In vitro and intracellular activity of vaborbactam combined with ß-lactams against Mycobacterium abscessus.

OBJECTIVES: Mycobacterium abscessus has emerged as an opportunistic pathogen responsible for lung infections, especially in cystic fibrosis patients. In spite of the production of the broad-spectrum ß-lactamase BlaMab, the carbapenem imipenem is recommended in the initial phase of the treatment of pulmonary infections. Here, we determine whether the addition of vaborbactam, a second-generation ß-lactamase inhibitor belonging to the boronate family, improves the activity of ß-lactams against M. abscessus. METHODS: The activity of ß-lactams, alone or in combination with vaborbactam, was evaluated against M. abscessus CIP104536 by determining MICs, time-killing and intramacrophage activity. Kinetic parameters for the inhibition of BlaMab by vaborbactam were determined by spectrophotometry. RESULTS: The combination of vaborbactam (8 mg/L) with ß-lactams decreased more than 8 times the MIC of amoxicillin (from >1024 to 128 mg/L) and 2 times the MICs of meropenem (from 16 to 8 mg/L) and imipenem (from 4 to 2 mg/L). The reduction of the MICs was less than that obtained with avibactam at 4 mg/L for amoxicillin (from >1024 to 16 mg/L, more than 64 times less) and for meropenem (from 16 to 4 mg/L, 4 times less). In vitro and intracellularly, M. abscessus was not killed by the meropenem/vaborbactam combination, in spite of significant in vitro inhibition of BlaMab by vaborbactam. CONCLUSIONS: Inhibition of BlaMab by vaborbactam decreases the MIC of ß-lactams, including that of meropenem. As meropenem/vaborbactam is clinically available, this combination offers an alternative therapeutic option that should be evaluated for the treatment of pulmonary infections due to M. abscessus.

Efficacy of single antibiotic therapy versus antibiotic combination in implant-free staphylococcal post-surgical spinal infections: a retrospective observational study.

BACKGROUND: Post-surgical spinal infections (pSSIs) are a serious complication of spinal surgeries, with Staphylococcus spp. being one of the most prominent bacteria identified. Optimal antimicrobial therapy for staphylococcal spinal infections without spinal implants is not well documented. METHODS: This single center retrospective 7-year observational study described and compared the outcome (treatment failure or mortality rate one year after diagnosis) of 20 patients with staphylococcal-implant-free pSSI treated with single or combination antibiotics. RESULTS: Median duration of treatment was 40 days (IQR 38-42), with 6 days (IQR 5-7) on intravenous antibiotics and 34 days (IQR 30-36) on oral therapy. Four patients (20%) underwent new surgical debridement, all due to surgical failure, and 1 patient died within the first year without significant differences between both treatment group. CONCLUSION: This study raises the possibility of single antibiotic therapy for patients with implant-free post-surgical spinal infections due to Staphylococcus spp.

Evaluating the heart valve tissue diffusion of amoxicillin in infective endocarditis: a pilot prospective observational non-comparative study.

OBJECTIVES: Treating patients with infective endocarditis (IE) due to streptococci and enterococci currently involves high-dosage antibiotics. Recent literature suggests a 30%-70% diffusion rate could be extrapolated to human heart valve tissue. The objective of this study was to evaluate the diffusion coefficient of amoxicillin in heart valve tissue of patients operated for IE. METHODS: Adult patients were prospectively included that underwent surgery at the European Hospital Georges Pompidou for IE due to streptococci and enterococci and had previous IV amoxicillin treatment. Plasma (taken 48 h preoperatively) and heart valve tissue amoxicillin concentrations were measured with a validated LC-MS/MS method. The MIC values of amoxicillin were measured for all available isolates. RESULTS: Seventeen patients were included. Eleven (64.7%) patients had native valve IE and six (35.3%) had prosthetic valve IE. Fourteen IE cases (82.4%) were due to streptococci, one (5.9%) was due to enterococci and two (11.8%) were Haemophilus spp, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella kingae group infections. Median (IQR) amoxicillin dose administered was 10.5 (8.0-12.0) g/day corresponding to 138.2 (112.5-160.0) mg/kg/day. The median amoxicillin plasma concentrations pre-surgery and intra-tissular weighted concentrations were 31.9 (25.9-51.9) mg/L and 19.0 (7.9-31.4) µg/g, respectively. Median tissue/plasma concentration ratio was 0.47 (0.24-0.67), with a median amoxicillin plasma/MIC ratio of 487 (179-745), and median amoxicillin tissue/MIC ratio of 42 (14-116). CONCLUSIONS: With a significant diffusion coefficient, amoxicillin dosage in heart valve tissues showed a concentration/MIC ratio well above current recommendations for bactericidal activity. Our study suggests that lower doses can be considered for susceptible bacteria.

