Structural insights into the loss of penicillinase and the gain of ceftazidimase activities by OXA-145 ß-lactamase in Pseudomonas aeruginosa.

OBJECTIVES: We previously described extended-spectrum oxacillinase OXA-145 from Pseudomonas aeruginosa, which differs from narrow-spectrum OXA-35 by loss of Leu-155. The deletion results in loss of benzylpenicillin hydrolysis and acquisition of activity against ceftazidime. We report the crystal structure of OXA-145 and provide the basis of its switch in substrate specificity. METHODS: OXA-145 variants were generated by site-directed mutagenesis and purified to homogeneity. The crystal structure of OXA-145 was determined and molecular dynamics simulations were performed. Kinetic parameters were investigated in the absence and in the presence of sodium hydrogen carbonate (NaHCO3) for representative substrates. RESULTS: The structure of OXA-145 was obtained at a resolution of 2.3 Å and its superposition with that of OXA-10 showed that Trp-154 was shifted by 1.8 Å away from the catalytic Lys-70, which was not N-carboxylated. Addition of NaHCO3 significantly increased the catalytic efficiency against penicillins, but not against ceftazidime. The active-site cavity of OXA-145 was larger than that of OXA-10, which may favour the accommodation of large molecules such as ceftazidime. Molecular dynamics simulations of OXA-145 in complex with ceftazidime revealed two highly coordinated water molecules on the α- or ß-face of the acyl ester bond, between Ser-67 and ceftazidime, which could be involved in the catalytic process. CONCLUSIONS: Deletion of Leu-155 resulted in inefficient positioning of Trp-154, leading to a non-carboxylated Lys-70 and thus to loss of hydrolysis of the penicillins. Ceftazidime hydrolysis could be attributed to enlargement of the active site and to a catalytic mechanism independent of the carboxylated Lys-70.

Antimicrobial resistance of Listeria monocytogenes strains isolated from humans in France.

Susceptibility to antibiotics of 4,816 clinical L. monocytogenes strains isolated since 1926 was studied, and the temporal evolution of susceptibility to antibiotics was analyzed through several decades. The mechanisms of resistance in each resistant strain were studied. The prevalence of resistant strains was estimated at 1.27% among isolates from humans. Resistance to tetracyclines+ and fluoroquinolones was more common and has recently emerged. Although acquired resistance in clinical L. monocytogenes did not implicate clinically relevant antibiotics, the possibility of resistance gene transfers, the description of the first clinical isolate with high-level resistance to trimethoprim, and the recent increase in penicillin MICs up to 2 microg/ml reinforce the need for microbiological surveillance.

New combinations of mutations in VanD-Type vancomycin-resistant Enterococcus faecium, Enterococcus faecalis, and Enterococcus avium strains.

We studied the clinical isolates Enterococcus faecium NEF1, resistant to high levels of vancomycin (MIC, 512 microg/ml) and teicoplanin (MIC, 64 microg/ml); Enterococcus faecium BM4653 and BM4656 and Enterococcus avium BM4655, resistant to moderate levels of vancomycin (MIC, 32 microg/ml) and to low levels of teicoplanin (MIC, 4 microg/ml); and Enterococcus faecalis BM4654, moderately resistant to vancomycin (MIC, 16 microg/ml) but susceptible to teicoplanin (MIC, 0.5 microg/ml). The strains were distinct, were constitutively resistant via the synthesis of peptidoglycan precursors ending in D-alanyl-D-lactate, and harbored a chromosomal vanD gene cluster that was not transferable. New mutations were found in conserved domains of VanS(D): at T(170)I near the phosphorylation site in NEF1, at V(67)A at the membrane surface in BM4653, at G(340)S in the G2 ATP-binding domain in BM4655, in the F domain in BM4656 (a 6-bp insertion), and in the G1 and G2 domains of BM4654 (three mutations). The mutations resulted in constitutivity, presumably through the loss of the phosphatase activity of the sensor. The chromosomal Ddl D-Ala:D-Ala ligase had an IS19 copy in NEF1, a mutation in the serine (S(185)F) or near the arginine (T(289)P) involved in D-Ala1 binding in BM4653 or BM4655, respectively, and a mutation next to the lysine (P(180)S) involved in D-Ala2 binding in BM4654, leading to the production of an impaired enzyme. In BM4653 vanY(D), a new insertion sequence, ISEfa9, belonging to the IS3 family, resulted in the absence of D,D-carboxypeptidase activity. Strain BM4656 had a functional D-Ala:D-Ala ligase, associated with high levels of both VanX(D) and VanY(D) activities, and is the first example of a VanD-type strain with a functional Ddl enzyme. Study of these five clinical isolates, displaying various assortments of mutations, confirms that all VanD-type strains isolated so far have undergone mutations in the vanS(D) or vanR(D) gene, leading to constitutive resistance, but that the Ddl host ligase is not always impaired. Based on sequence differences, the vanD gene clusters could be assigned to two subtypes: vanD-1 and vanD-4.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Real-time PCR detection of gyrA and parC mutations in Streptococcus pneumoniae.

