Metallo-beta-lactamases of Pseudomonas aeruginosa--a novel mechanism resistance to beta-lactam antibiotics

Since about twenty years, following the introduction into therapeutic of news beta-lactam antibiotics (broad-spectrum cephalosporins, monobactams and carbapenems), a very significant number of new beta-lactamases appeared. These enzymes confer to the bacteria which put them, the means of resisting new molecules. The genetic events involved in this evolution are of two types: evolution of old enzymes by mutation and especially appearance of new genes coming for some, from bacteria of the environment. Numerous mechanisms of enzymatic resistance to the carbapenems have been described in Pseudomonas aeruginosa. The important mechanism of inactivation carbapenems is production variety of b-lactam hydrolysing enzymes associated to carbapenemases. The metallo-beta-enzymes (IMP, VIM, SPM, GIM types) are the most clinically significant carbapenemases. P. aeruginosa posses MBLs and seem to have acquired them through transmissible genetic elements (plasmids or transposons associated with integron) and can be transmission to other bacteria. They have reported worldwide but mostly from South East Asia and Europe. The enzymes, belonging to the molecular class B family, are the most worrisome of all beta-lactamases because they confer resistance to carbapenems and all the beta-lactams (with the exception of aztreonam) and usually to aminoglycosides and quinolones. The dissemination of MBLs genes is thought to be driven by regional consumption of extended--spectrum antibiotics (e.g. cephalosporins and carbapenems), and therefore care must be taken that these drugs are not used unnecessarily.     

Sensitivity to selected beta-lactam antibiotics of clinical strains of Escherichia coli and Klebsiella pneumoniae

The studies aimed at analysing the resistance to some beta-lactam antibiotics among E. coli and K. pneumoniae clinical isolates and at evaluating. The extended spectrum of beta-lactamases (ESBL) production in the isolates. The analysis included 137 E. coli strains and 52 K. pneumoniae strains, isolated from hospitalized patients and out-patients treated in the first trimester of 1998. The strains were identified using the ATB computer system. Antibiotic sensitivity of the isolates was determined by disc-diffusion tests. ESBL production capacity of E. coli and K. pneumoniae strains was estimated by double-disc and ATB BLSA tests. Most of the analysed E. coli strains were found to exhibit significant sensitivity to compound penicillin preparations containing beta-lactam inhibitor (Augmentin, Tazocin) and to the third generation cefalosporins, in contrast, K. pneumoniae strains much more frequently were resistant to the drugs. Among the obtained isolates, 3 (2.2%) E. coli strains and 21 (40.4%) K. pneumoniae strains produced ESBL but all the isolates proved sensitive to imipenem. In evaluation of ESBL production-detecting tests, the double-disc test was found to be more reliable than ATB BLSA test.     

Human-liver alanine aminopeptidase. A kinin-converting enzyme sensitive to beta-lactam antibiotics

Human liver alanine aminopeptidase (EC 3.4.11.14; L-alpha-aminoacyl-peptide hydrolase) catalyzes the stepwise hydrolysis of methionyl-lysyl-bradykinin to yield methionine, lysine, and the limit nonapeptide, bradykinin which is resistant to further hydrolytic cleavage by this enzyme. Alanine aminopeptidase also catalyzes the hydrolysis of various neutral amino acid beta-naphthylamides. This enzyme cleaves N-terminal arginyl residues unless the adjacent penultimate residue is proline as is the case for bradykinin. The properties are consistent with the requirements of a kinin converting enzyme. Human alanine aminopeptidase activity is reduced by several beta-lactam antibiotics, with the cloxacillin, oxacillin, and methicillin Ki values being 0.51 mM, 1.6 mM, and 2.4 mM respectively. Our experiments with radioactively labelled penicillin indicate that two moles of antibiotic are bound per mole of enzyme. Neither chromatography of the penicillin-treated enzyme on G-25 Sephadex, treatment of penicillin-G-treated enzyme with penicillinase, nor extensive dilution of cloxacillin-treated enzyme diminished the degree of inactivation produced. Inhibition was obtained with 6-aminopenicillanic acid, which indicated that the penicillin nucleus itself was being bound, but substitutions, as in cloxacillin, could enhance the binding.     

