Impact of ß-lactamase inhibition on the activity of ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus.

The production of ß-lactamases Bla(Mab) and BlaC contributes to ß-lactam resistance in Mycobacterium abscessus and Mycobacterium tuberculosis, respectively. Ceftaroline was efficiently hydrolyzed by these enzymes. Inhibition of M. tuberculosis BlaC by clavulanate decreased the ceftaroline MIC from ≥ 256 to 16 to 64 µg/ml, but these values are clinically irrelevant. In contrast, the ceftaroline-avibactam combination should be evaluated against M. abscessus since it inhibited growth at lower and potentially achievable drug concentrations.

ß-Lactamase inhibition by avibactam in Mycobacterium abscessus.

OBJECTIVES: Two ß-lactams, cefoxitin and imipenem, are part of the reference treatment for pulmonary infections with Mycobacterium abscessus. M. abscessus has recently been shown to produce a broad-spectrum ß-lactamase, BlaMab, indicating that the combination of ß-lactams with a BlaMab inhibitor may improve treatment efficacy. The objectives of this study were to evaluate the impact of BlaMab production on the efficacy of ß-lactams in vitro and to assess the benefit of BlaMab inhibition on the activity of ß-lactams intracellularly and in an animal model. METHODS: We analysed the mechanism and kinetics of BlaMab inactivation by avibactam, a non-ß-lactam ß-lactamase inhibitor currently in Phase III of development, in combination with ceftazidime for the treatment of serious infections due to Gram-negative bacteria. We then deleted the gene encoding BlaMab to assess the extent of BlaMab inhibition by avibactam based on a comparison of the impact of chemical and genetic inactivation. Finally, the efficacy of amoxicillin in combination with avibactam was evaluated in cultured human macrophages and in a zebrafish model of M. abscessus infection. RESULTS: We showed that avibactam efficiently inactivated BlaMab via the reversible formation of a covalent adduct. An inhibition of BlaMab by avibactam was observed in both infected macrophages and zebrafish. CONCLUSIONS: Our data identify avibactam as the first efficient inhibitor of BlaMab and strongly suggest that ß-lactamase inhibition should be evaluated to provide improved therapeutic options for M. abscessus infections.

Characterization of broad-spectrum Mycobacterium abscessus class A ß-lactamase.

OBJECTIVES: Imipenem and cefoxitin are used to treat Mycobacterium abscessus infections and have moderate activity against this fast-growing mycobacterium (MIC50 of 16 and 32 mg/L, respectively). M. abscessus is highly resistant to most other ß-lactams, although the underlying mechanisms have not been explored. Here, we characterized M. abscessus class A ß-lactamase (Bla(Mab)) and investigated its role in ß-lactam resistance. METHODS: Hydrolysis kinetic parameters of purified Bla(Mab) were determined by spectrophotometry for various ß-lactams and compared with those of related BlaC from Mycobacterium tuberculosis. MICs of ß-lactams were determined for M. abscessus CIP104536 and for Escherichia coli producing Bla(Mab) and BlaC. RESULTS: Bla(Mab) had a broad hydrolysis spectrum, similar to that of BlaC, but with overall higher catalytic efficiencies, except for cefoxitin. As expected from its in vivo efficacy, cefoxitin was very slowly hydrolysed by Bla(Mab) (k(cat)/K(m) = 6.7 M(-1) s(-1)). Bla(Mab) hydrolysed imipenem more efficiently (k(cat)/K(m) = 3.0 × 10(4) M(-1) s(-1)), indicating that the in vivo activity of this drug might be improved by combination with a ß-lactamase inhibitor. ß-Lactamase inhibitors clavulanate, tazobactam and sulbactam did not inhibit Bla(Mab). This enzyme efficiently hydrolysed clavulanate, in contrast to BlaC, which is irreversibly acylated by this inhibitor. Bla(Mab) and BlaC were functional in E. coli and the resistance profiles mediated by these enzymes were in agreement with the kinetic parameters. CONCLUSIONS: M. abscessus produces a clavulanate-insensitive broad-spectrum ß-lactamase that limits the in vivo efficacy of ß-lactams.