TIVAP-related infection due to Gram-negative aerobic bacilli: should TIVAP stay or should it go?

We aimed to describe the outcome of totally implantable venous-access port (TIVAP)-related infections due to Gram-negative aerobic bacilli (Pseudomonas aeruginosa and other Pseudomonas spp., Acinetobacter spp., and Stenotrophomonas maltophilia), or GNAB, and assess the safety of conservative treatment. We conducted a retrospective study in a French teaching hospital, from January 2016 to December 2020, including adult patients treated for TIVAP-related infection due to GNAB. Success of conservative treatment was defined as a functional TIVAP 3 months after infection with no recurrence. We performed a bivariate analysis and analyzed causes for treatment failure. We included 68 patients (53 TIVAP-related bloodstream infections, 11 TIVAP-related infections, and 4 probable TIVAP-related infections) due to GNAB, mostly P. aeruginosa (50/68, 74%). TIVAP removal was initially decided for 49/68 patients (72%). Among the 19/68 (28%) patients with conservative treatment (all for infections caused by P. aeruginosa), 5/19 (26%) had successful treatment, 7/19 (37%) experienced failure (without sepsis or septic shock), 6/19 (32%) died within 3 months without TIVAP removal and no signs of infection recurrence, and 1 patient had TIVAP removal as it was no longer required. TIVAP-related infections caused by GNAB frequently require TIVAP removal. Conservative treatment can be performed in selected patients with a non-complicated infection caused by P. aeruginosa, who can benefit from the continuation of antineoplastic chemotherapy or palliative care. Treatment failures were not associated with sepsis or septic shock.

Clinical and biological diagnosis and follow-up of patients treated for endovascular infections due to Coxiellaburnetii.

The objective of this study was to evaluate the effectiveness of the recommended treatment for endovascular infections due to Coxiella burnetii. This single-center retrospective study was conducted in 13 patients with endovascular infection due to C. burnetii between January 2001 and December 2020 for a definite or possible endovascular infection due to C. burnetii with a minimum follow-up of 18 months post-infection. Clinical and biological data, including serology, blood and tissue PCR results, doxycycline and hydroxychloroquine assays were collected. Among the 13 patients, 11 had endocarditis (8 definite and 3 possible) and 2 had a vascular infection. At the time of diagnosis, fever was present in only 46% of cases. In case of endocarditis, 73% of patients had a pathological echocardiography. Biologically, the CRP level was low (52 mg/l ± 44). Autoimmune antibodies (antinuclear factor, neutrophil anticytoplasm) were present in 23% of patients. At the time of diagnosis, tissue PCR was very sensitive (100%) unlike blood or serum (29%). Blood levels of doxycycline and hydroxychloroquine were within expected values. Only one patient experienced treatment failure at two years, requiring surgery. For the 7 patients whose phase I IgG titres fell below 1/800, a minimum of 18 months of treatment was necessary. In the long term, the clinical and biological cure was 100% and 92% respectively, underlining the importance of monitoring the serum dosages of doxycycline and hydroxychloroquine. Given its sensitivity, tissue PCR could be added to the major Duke criteria.

Modulation of the Specificity of Carbapenems and Diazabicyclooctanes for Selective Activity against Mycobacterium tuberculosis.

Treatment of multidrug-resistant tuberculosis with combinations of carbapenems and ß-lactamase inhibitors carries risks for dysbiosis and for the development of resistances in the intestinal microbiota. Using Escherichia coli producing carbapenemase KPC-2 as a model, we show that carbapenems can be modified to obtain drugs that are inactive against E. coli but retain antitubercular activity. Furthermore, functionalization of the diazabicyclooctanes scaffold provided drugs that did not effectively inactivate KPC-2 but retained activity against Mycobacterium tuberculosis targets.

Impact of relebactam-mediated inhibition of Mycobacterium abscessus BlaMab ß-lactamase on the in vitro and intracellular efficacy of imipenem.