Fluoroquinolone resistance in Streptococcus pneumoniae mainly involves stepwise mutations predominantly in the parC and gyrA genes. We have developed a single-run real-time PCR assay for detection of the four most common mutations in the quinolone resistance-determining regions of these genes. This assay provides a useful tool for both clinical and epidemiological use.

Predictable and unpredictable evolution of antibiotic resistance.

Evolution of bacteria towards antibiotic resistance is unavoidable as it represents a particular aspect of the general evolution of bacteria. Thus, at the very best, the only hope we can have in the field of resistance is to delay dissemination of resistant bacteria or resistance genes. Resistance to antibiotics in bacteria can result from mutations in resident structural or regulatory genes or from horizontal acquisition of foreign genetic information. In this review, we will consider the predictable future of the relationship between bacteria and antibiotics.

Functional gene transfer from intracellular bacteria to mammalian cells.

We provide evidence of direct transfer of functional DNA from bacteria to mammalian cells. An Escherichia coli K12 diaminopimelate auxotroph made invasive by cloning the invasin gene from Yersinia pseudotuberculosis transfers DNA after simple co-incubation, into a variety of mammalian cell lines. Transfer efficiency was enhanced in some cells by coexpression of the gene for listeriolysin from Listeria monocytogenes. Expression of the acquired genes occurs in both dividing and quiescent cells. The only requirement for bacteria to transfer genetic material into nonprofessional phagocytic cells and macrophages is the ability to invade the host cell.

Why is antibiotic resistance a deadly emerging disease?

Evolution of bacteria towards resistance to antimicrobial agents, including multidrug resistance, is unavoidable because it represents a particular aspect of the general evolution of bacteria that is unstoppable. Therefore, the only means of dealing with this situation is to delay the emergence and subsequent dissemination of resistant bacteria or resistance genes. In this review, we will consider the biochemical mechanisms and the genetics that bacteria use to offset antibiotic selective pressure. The data provided are mainly, if not exclusively, taken from the work carried out in the laboratory, although there are numerous other examples in the literature.

A penicillin- and metronidazole-resistant Clostridium botulinum strain responsible for an infant botulism case.

The clinical course of a case of infant botulism was characterized by several relapses despite therapy with amoxicillin and metronidazole. Botulism was confirmed by identification of botulinum toxin and Clostridium botulinum in stools. A C. botulinum A2 strain resistant to penicillins and with heterogeneous resistance to metronidazole was isolated from stool samples up to 110 days after onset. Antibiotic susceptibility was tested by disc agar diffusion and MICs were determined by Etest. Whole genome sequencing allowed detection of a gene cluster composed of blaCBP for a novel penicillinase, blaI for a regulator, and blaR1 for a membrane-bound penicillin receptor in the chromosome of the C. botulinum isolate. The purified recombinant penicillinase was assayed. Resistance to ß-lactams was in agreement with the kinetic parameters of the enzyme. In addition, the ß-lactamase gene cluster was found in three C. botulinum genomes in databanks and in two of 62 genomes of our collection, all the strains belonging to group I C. botulinum. This is the first report of a C. botulinum isolate resistant to penicillins. This stresses the importance of antibiotic susceptibility testing for adequate therapy of botulism.

Glycopeptide resistance in enterococci.

Glycopeptide resistance in enterococci results from the production of peptidoglycan precursors with low affinity for these antibiotics. The mobility of the resistance genes by transposition and conjugation and the ability of the resistance proteins to interfere with synthesis of normal precursors in different hosts indicate that dissemination into other bacterial species should be anticipated.

TEM beta-lactamase mutants hydrolysing third-generation cephalosporins. A kinetic and molecular modelling analysis.

The catalytic properties of six "natural" mutants of the TEM-1 beta-lactamase have been studied in detail, with special emphasis on their activity versus third-generation cephalosporins. On the basis of the recently determined high-resolution structure of the wild-type enzyme, and of the substrates' structures optimized by the AMI quantum chemistry method, we have attempted to explain the influences of the mutations on the substrate profiles of the enzymes. Some of the kinetic results have thus received a satisfactory, semi-quantitative interpretation, especially in the case of single mutations. Analysis of the double mutants proved more hazardous. Extending the comparison to some other class A beta-lactamases showed that similar properties could result from different sequences, supplying an interesting example of convergent evolution within a generally diverging family.

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium.

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.

Aminoglycoside-modifying enzymes of Staphylococcus aureus; expression in Escherichia coli.