Assessing pneumococcal meningitis association with viral respiratory infections and antibiotics: insights from statistical and mathematical models.

Pneumococcus is an important human pathogen, highly antibiotic resistant and a major cause of bacterial meningitis worldwide. Better prevention requires understanding the drivers of pneumococcal infection incidence and antibiotic susceptibility. Although respiratory viruses (including influenza) have been suggested to influence pneumococcal infections, the underlying mechanisms are still unknown, and viruses are rarely considered when studying pneumococcus epidemiology. Here, we propose a novel mathematical model to examine hypothetical relationships between Streptococcus pneumoniae meningitis incidence (SPMI), acute viral respiratory infections (AVRIs) and antibiotic exposure. French time series of SPMI, AVRI and penicillin consumption over 2001-2004 are analysed and used to assess four distinct virus-bacteria interaction submodels, ascribing the interaction on pneumococcus transmissibility and/or pathogenicity. The statistical analysis reveals strong associations between time series: SPMI increases shortly after AVRI incidence and decreases overall as the antibiotic-prescription rate rises. Model simulations require a combined impact of AVRI on both pneumococcal transmissibility (up to 1.3-fold increase at the population level) and pathogenicity (up to threefold increase) to reproduce the data accurately, along with diminished epidemic fitness of resistant pneumococcal strains causing meningitis (0.97 (0.96-0.97)). Overall, our findings suggest that AVRI and antibiotics strongly influence SPMI trends. Consequently, vaccination protecting against respiratory virus could have unexpected benefits to limit invasive pneumococcal infections.

Inactivation kinetics of a new target of beta-lactam antibiotics.

Peptidoglycan is predominantly cross-linked by serine DD-transpeptidases in most bacterial species. The enzymes are the essential targets of ß-lactam antibiotics. However, unrelated cysteine LD-transpeptidases have been recently recognized as a predominant mode of peptidoglycan cross-linking in Mycobacterium tuberculosis and as a bypass mechanism conferring resistance to all ß-lactams, except carbapenems such as imipenem, in Enterococcus faecium. Investigation of the mechanism of inhibition of this new ß-lactam target showed that acylation of the E. faecium enzyme (Ldt(fm)) by imipenem is irreversible. Using fluorescence kinetics, an original approach was developed to independently determine the catalytic constants for imipenem binding (k(1) = 0.061 µM(-1) min(-1)) and acylation (k(inact) = 4.5 min(-1)). The binding step was limiting at the minimal drug concentration required for bacterial growth inhibition. The Michaelis complex was committed to acylation because its dissociation was negligible. The emergence of imipenem resistance involved substitutions in Ldt(fm) that reduced the rate of formation of the non-covalent complex but only marginally affected the efficiency of the acylation step. The methods described in this study will facilitate development of new carbapenems active on extensively resistant M. tuberculosis.

Inactivation of Mycobacterium tuberculosis l,d-transpeptidase LdtMt1 by carbapenems and cephalosporins.

The structure of Mycobacterium tuberculosis peptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized by l,d-transpeptidases that replace 4→3 cross-links formed by the d,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these l,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistant M. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypic M. tuberculosis l,d-transpeptidase Ldt(Mt1) by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of Ldt(Mt1), which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited Ldt(Mt1), with a binding rate constant of 0.08 µM(-1) min(-1). The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min(-1)) in comparison to the acylation rate constant (3.1 min(-1)). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10(-3) min(-1). Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient Ldt(Mt1) inactivator. Cephalosporins also formed covalent adducts with Ldt(Mt1), although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition in M. tuberculosis.

Molecular basis for different levels of tet(M) expression in Streptococcus pneumoniae clinical isolates.

Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet(M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 µg/ml) were found to harbor truncated tet(M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet(M) promoter region, among which four displayed increased Tc MICs (16 to 64 µg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet(M). Expression in Enterococcus faecalis of a tet(M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet(M) and by alterations of the upstream transcriptional attenuator.

Kinetic analysis of Enterococcus faecium L,D-transpeptidase inactivation by carbapenems.