OBJECTIVES: Imipenem is one of the recommended ß-lactams for the treatment of Mycobacterium abscessus pulmonary infections in spite of the production of BlaMab ß-lactamase. Avibactam, a second-generation ß-lactamase inhibitor, was previously shown to inactivate BlaMab, but its partner drug, ceftazidime, is devoid of any antibacterial activity against M. abscessus. Here, we investigate whether relebactam, a novel second-generation inhibitor developed in combination with imipenem, improves the activity of this carbapenem against M. abscessus. METHODS: The impact of BlaMab inhibition by relebactam was evaluated by determining MICs, time-kill curves and M. abscessus intracellular proliferation in human macrophages. Kinetic parameters for the inhibition of BlaMab by relebactam were determined by spectrophotometry using nitrocefin as the substrate. The data were compared with those obtained with avibactam. RESULTS: Combination of relebactam (4 mg/L) with ß-lactams led to >128- and 2-fold decreases in the MICs of amoxicillin (from >4096 to 32 mg/L) and imipenem (from 8 to 4 mg/L). In vitro, M. abscessus was not killed by the imipenem/relebactam combination. In contrast, relebactam increased the intracellular activity of imipenem, leading to 88% killing. Relebactam and avibactam similarly potentiated the antibacterial activities of ß-lactams although BlaMab was inactivated 150-fold less effectively by relebactam than by avibactam. CONCLUSIONS: Inhibition of BlaMab by relebactam improves the efficacy of imipenem against M. abscessus in macrophages, indicating that the imipenem/relebactam combination should be clinically considered for the treatment of infections due to M. abscessus.

Successful 6-Week Antibiotic Treatment for Early Surgical-site Infections in Spinal Surgery.

BACKGROUND: The incidence of spinal surgical site infections (SSIs) remains stable at less than 10%. Surgical reinterventions may be hampered by decubitus, treatment-related adverse events, and cost. In the context of emergence of bacterial resistance, a short duration of antimicrobial treatment is of critical importance. If the duration of treatment for SSI is currently 12 weeks, the aim of our study was to assess the efficacy of an antimicrobial treatment shortened to 6 weeks. METHODS: This prospective study was carried out from November 2014 to July 2016 in an 827-bed teaching hospital. After surgical management of SSIs, patients received broad-spectrum antibiotics intravenously for 10 days and orally for the remainder, for a total of 6 weeks. Success was defined as absence of relapse, superinfection, or surgical failure at the end of treatment and at 1-year follow-up. RESULTS: Eighty-five patients were included in this study. The median delay between initial surgery and diagnosis of SSI was 16 days. In 65 cases (76.4%), SSIs were monomicrobial; among these, Staphylococcus aureus was found in 30 cases (46%). Failure was observed in 7 cases (8.2%), with more than half caused by anaerobic bacteria. CONCLUSIONS: Surgical management of SSI followed by a 6-week antibiotic treatment is associated with favorable outcome. Anaerobic bacteria seem to play a role in the occurrence of relapses. A 6-week reduction in antibiotic treatment leads to reduction in cost and, likely, also to reduction in the emergence and spread of resistant microorganisms.

In Vitro and Intracellular Activity of Imipenem Combined with Tedizolid, Rifabutin, and Avibactam against Mycobacterium abscessus.

Mycobacterium abscessus infections are difficult to treat because of their resistance to many antibiotics. In vitro, tedizolid combined with imipenem displayed a moderate synergistic effect (fractional inhibitory concentration index, 0.41) but no bactericidal activity. Intracellularly, tedizolid 2 µg/ml (half of the MIC), corresponding to the peak serum concentration, increased the efficacy of imipenem at 8 and 32 µg/ml. Addition of avibactam and rifabutin, alone or in combination, improved the activity of the imipenem-tedizolid combination.

In Vitro and Intracellular Activity of Imipenem Combined with Rifabutin and Avibactam against Mycobacterium abscessus.