Escherichia coli murein-DD-endopeptidase insensitive to beta-lactam antibiotics.

A novel endopeptidase degrading the peptide cross-links in sacculi has been isolated from Escherichia coli and purified to homogeneity. The enzyme has a molecular weight of 30,000 and, in contrast to already known enzymes of similar specificity, remains fully active in the presence of beta-lactam antibiotics. In addition, it is exceptional in being inhibited by single-stranded deoxyribonucleic acid and by some polynucleotides. The possible role of the enzyme in cell division is discussed.     

Group 1 beta-lactamases of Aeromonas caviae and their resistance to beta-lactam antibiotics

The contribution of beta-lactamase production to beta-lactam antibiotic resistance was examined in an Aeromonas caviae mutant strain, selected in vitro by cefotaxime and derived from a wild-type strain isolated in our laboratory from crude sewage. Both strains produced beta-lactamase. The mutant strain (AC7m) produced beta-lactamase constitutively, in contrast to the parental strain (AC7), which was inducible by cefoxitin. AC7m was regarded as a mutant from AC7, which over-expressed beta-lactamase. The mutant strain showed a remarkable reduction in sensitivity to most of the beta-lactam antibiotics tested, such as (i) aminopenicillins and their combinations with clavulanic acid and sulbactam, (ii) carboxypenicillins, (iii) ureidopenicillins, and (iv) cephalosporins. This strain remained susceptible to ceftazidime, imipenem, and aztreonam. Isoelectric focusing of sonic extracts revealed that both strains AC7 and AC7m shared a common major beta-lactamase band at pI 6.5. The plasmid DNA assays showed that the beta-lactamases expressed by each A. caviae strain were chromosomally encoded. Based on substrate and inhibitor profiles determined in sonic extracts for AC7 and AC7m, the enzymes displayed on isoelectric focusing at pI 6.5 were assigned to chromosomal Group 1 beta-lactamases. Imipenem would therefore be the appropriate choice for therapy of infections caused by A. caviae beta-lactamase over-expressing mutants.     

Relationship between beta-lactamase activity and resistance to beta-lactam antibiotics in Mycobacterium smegmatis

Penicillin-susceptible mutants and beta-lactamase-negative mutants were isolated from Mycobacterium smegmatis after nitrosoguanidine mutagenesis. Both the mutants were found to be susceptible to low levels of penicillin and cephalosporins by twofold dilution testing. Clavulanic acid reduced the minimal inhibitory concentrations of beta-lactamase-labile beta-lactams for the penicillin-susceptible mutants and the parent strain, but had no effect on the susceptibility of the beta-lactamase-negative mutants. Comparison of the beta-lactamase activities found in these mutants and the parent strain indicated that there was a rough correlation between the beta-lactamase level in these organisms and their susceptibility to beta-lactams.     

Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.     

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.     

Chemiluminescence flow-injection analysis of beta-lactam antibiotics using the luminol-permanganate reaction.

A new flow injection chemiluminescence (CL) method has been developed for the determination of six beta-lactam antibiotics, including amoxicillin, cefadroxil, cefoperazone sodium, cefazolin sodium, cefradine and ceftriaxone sodium. When the antibiotic was injected into a stream of KMnO4 with alkaline luminol, a strong CL signal was produced. The method allows the measurements of 0.1-50.0 mg/L amoxicillin, 0.1-80.0 mg/L cefadroxil, 1.0-30.0 mg/L cefoperazone sodium, 1.0-30.0 mg/L cefazolin sodium, 3.0-50.0 mg/L cefradine and 3.0-50.0 mg/L ceftriaxone sodium. The detection limits are 0.05 mg/L for amoxycillin, 0.05 mg/L for cefadroxil, 0.4 mg/L for cefoperazonum sodium, 0.4 mg/L for cefazolin sodium, 0.8 mg/L for cefradine and 0.8 mg/L for ceftriaxone sodium. The relative standard deviations in 11 repeated measurements are 0.6%, 0.8%, 1.5%, 1.2%, 0.4% and 0.3% for 3.0 mg/L amoxicillin, 1.0 mg/L cefadroxil, 10.0 mg/L cefoperazone sodium, 10.0 mg/L cefazolin sodium, 10.0 mg/L cefradine and 10.0 mg/L ceftriaxone sodium, respectively. The method was successfully applied to the determination of amoxicillin in pharmaceutical preparations. A possible CL reaction mechanism is also discussed.     