Bypass of classical penicillin-binding proteins by the L,D-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high-level ampicillin resistance in E. faecium mutants, whereas carbapenems remain the lone highly active ß-lactams. Kinetics of Ldt(fm) inactivation was determined for four commercial carbapenems and a derivative obtained by introducing a minimal ethyl group at position 2. We show that the bulky side chains of commercial carbapenems have both positive and negative effects in preventing hydrolysis of the acyl enzyme and impairing drug binding.

The peptidoglycan of Mycobacterium abscessus is predominantly cross-linked by L,D-transpeptidases.

Few therapeutic alternatives remain for the treatment of infections due to multiresistant Mycobacterium abscessus. Here we show that the peptidoglycans of the "rough" and "smooth" morphotypes contain predominantly 3→3 cross-links generated by l,d-transpeptidases, indicating that these enzymes are attractive targets for the development of efficient drugs.

The beta-lactam-sensitive D,D-carboxypeptidase activity of Pbp4 controls the L,D and D,D transpeptidation pathways in Corynebacterium jeikeium.

Corynebacterium jeikeium is an emerging nosocomial pathogen responsible for vascular catheters infections, prosthetic endocarditis and septicemia. The treatment of C. jeikeium infections is complicated by the multiresistance of clinical isolates to antibiotics, in particular to beta-lactams, the most broadly used class of antibiotics. To gain insight into the mechanism of beta-lactam resistance, we have determined the structure of the peptidoglycan and shown that C. jeikeium has the dual capacity to catalyse formation of cross-links generated by transpeptidases of the d,d and l,d specificities. Two ampicillin-insensitive cross-linking enzymes were identified, Ldt(Cjk1), a member of the active site cysteine l,d-transpeptidase family, and Pbp2c, a low-affinity class B penicillin-binding protein (PBP). In the absence of beta-lactam, the PBPs and the l,d-transpeptidase contributed to the formation of 62% and 38% of the cross-links respectively. Although Ldt(Cjk1) and Pbp2C were not inhibited by ampicillin, the participation of the l,d-transpeptidase to peptidoglycan cross-linking decreased in the presence of the drug. The specificity of Ldt(Cjk1) for acyl donors containing a tetrapeptide stem accounts for this effect of ampicillin since the essential substrate of Ldt(Cjk1) was produced by an ampicillin-sensitive d,d-carboxypeptidase (Pbp4(Cjk)). Acquisition and mutational alterations of pbp2C accounted for high-level beta-lactam resistance in C. jeikeium.

Postoperative mediastinitis due to Finegoldia magna with negative blood cultures.

We report a case of Finegoldia magna (formerly known as Peptostreptococcus magnus) mediastinitis following coronary artery bypass in a 50-year-old patient. Even if staphylococci remain the main causative organism of postoperative mediastinitis, the responsibility of anaerobic bacteria must be considered in cases of fever and sternal drainage with negative blood cultures.

Identification of the L,D-transpeptidases for peptidoglycan cross-linking in Escherichia coli.

Three active-site cysteine L,D-transpeptidases can individually anchor the Braun lipoprotein to the Escherichia coli peptidoglycan. We show here that two additional enzymes of the same family form peptide bonds between the third residues of peptidoglycan stems, generating meso-DAP(3)-->meso-DAP(3) unusual cross-links. This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins.

The peptidoglycan of stationary-phase Mycobacterium tuberculosis predominantly contains cross-links generated by L,D-transpeptidation.

Our understanding of the mechanisms used by Mycobacterium tuberculosis to persist in a "dormant" state is essential to the development of therapies effective in sterilizing tissues. Gene expression profiling in model systems has revealed a complex adaptive response thought to endow M. tuberculosis with the capacity to survive several months of combinatorial antibiotic treatment. We show here that this adaptive response may involve remodeling of the peptidoglycan network by substitution of 4-->3 cross-links generated by the D,D-transpeptidase activity of penicillin-binding proteins by 3-->3 cross-links generated by a transpeptidase of L,D specificity. A candidate gene, previously shown to be upregulated upon nutrient starvation, was found to encode an L,D-transpeptidase active in the formation of 3-->3 cross-links. The enzyme, Ldt(Mt1), was inactivated by carbapenems, a class of beta-lactam antibiotics that are poorly hydrolyzed by the M. tuberculosis beta-lactamases. Ldt(Mt1) and carbapenems may therefore represent a target and a drug family relevant to the eradication of persistent M. tuberculosis.