Repurposing drugs may be useful as an add-on in the treatment of Mycobacterium abscessus pulmonary infections, which are particularly difficult to cure. M. abscessus naturally produces a ß-lactamase, BlaMAb, which is inhibited by avibactam. The recommended regimens include imipenem, which is hydrolyzed by BlaMAb and used without any ß-lactamase inhibitor. Here, we determine whether the addition of rifabutin improves the activity of imipenem alone or in combination with avibactam against M. abscessus CIP104536. Rifabutin at 16 µg/ml was only bacteriostatic (MIC of 4 µg/ml) and was moderately synergistic in combination with imipenem (fractional inhibitory concentration [FIC] index of 0.38). Addition of rifabutin (16 µg/ml) moderately increased killing by a low (8 µg/ml) but not by a high (32 µg/ml) concentration of imipenem. Addition of avibactam (4 µg/ml) did not further increase killing by the former combination. In infected macrophages, rifabutin (16 µg/ml) increased the activity of imipenem at 8 and 32 µg/ml, achieving 3- and 100-fold reductions in the numbers of intracellular bacteria, respectively. Avibactam (16 µg/ml) improved killing by imipenem at 8 µg/ml. A 5-fold killing was obtained for a triple combination comprising avibactam (16 µg/ml) and therapeutically achievable doses of imipenem (8 µg/ml) and rifabutin (1 µg/ml). These results indicate that the imipenem-rifabutin combination should be further considered for the treatment of M. abscessus pulmonary infections in cystic fibrosis patients and that addition of a ß-lactamase inhibitor might improve its efficacy. Mechanistically, the impact of BlaMAb inhibition by avibactam on antibiotic activity was assessed by comparing CIP104536 and a ß-lactamase-deficient derivative.

Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by ß-Lactams.

In most bacteria, the essential targets of ß-lactam antibiotics are the d,d-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l,d-transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l,d-transpeptidase paralogues LdtCd1, LdtCd2, and LdtCd3, which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. LdtCd2 and LdtCd3 catalyzed the formation of 3→3 cross-links (l,d-transpeptidase activity), the hydrolysis of the C-terminal d-Ala residue of the disaccharide-tetrapeptide substrate (l,d-carboxypeptidase activity), and the exchange of the C-terminal d-Ala for d-Met. LdtCd1 displayed only l,d-carboxypeptidase activity. Mass spectrometry analyses indicated that LdtCd1 and LdtCd2 were acylated by ß-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of LdtCd3 by these ß-lactams was not detected. The acylation efficacy of LdtCd1 and LdtCd2 was higher for the carbapenems (480 to 6,600 M-1 s-1) than for ampicillin and ceftriaxone (3.9 to 82 M-1 s-1). In contrast, the efficacy of the hydrolysis of ß-lactams by LdtCd1 and LdtCd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that LdtCd1 and LdtCd2 are inactivated only by ß-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.

Synthesis of Avibactam Derivatives and Activity on ß-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria.

There is a renewed interest for ß-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their ß-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg mL-1 for both species). Mechanistically, ß-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.

In Vitro Activity of Ceftolozane-Tazobactam against Multidrug-Resistant Nonfermenting Gram-Negative Bacilli Isolated from Patients with Cystic Fibrosis.

Ceftolozane-tazobactam was tested against 58 multidrug-resistant nonfermenting Gram-negative bacilli (35 Pseudomonas aeruginosa, 11 Achromobacter xylosoxydans, and 12 Stenotrophomonas maltophilia isolates) isolated from cystic fibrosis patients and was compared to ceftolozane alone, ceftazidime, meropenem, and piperacillin-tazobactam. Ceftolozane-tazobactam was the most active agent against P. aeruginosa but was inactive against A. xylosoxydans and S. maltophilia In time-kill experiments, ceftolozane-tazobactam had complete bactericidal activity against 2/6 clinical isolates (33%).

Inhibition of the ß-Lactamase BlaMab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus.