Staphylococcus aureus plasmids PSH2, RN1956 and pWA1 code for an aminoglycoside phosphotransferase; plasmid pWA1 also encodes an aminoglycoside-aminocyclitol adenylyltransferase. S. aureus plasmid pWA2 confers resistance to erythromycin and sulfonamide. Using plasmid ColE1-ApR (RSF2124) as a vehicle, we have transferred the genes determining aminoglycoside phosphotransferase and aminoglycoside-aminocyclitol adenylyltransferase activities from S. aureus to Escherichia coli. The new plasmids obtained confer aminoglycoside-aminocyclitol resistant phenotypes to E. coli, similar to, and by the same mechanisms as "naturally" occurring plasmids. By contrast, the results obtained after cloning of plasmid pWA2 indicate that certain S. aureus antibiotic resistance determinants (e.g. for erythromycin (Em) and sulfonamide (Su) cannot be phenotypically expressed in E. coli. The DNA of the constructed hybrid plasmids has been analysed by agarose gel electrophoresis following digestion with restriction endonucleases, by ultracentrifugation in cesium chloride, by hybridization, and by electron microscopy. Each hybrid is a cointegrate replicon, composed of an entire S. aureus plasmid covalently joined to ColE1-ApR.

Characterization of Tn1547, a composite transposon flanked by the IS16 and IS256-like elements, that confers vancomycin resistance in Enterococcus faecalis BM4281.

A 64-kb genetic element harboring a vanB vancomycin-resistance (VmR) gene cluster was shown to translocate from the chromosome of Enterococcus faecalis BM4281 into the hemolysin (Hly) plasmid, pIP964. Sequence analysis of the resulting junction fragments indicated that the VmR genes were carried by a composite transposon, Tn1547, bounded by two distantly related insertion sequences (IS), designated IS256-like and IS16, in a direct orientation. IS256-like (1324 bp) was identical to IS256, except for two nucleotide (nt) transitions in the putative transposase gene. IS16 (1466 bp) contained a large open reading frame (ORF) that was 61% identical to the gene encoding the putative transposase of IS256. There was 58% identity between the deduced amino acid (aa) sequences of the putative transposases of IS256-like (390 aa) and IS16 (395 aa). IS16 was delineated by imperfect inverted repeats (IR) (18 out of 26 bp) which were related (23/26 bp identity) to their respective imperfect IR counterparts in IS256. The difference between the IS was not a barrier for transposition of Tn1547 which generated an 8-bp duplication at the target site. Dissemination of VanB-type resistance among enterococci results from two mechanisms: (i) large conjugative elements translocate from chromosome to chromosome following inter-strain transfer; and (ii) as described in this report, the VmR genes transpose from replicon to replicon within the same strain as part of composite transposons that are internal to the conjugative elements.

Sequence of the genes blaT-1B and blaT-2.

We have completed the nucleotide sequence of the genes blaT-1B from transposon Tn2, and blaT-2 from Tn1, which encode the penicillinases TEM-1 and TEM-2, respectively.

Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli.

We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.

Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp.

Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.

Nucleotide sequence of gene linA encoding resistance to lincosamides in Staphylococcus haemolyticus.

We have determined the nucleotide sequence of gene linA in plasmid pIP855, which confers resistance to lincomycin by inactivating it in Staphylococcus haemolyticus. The gene was defined by start and stop codons and an open reading frame of 483 bp corresponding to a product with an Mr of 19020. The apparent size of the resistance-conferring protein was 21 kDa, when a minicell extract was electrophoresed in the sodium dodecyl sulfate-polyacrylamide gels. Full expression of linA was obtained both in Escherichia coli and Bacillus subtilis.

Characterization of a new class of tetracycline-resistance gene tet(S) in Listeria monocytogenes BM4210.

The nucleotide sequence of the tetracycline (Tc)-minocycline (Mc)-resistance determinant of plasmid pIP811 from Listeria monocytogenes BM4210 has been determined. The gene, designated tet(S), was identified by analysis of the start and stop codons as a coding sequence of 1923 bp, corresponding to a protein with a calculated M(r) of 72,912. The apparent 68-kDa size estimated by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the protein characterized in a cell-free coupled transcription-translation system was in good agreement with the calculated value. The tet(S) gene product exhibits 79 and 72% amino acid identity with Tet(M) from Streptococcus pneumoniae and Tet(O) from Campylobacter coli, respectively. The distribution of tet(S) in strains of Gram+ and Gram- genera resistant to Tc (TcR) and Mc (McR) was studied by hybridization under high stringency using a 590-bp intragenic probe. Homology with tet(S) was detected in two clinical isolates of L. monocytogenes isolated in different geographical areas.

Sequence of the vanY gene required for production of a vancomycin-inducible D,D-carboxypeptidase in Enterococcus faecium BM4147.

Cloning and nucleotide sequencing identified the vanY gene as a member of the vancomycin-resistance van gene cluster of enterococcal plasmid, pIP816. The vanY gene was necessary for synthesis of the vancomycin-inducible D,D-carboxypeptidase activity previously proposed to be responsible for glycopeptide resistance. However, this activity was not required for peptidoglycan synthesis in the presence of glycopeptides. The deduced product of vanY did not display significant similarity with other D,D-carboxypeptidases.