A plasmid-borne Shewanella algae Gene, qnrA3, and its possible transfer in vivo between Kluyvera ascorbata and Klebsiella pneumoniae.

The plasmid-borne quinolone resistance gene qnrA1 is prevalent in multidrug-resistant Enterobacteriaceae. A chromosomally encoded homologue in Shewanella algae, qnrA3, has been described. We isolated two qnrA3-positive strains, one of Klebsiella pneumoniae (He96) and one of Kluyvera ascorbata (Kas96), from the feces of an immunocompromised outpatient. The qnrA3 allele was identical to that of S. algae except for 5 nucleotides and differed from qnrA1 by 29 nucleotides affecting three amino acids. The analysis of the qnrA3 genetic environment showed that qnrA3 was inserted downstream from an ISCR1 element at a recombination crossover site described for other resistance genes, including qnrA1, and immediately upstream from IS26, a situation not described before. IS26 preceded an incomplete class 1 integron which contained, among other genes, aac(6')-Ib-cr, another transferable quinolone resistance gene, and the beta-lactamase gene bla(OXA-1/30). The 10-kb fragment encompassing qnrA3 was compared to previously described qnrA1-containing plasmids and multidrug-resistant plasmids; it shares identical sequences with pC15a, pHSH2, pQR1, pQKp311H, and pSAL-1 but with rearrangements, deletions, and mutations. Conjugal transfer of qnrA3 was highly efficient (10(-2)) from K. pneumoniae He96 or K. ascorbata Kas96 to Escherichia coli J53 but less so (10(-5)) from either donor to a clinical strain of Enterobacter cloacae. This first description of a plasmid-borne copy and of the in vitro transfer of qnrA3 is taken to illustrate its likely in vivo transfer from S. algae to the Enterobacteriaceae.

Crystal structure of a novel beta-lactam-insensitive peptidoglycan transpeptidase.

During the final stages of cell-wall synthesis in bacteria, penicillin-binding proteins (PBPs) catalyse the cross-linking of peptide chains from adjacent glycan strands of nascent peptidoglycan. We have recently shown that this step can be bypassed by an L,D-transpeptidase, which confers high-level beta-lactam-resistance in Enterococcus faecium. The resistance bypass leads to replacement of D-Ala4-->D-Asx-L-Lys3 cross-links generated by the PBPs by L-Lys3-->D-Asx-L-Lys3 cross-links generated by the L,D-transpeptidase. As the first structure of a member of this new transpeptidase family, we have determined the crystal structure of a fragment of the L,D-transpeptidase from E.faecium (Ldt(fm217)) at 2.4A resolution. Ldt(fm217) consists of two domains, the N-terminal domain, a new mixed alpha-beta fold, and the ErfK_YbiS_YhnG C-terminal domain, a representative of the mainly beta class of protein structures. Residue Cys442 of the C-terminal domain has been proposed to be the catalytic residue implicated in the cleavage of the L-Lys-D-Ala peptide bond. Surface analysis of Ldt(fm217) reveals that residue Cys442 is localized in a buried pocket and is accessible by two paths on different sides of the protein. We propose that the two paths to the catalytic residue Cys442 are the binding sites for the acceptor and donor substrates of the L,D-transpeptidase.

Insight Into Resistance Phenotypes of Emergent Non 13-valent Pneumococcal Conjugate Vaccine Type Pneumococci Isolated From Invasive Disease After 13-valent Pneumococcal Conjugate Vaccine Implementation in France.