Mycobacterium abscessus pulmonary infections are treated with a macrolide (clarithromycin or azithromycin), an aminoglycoside (amikacin), and a ß-lactam (cefoxitin or imipenem). The triple combination is used without any ß-lactamase inhibitor, even though Mabscessus produces the broad-spectrum ß-lactamase BlaMab We determine whether inhibition of BlaMab by avibactam improves the activity of imipenem against M. abscessus The bactericidal activity of drug combinations was assayed in broth and in human macrophages. The in vivo efficacy of the drugs was tested by monitoring the survival of infected zebrafish embryos. The level of BlaMab production in broth and in macrophages was compared by quantitative reverse transcription-PCR and Western blotting. The triple combination of imipenem (8 or 32 µg/ml), amikacin (32 µg/ml), and avibactam (4 µg/ml) was bactericidal in broth (<0.1% survival), with 3.2- and 4.3-log10 reductions in the number of CFU being achieved at 72 h when imipenem was used at 8 and 32 µg/ml, respectively. The triple combination achieved significant intracellular killing, with the bacterial survival rates being 54% and 7% with the low (8 µg/ml) and high (32 µg/ml) dosages of imipenem, respectively. In vivo inhibition of BlaMab by avibactam improved the survival of zebrafish embryos treated with imipenem. Expression of the gene encoding BlaMab was induced (20-fold) in the infected macrophages. Inhibition of BlaMab by avibactam improved the efficacy of imipenem against M. abscessusin vitro, in macrophages, and in zebrafish embryos, indicating that this ß-lactamase inhibitor should be clinically evaluated. The in vitro evaluation of imipenem may underestimate the impact of BlaMab, since the production of the ß-lactamase is inducible in macrophages.

Inhibition of ß-lactamases of mycobacteria by avibactam and clavulanate.

Objectives: Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A ß-lactamases, BlaC and Bla Mab , which are inhibited by clavulanate and avibactam, respectively. BlaC differs from Bla Mab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of ß-lactamases from slowly and rapidly growing mycobacteria. Methods: Enzyme kinetics were determined to assess the impact of the substitutions G 132 N in BlaC and N 132 G in Bla Mab on ß-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the ß-lactamases in Escherichia coli . Results: The substitution G 132 N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. Bla Mab efficiently hydrolysed clavulanate, but the substitution N 132 G led to a 5600-fold reduction in the hydrolysis rate constant k cat due to stabilization of Bla Mab -clavulanate covalent adducts. The N 132 G substitution also led to a 610-fold reduction in the efficacy of Bla Mab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the ß-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa. Conclusions: G 132 N and N 132 G had opposite effects on the inhibition of BlaC and Bla Mab , indicating that these substitutions might lead to acquisition of resistance to either of the ß-lactamase inhibitors, but not to both of them.

Evaluation of early antimicrobial therapy adaptation guided by the BetaLACTA® test: a case-control study.

BACKGROUND: Rapid diagnostic tests detecting microbial resistance are needed for limiting the duration of inappropriateness of empirical antimicrobial therapy (EAT) in intensive care unit patients, besides reducing the use of broad-spectrum antibiotics. We hypothesized that the betaLACTA® test (BLT) could lead to early increase in the adequacy of antimicrobial therapy. METHODS: This was a case-control study. Sixty-one patients with BLT-guided adaptation of EAT were prospectively included, and then matched with 61 "controls" having similar infection characteristics (community or hospital-acquired, and source of infection), in whom EAT was conventionally adapted to antibiogram results. Endpoints were to compare the proportion of appropriate (primary endpoint) and optimal (secondary endpoint) antimicrobial therapies with each of the two strategies, once microbiological sample culture results were available. RESULTS: Characteristics of patients, infections and EAT at inclusion were similar between groups. Nine early escalations of EAT occurred in the BLT-guided adaptation group, reaching 98% appropriateness vs. 77% in the conventional adaptation group (p < 0.01). The BLT reduced the time until escalation of an inappropriate EAT from 50.5 (48-73) to 27 (24-28) hours (p < 0.01). Seventeen early de-escalations occurred in the BLT-guided adaptation group, compared to one in the conventional adaptation group, reducing patients' exposure to broad-spectrum beta-lactam such as carbapenems. In multivariate analysis, use of the BLT was strongly associated with early appropriate (OR = 18 (3.4-333.8), p = 0.006) and optimal (OR = 35.5 (9.6-231.9), p < 0.001) antimicrobial therapies. Safety parameters were similar between groups. CONCLUSIONS: Our study suggests that a BLT-guided adaptation strategy may allow early beta-lactam adaptation from the first 24 hours following the beginning of sepsis management.

Bactericidal and intracellular activity of ß-lactams against Mycobacterium abscessus.

