Epidemiology of Carbapenem Resistance Determinants Identified in Meropenem-Nonsusceptible Enterobacterales Collected as Part of a Global Surveillance Program, 2012 to 2017.

To estimate the incidence of carbapenem-resistant Enterobacterales (CRE), a global collection of 81,781 surveillance isolates of Enterobacterales collected from patients in 39 countries in five geographic regions from 2012 to 2017 was studied. Overall, 3.3% of isolates were meropenem-nonsusceptible (MIC ≥2 µg/ml), ranging from 1.4% (North America) to 5.3% (Latin America) of isolates by region. Klebsiella pneumoniae accounted for the largest number of meropenem-nonsusceptible isolates (76.7%). The majority of meropenem-nonsusceptible Enterobacterales carried KPC-type carbapenemases (47.4%), metallo-ß-lactamases (MBLs; 20.6%) or OXA-48-like ß-lactamases (19.0%). Forty-three carbapenemase sequence variants (8 KPC-type, 4 GES-type, 7 OXA-48-like, 5 NDM-type, 7 IMP-type, and 12 VIM-type) were detected, with KPC-2, KPC-3, OXA-48, NDM-1, IMP-4, and VIM-1 identified as the most common variants of each carbapenemase type. The resistance mechanisms responsible for meropenem-nonsusceptibility varied by region. A total of 67.3% of all carbapenemase-positive isolates identified carried at least one additional plasmid-mediated or intrinsic chromosomally encoded extended-spectrum ß-lactamase, AmpC ß-lactamase, or carbapenemase. The overall percentage of meropenem-nonsusceptible Enterobacterales increased from 2.7% in 2012 to 2014 to 3.8% in 2015 to 2017. This increase could be attributed to the increasing proportion of carbapenemase-positive isolates that was observed, most notably among isolates carrying NDM-type MBLs in Asia/South Pacific, Europe, and Latin America; OXA-48-like carbapenemases in Europe, Middle East/Africa, and Asia/South Pacific; VIM-type MBLs in Europe; and KPC-type carbapenemases in Latin America. Ongoing CRE surveillance combined with a global antimicrobial stewardship strategy, sensitive clinical laboratory detection methods, and adherence to infection control practices will be needed to interrupt the spread of CRE.

Longitudinal analysis of ESBL and carbapenemase carriage among Enterobacterales and Pseudomonas aeruginosa isolates collected in Europe as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme, 2013-17.

OBJECTIVES: To determine the spread of ESBLs and carbapenemases in Enterobacterales and Pseudomonas aeruginosa in Europe. METHODS: 45 335 Gram-negative bacilli were collected in 18 European countries as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme from 2013 to 2017. Antimicrobial susceptibility was determined using broth microdilution, and 9546 isolates were screened for ß-lactamase genes by PCR and sequencing. RESULTS: ESBLs were identified in 35.5% of Klebsiella pneumoniae and 18.5% of Escherichia coli. ESBL carriage was lowest among isolates in Northern/Western Europe and highest in Eastern Europe. CTX-M-15 was the dominant ESBL in all countries except Greece, where SHV-type ESBLs were more common. Carbapenemases (KPC, OXA-48-like, GES, NDM and VIM) were found in 3.4% of Enterobacterales and were most common among K. pneumoniae (10.5% of those collected). Carbapenemase carriage was lowest in Northern/Western and highest in Southern Europe. KPC-positive Enterobacterales were most abundant but the percentages of OXA-48-like-, NDM- and VIM-positive isolates increased over time and were correlated with an increase in meropenem non-susceptibility. Carbapenemases (VIM, IMP, NDM and GES) were also identified in 5.1% of P. aeruginosa and were commonly found in Eastern Europe. Carbapenemase carriage and meropenem non-susceptibility among P. aeruginosa fluctuated over the 5 years studied and were not well correlated. CONCLUSIONS: ESBL and carbapenemase carriage varied by species and European subregion. Meropenem non-susceptibility in European isolates of Enterobacterales can be attributed to carbapenemase carriage and is increasingly caused by MBLs and OXA-48-like carbapenemases. Carbapenemases or other ß-lactamases are not a common cause of meropenem non-susceptibility in P. aeruginosa in Europe.

In Vitro Activity of Ceftazidime-Avibactam against Clinical Isolates of Enterobacteriaceae and Pseudomonas aeruginosa Collected in Latin American Countries: Results from the INFORM Global Surveillance Program, 2012 to 2015.

The International Network for Optimal Resistance Monitoring (INFORM) global surveillance program collected clinical isolates of Enterobacteriaceae (n = 7,665) and Pseudomonas aeruginosa (n = 1,794) from 26 medical centers in six Latin American countries from 2012 to 2015. The in vitro activity of ceftazidime-avibactam and comparators was determined for the isolates using the Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution method. Enterobacteriaceae were highly susceptible (99.7%) to ceftazidime-avibactam, including 99.9% of metallo-ß-lactamase (MBL)-negative isolates; 87.4% of all P. aeruginosa isolates and 92.8% of MBL-negative isolates were susceptible to ceftazidime-avibactam. Susceptibility to ceftazidime-avibactam ranged from 99.4% to 100% for Enterobacteriaceae and from 79.1% to 94.7% for P. aeruginosa when isolates were analyzed by country of origin. Ceftazidime-avibactam inhibited 99.6% to 100% of Enterobacteriaceae isolates that carried serine ß-lactamases, including extended-spectrum ß-lactamases (ESBLs), AmpC cephalosporinases, and carbapenemases (KPC and OXA-48-like) as well as 99.7%, 99.6%, 99.5%, and 99.2% of MBL-negative isolates demonstrating ceftazidime-nonsusceptible, multidrug-resistant (MDR), meropenem-nonsusceptible, and colistin-resistant phenotypes, respectively. Among carbapenem-nonsusceptible isolates of P. aeruginosa (n = 750), 14.7% carried MBLs with or without additional acquired serine ß-lactamases, while in the majority of isolates (70.0%), no acquired ß-lactamase was identified. Ceftazidime-avibactam inhibited 89.5% of carbapenem-nonsusceptible P. aeruginosa isolates in which no acquired ß-lactamase was detected. Overall, clinical isolates of Enterobacteriaceae collected in Latin America from 2012 to 2015 were highly susceptible to ceftazidime-avibactam, including isolates that exhibited resistance to ceftazidime, meropenem, colistin, or an MDR phenotype. Country-specific variations were noted in the susceptibility of P. aeruginosa isolates to ceftazidime-avibactam.