Background. In 2010, the pneumococcal 13-valent conjugate vaccine (PCV13), containing 6 additional serotypes including the multidrug-resistant 19A, replaced the PCV7 in France. This study aimed at analyzing trends in antibiotic resistance in invasive pneumococcal disease (IPD) isolates in France after PCV13 introduction. Methods. A total of 5243 pneumococci isolated from IPD in 2008-2009 (late PCV7 era) and 2011-2012 (PCV13 era) were studied according to their serotype and antibiotic resistance profile. Multilocus sequence typing analysis was performed on strains of the predominant serotypes (12F and 24F) isolated from young children. Results. Overall, the prevalence of antibiotic resistance decreased in France (-21.5% for penicillin from 2008-2009 to 2011-2012), mainly driven by the decline of the 19A serotype. Among non-PCV13 serotypes that concomitantly emerged, serotypes 12F, 24F, 15A, and 35B were consistently associated with resistance to 1 or more antibiotics. In children under 2 years, serotypes 15A, 35B, and 24F accounted together for 37.8% and 31.9% of penicillin-nonsusceptible and erythromycin-resistant isolates, respectively. Chloramphenicol and cotrimoxazole resistance were mainly associated with serotypes 12F and 24F, respectively. Genetic analysis showed that although emergence of serotype 12F pneumococci resulted from the expansion of various pre-existing lineages, increase in serotype 24F was related to the clonal expansion of the ST162 penicillin-susceptible cotrimoxazole-resistant lineage. Conclusions. We showed that decline of PCV13-related IPD was associated with a decline in antibiotic resistance in France, but that it likely favored the spread of several resistant nonvaccine serotypes. However, antibiotic resistance does not seem to be the only element that may drive this phenomenon.

Molecular epidemiology of extended-spectrum beta-lactamase-producing Enterobacteriaceae isolated from environmental and clinical specimens in a cardiac surgery intensive care unit.

OBJECTIVES: To investigate environmental contamination by extended-spectrum beta-lactamase-producing Enterobacteriaceae and to perform a comparative molecular analysis of clinical and environmental strains. SETTING: A 17-bed cardiac surgery intensive care unit of a 480-bed university teaching hospital. METHODS: Following an outbreak of extended-spectrum beta-lactamase-producing Enterobacteriaceae, an environmental survey revealed extensive contamination of the environment (particularly faucets, sink drains, and the joints of the countertops) by extended-spectrum beta-lactamase-producing Enterobacteriaceae. Environmental strains were compared with clinical strains by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA. RESULTS: A total of 62 environmental strains belonging to 4 species of extended-spectrum beta-lactamase-producing Enterobacteriaceae were analyzed and compared with 43 clinical strains obtained from 34 patients. Comparative molecular analysis revealed 4 identical or closely related patterns (3 from Klebsiella oxytoca and 1 from Enterobacter cloacae) between environmental and clinical strains. CONCLUSIONS: Moist surfaces may serve as sources of multiply resistant Enterobacteriaceae in the intensive care unit. Identification and disinfection of such sources may therefore be helpful in prevention and control of outbreaks.

Airway colonisation in long-term mechanically ventilated patients. Effect of semi-recumbent position and continuous subglottic suctioning.

OBJECTIVE: To evaluate the impact of continuous subglottic suctioning and semi-recumbent body position on bacterial colonisation of the lower respiratory tract. DESIGN: A randomised controlled trial. SETTING: The ten-bed medical ICU of a French university hospital. PATIENTS: Critically ill patients expected to require mechanical ventilation for more than 5 days. INTERVENTIONS: Patients were randomly assigned to receive either continuous suctioning of subglottic secretions and semi-recumbent body position or to receive standard care and supine position. MEASUREMENTS AND RESULTS: Oropharyngeal and tracheal secretions were sampled daily and quantitatively cultured. All included patients were followed up from day 1 (intubation) to day 10, extubation or death. Ninety-seven samples of oropharynx and trachea were analysed (40 for the suctioning group and 57 for the control group). The median bacterial counts in trachea were 6.6 Log10 CFU/ml (interquartile range, IQR, 4.4-8.3) in patients who received continuous suctioning and 5.1 Log10 CFU/ml (IQR 3.6-5.5) in control patients. Most of the patients were colonised in the trachea after 1 day of mechanical ventilation (75% in the suctioning group, 80% in the control group). No significant difference was found in the daily bacterial counts in the oropharynx and in the trachea between the two groups of patients. CONCLUSION: Tracheal colonisation in long-term mechanically ventilated ICU patients was not modified by the use of continuous subglottic suctioning and semi-recumbent body position.