OBJECTIVES: Cefoxitin and imipenem are the sole recommended ß-lactams for the treatment of Mycobacterium abscessus pulmonary infections. Here, we investigated whether one of these drugs displays superiority in terms of killing and intracellular activity. We have also evaluated whether the use of a ß-lactamase inhibitor could improve their activity. METHODS: The impact of the ß-lactamase BlaMab on the activity of ß-lactams was assessed by comparing M. abscessus CIP104536 and its ß-lactamase-deficient ΔblaMab derivative, as well as by using the ß-lactamase inhibitor avibactam. The activity of cefoxitin, imipenem, amoxicillin and ceftaroline, alone and in various combinations including amikacin, was compared based on determination of time-kill curves and of intracellular proliferation in human macrophages. RESULTS: Imipenem was superior to cefoxitin in both the time-kill and macrophage assays. Production of BlaMab limited the activity of imipenem. The combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. Deletion of blaMab extended the spectrum of ß-lactams active against M. abscessus to include amoxicillin and ceftaroline. In the absence of BlaMab, amoxicillin was as active as imipenem. These drugs were more active than ceftaroline and cefoxitin was the least active. Avibactam increased the intracellular activity of ceftaroline, but inhibition of BlaMab was only partial, as previously reported for amoxicillin. CONCLUSIONS: Evaluation of the killing and intracellular activities of ß-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drugs potentially useful to treat pulmonary infections.

Mutation landscape of acquired cross-resistance to glycopeptide and ß-lactam antibiotics in Enterococcus faecium.

Bypass of the d,d-transpeptidase activity of penicillin-binding proteins by an l,d-transpeptidase (Ldtfm) results in resistance to ampicillin and glycopeptides in Enterococcus faecium M9, a mutant obtained by nine consecutive selection steps. Resistance requires activation of a cryptic locus for production of the essential tetrapeptide-containing substrate of Ldtfm and impaired activity of protein phosphatase StpA. Here, whole-genome sequencing revealed a high mutation rate for the entire selection procedure (79 mutations in 900 generations). Acquisition of a mutation in the mismatch repair gene mutL had little impact on the frequency of rifampin-resistant mutants although the mutation spectrum of M9 was typical of impaired MutL with high transversion to transition (40/11) and substitution to deletion (51/28) ratios. M9 did not mainly accumulate neutral mutations since base substitutions occurred more frequently in coding sequences than expected (χ(2) = 5.0; P < 0.05) and silent mutations were underrepresented (χ(2) = 5.72; P < 0.02). None of the mutations directly affected recognition of the tetrapeptide substrate of Ldtfm by peptidoglycan synthesis enzymes. Instead, mutations appear to remodel regulatory circuits involving two-component regulatory systems and sugar metabolism. The high number of mutations required for activation of the l,d-transpeptidase pathway may strongly limit emergence of cross-resistance to ampicillin and glycopeptides by this mechanism.

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.

Hydrolysis of clavulanate by Mycobacterium tuberculosis ß-lactamase BlaC harboring a canonical SDN motif.

Combinations of ß-lactams with clavulanate are currently being investigated for tuberculosis treatment. Since Mycobacterium tuberculosis produces a broad spectrum ß-lactamase, BlaC, the success of this approach could be compromised by the emergence of clavulanate-resistant variants, as observed for inhibitor-resistant TEM variants in enterobacteria. Previous analyses based on site-directed mutagenesis of BlaC have led to the conclusion that this risk was limited. Here, we used a different approach based on determination of the crystal structure of ß-lactamase BlaMAb of Mycobacterium abscessus, which efficiently hydrolyzes clavulanate. Comparison of BlaMAb and BlaC allowed for structure-assisted site-directed mutagenesis of BlaC and identification of the G(132)N substitution that was sufficient to switch the interaction of BlaC with clavulanate from irreversible inactivation to efficient hydrolysis. The substitution, which restored the canonical SDN motif (SDG→SDN), allowed for efficient hydrolysis of clavulanate, with a more than 10(4)-fold increase in k cat (0.41 s(-1)), without affecting the hydrolysis of other ß-lactams. Mass spectrometry revealed that acylation of BlaC and of its G(132)N variant by clavulanate follows similar paths, involving sequential formation of two acylenzymes. Decarboxylation of the first acylenzyme results in a stable secondary acylenzyme in BlaC, whereas hydrolysis occurs in the G(132)N variant. The SDN/SDG polymorphism defines two mycobacterial lineages comprising rapidly and slowly growing species, respectively. Together, these results suggest that the efficacy of ß-lactam-clavulanate combinations may be limited by the emergence of resistance. ß-Lactams active without clavulanate, such as faropenem, should be prioritized for the development of new therapies.