eight weeks. The isolate was resistant to ceftazidime but susceptible to other third-generation cephalosporins. Alterations in outer membrane proteins were implicated in this selective ceftazidime resistance. As ceftazidime susceptibility was regained, the quantity of outer membrane proteins of 37,000 and 39,000 molecular weight increased. Although the isolate possessed a TEM-1 beta-lactamase, this enzyme was not involved in the selective resistance to ceftazidime; it did not disappear on reacquisition of ceftazidime susceptibility and did not hydrolyze the drug. Potassium clavulanate enhanced the activity of ceftazidime against E. coli strain MG/32, but this enhancement was due to a direct effect on outer membrane proteins and not to beta-lactamase inhibition.

Emergence of Resistance to Imipenem During Therapy for Pseudomonas aeruginosa Infections

We studied the mechanism of resistance to imipenem in three clinical isolates of Pseudomonas aeruginosa. Two of these isolates arose from imipenem-susceptible strains isolated during therapy with imipenem and were associated with treatment failure. One of these two strains had previously been broadly resistant to beta-lactams; the second acquired resistance to imipenem alone. One isolate of the third strain was resistant to imipenem but susceptible to other antipseudomonal beta-lactams. No isolate contained beta-lactamase activity capable of hydrolyzing imipenem at a detectable rate. Studies of the penicillin-binding proteins of all isolates revealed no differences in the number of proteins, molecular weight of, affinity for penicillin, or affinity for imipenem in any isolate. In each case the resistant isolate lacked one or more outer membrane proteins that were present in a susceptible isolate of the same strain. The observed alterations in outer membrane proteins may be associated with diminished permeability of the bacterial outer membrane to imipenem and may be the major factor responsible for resistance in these isolates.

Cross-resistance of nalidixic acid resistant Enterobacteriaceae to new quinolones and other antimicrobials.

One hundred urine isolates Enterobacteriaceae screened for resistance to 30 micrograms nalidixic acid by disc diffusion test were examined by MIC determination for in vitro susceptibility to nalidixic acid, ciprofloxacin, enoxacin, gentamicin, nitrofurantoin, trimethoprim, cephalexin and ceftazidime. Those resistant to nalidixic acid and also gentamicin or a cephalosporin were further examined to determine the mechanism of resistance. Compared to the total urine isolates of Enterobacteriaceae from the same time period, this population as a whole was less susceptible to all antimicrobials tested except gentamicin. Strains that exhibited multiple resistance had the conventional mechanisms of resistance to those antimicrobials. No multiply resistant strains had a permeability barrier due to outer membrane protein alterations causing cross-resistance to chemically unrelated classes of antimicrobials.

Role of beta-lactamases and outer membrane proteins in multiple beta-lactam resistance of Enterobacter cloacae.

The chromosomal beta-lactamase and outer membrane proteins of Enterobacter cloacae were examined to determine their relative contributions to multiple antibiotic resistance in this organism. Mutants altered in beta-lactamase expression, whether derived in the laboratory or recovered from patients treated with one of the new beta-lactam antibiotics, were found to have no detectable alterations in outer membrane proteins. Derepression of beta-lactamase in these mutants was associated with high-level resistance to multiple beta-lactam antibiotics, while loss of inducible beta-lactamase (i.e., production of basal enzyme levels only) was associated with acquisition of susceptibility to many beta-lactam antibiotics, including cephalothin. In contrast, alteration in outer membrane proteins was associated with only moderate-level resistance to beta-lactam antibiotics. However, this included resistance to such drugs as amdinocillin and Sch 34343, which were unaffected by derepression of beta-lactamase. Resistance to chloramphenicol and tetracycline also accompanied changes in outer membrane proteins. Although the outer membrane proteins of various strains of E. cloacae were similar, there did appear to be some major strain-to-strain variations. Thus, it appears that alterations in both beta-lactamase and outer membrane proteins can affect the susceptibility of E. cloacae to many antibiotics. However, alterations in beta-lactamase alone are sufficient to produce high-level multiple beta-lactam resistance in this organism.

Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella, Enterobacter, and Serratia.

We studied in vitro mutants of Klebsiella, Enterobacter, and Serratia cross-resistant to nalidixic acid, trimethoprim, and chloramphenicol that were similar to mutants found in vivo. The sole mechanism for this type of resistance appeared to be a reduction in permeability of the cell envelope. The mutants had significantly lower rates of uptake of glucose and chloramphenicol, but binding of chloramphenicol to ribosomes was normal. In addition, the amounts of dihydrofolate reductase were similar in both wild-type and cross-resistant mutants of Klebsiella. Examination of the bacterial outer membrane revealed that the amount of at least one major protein, with a molecular size of approximately 40 kilodaltons, was decreased in the mutants. Therefore the resistance seemed likely to be due to the reduction in quantity of these outer membrane proteins, possibly porins, in the mutant bacteria.