Enterobacterial sensitivity to beta-lactam antibiotics]

The susceptibility to betalactams of 868 enteric bacteria isolated from the patients at the hospital was studied. The isolated pathogens included: E. coli (549), Klebsiella sp. (195), Serratia sp. (124). Ampicillin and cefazoline demonstrated the lowest activity. Cefotaxime, ceftazidime and imipenem were active against 90 per cent of isolates. Among E. coli isolates the susceptibility to the above mentioned drugs was the following: 95.1, 96.9, 99.3 per cent, among Klebsiella sp.--89.7, 88.7, 97.9 per cent, among Proteus sp.--89.5, 90.3, 91.9 per cent respectively. Thus cefotaxime may be used in antibacterial empiric therapy if Pseudomonas aeruginosa infection is excluded.     

Studies on the mechanism of intrinsic resistance to beta-lactam antibiotics in group D streptococci

Six penicillin-binding proteins (PBPs) were detected in clinical isolates of each one of three group D streptococci: Streptococcus bovis, S. faecalis and S. faecium. When examined in whole organisms, the PBPs of S. faecium, the most penicillin-resistant species of group D streptococci, generally had lower affinities for the antibiotic than those of S. faecalis (intermediate penicillin resistance), which in turn were of lower affinity than those of S. bovis (penicillin-sensitive). On the other hand, no quantitative correlation could be established between the binding of penicillin to any one PBP or group of PBPs, and the penicillin MIC value for the corresponding micro-organism. Examination of the amounts of antibiotic bound and the rates of binding to PBPs of equal numbers of protoplasts and whole bacteria of S. faecalis and S. faecium, indicated that there was no permeability barrier to benzylpenicillin in the cell walls of these species. The lower antibacterial effectiveness of cephalothin compared with ampicillin in group D streptococci was paralleled by the higher concentrations of cephalothin needed in competition assays to inhibit the lower molecular size PBPs of these bacteria.     

Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of gamma-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1(-/-) mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo.     

Conjugative transfer of multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).     

Yeast resistance to polyene antibiotics. I. Production of Saccharomyces cerevisiae mutants resistant to nystatin and their genetic analysis]

239 nistatin-resistant mutants were selected after UV-irradiation of yeasts. Phenotypical analysis has revealed two main groups of the mutants: 1) resistant to nistatin and resistant or sensitive (in different combinations) to haptaens; 2) resistant to nistatin and having an increased resistance to haptens. It is found that the sensitivity dominates over the resistance and hyper-resistance. Genetic analysis of the mutant collection has shown that the resistance to nistatin is determined by 5 nuclear genes (hysr). Hyper-resistance is controlled by mutations in other genes, which are not connected with stable phenotype. Genes of hyper-resistance can be considered as minus-modificators of pleiothrophic cross-resistance, characteristic of hysr genes. Plus-modificator genes of polyenic resistance are described. The gene hysr1 is linked with its chromosome.     

Resistance of hospital Staphylococci to beta-lactam antibiotics]

Resistance of hospital Staphylococci to beta-lactam antibiotics was studied. The strains were isolated in two obstetric hospitals during an outbreak of purulent inflammatory infections in them. A modification of the "clover leaf" procedure providing elucidation of the resistance mechanism was used in the study. Development of the resistance to the beta-lactam antibiotics was shown to be mainly due the activity of beta-lactamases which makes it possible to discuss their role in the mechanism of resistance development.