In Vitro Activity of Ceftazidime-Avibactam and Aztreonam-Avibactam against OXA-48-Carrying Enterobacteriaceae Isolated as Part of the International Network for Optimal Resistance Monitoring (INFORM) Global Surveillance Program from 2012 to 2015.

Enterobacteriaceae producing the Ambler class D OXA-48 carbapenemase, combined with additional resistance mechanisms, such as permeability defects or cocarriage of class A, B, or C ß-lactamases, can become highly resistant to most ß-lactams currently in use, including carbapenems. A total of 45,872 Enterobacteriaceae clinical isolates collected in 39 countries as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance study in 2012 to 2015 were tested for susceptibility to ß-lactams and comparator agents using the Clinical and Laboratory Standards Institute broth microdilution methodology and screened for the presence of ß-lactamases. The blaOXA-48 and blaOXA-48-like genes were detected in 333 isolates across 14 species of Enterobacteriaceae collected in 20 countries across the globe. Few agents tested were effective in vitro against the overall collection of OXA-48-producers (n = 265), with tigecycline (MIC90, 2 µg/ml; 92.5% susceptible), ceftazidime-avibactam (MIC90, 4 µg/ml; 92.5% susceptible), and aztreonam-avibactam (MIC90, 0.5 µg/ml; 99.6% of isolates with MIC ≤8 µg/ml) demonstrating the greatest activity. Similarly, colistin (MIC90, 1 µg/ml; 94.2% susceptible), tigecycline (MIC90, 2 µg/ml; 92.6% susceptible), ceftazidime-avibactam (MIC90, >128 µg/ml; 89.7% susceptible), and aztreonam-avibactam (MIC90, 4 µg/ml; 100% of isolates with MIC ≤8 µg/ml) were most active against OXA-48-like-positive isolates (n = 68). The in vitro activity of ceftazidime-avibactam was improved against the subset of metallo-ß-lactamase (MBL)-negative, OXA-48- and OXA-48-like-positive isolates (99.2% and 100% susceptible, respectively). The data reported here support the continued investigation of ceftazidime-avibactam and aztreonam-avibactam for the treatment of infections caused by carbapenem-resistant Enterobacteriaceae carrying OXA-48 and OXA-48-like ß-lactamases in combination with serine- or metallo-ß-lactamases.

In vitro activity of ceftazidime/avibactam against isolates of Enterobacteriaceae collected in European countries: INFORM global surveillance 2012-15.

Objectives: The activity of ceftazidime/avibactam was assessed against 24 750 isolates of Enterobacteriaceae collected from 96 medical centres in 18 European countries as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme from 2012 to 2015. Activity was analysed against subsets of isolates based on resistant phenotypes and ß-lactamase content. Methods: Antimicrobial susceptibility testing was performed using broth microdilution and the presence of ß-lactamase genes in isolates of interest was determined using PCR and sequencing. Results: Ceftazidime/avibactam was the most active agent, compared with all other tested comparator agents, against the overall collection of Enterobacteriaceae isolates (99.4% susceptible) and against subsets of ceftazidime-non-susceptible (97.7% susceptible), colistin-resistant (98.2% susceptible), MDR (96.7% susceptible) and meropenem-non-susceptible, MBL-negative (98.5% susceptible) isolates. At the country level, susceptibility to ceftazidime/avibactam ranged from 96.3% to 100% among Enterobacteriaceae isolates, with decreased susceptibilities only observed in countries where MBLs were more frequently encountered (e.g. Greece and Romania). Ceftazidime/avibactam was active against 99.7% of Enterobacteriaceae isolates that carried serine ß-lactamases, including ESBLs, AmpC cephalosporinases and carbapenemases (KPC, GES and OXA-48-like) in all combinations. As expected, ceftazidime/avibactam was not active against isolates carrying MBLs. Conclusions: The data show that ceftazidime/avibactam is highly potent in vitro against clinical isolates of Enterobacteriaceae collected in European countries, including isolates that exhibit resistance to ceftazidime, meropenem and colistin and combined resistance to agents from multiple drug classes.

In vitro activity of ceftazidime/avibactam against isolates of Pseudomonas aeruginosa collected in European countries: INFORM global surveillance 2012-15.

Objectives: The activity of ceftazidime/avibactam was assessed against 5716 Pseudomonas aeruginosa isolates collected from 96 medical centres in 18 European countries as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme from 2012 to 2015. Activity was analysed against subsets of isolates based on resistance phenotypes and ß-lactamase content. Methods: Antimicrobial susceptibility testing was performed by broth microdilution and ß-lactamase genes were detected by PCR screening and sequencing. Results: Ceftazidime/avibactam was highly active in vitro against the overall collection of P. aeruginosa isolates and colistin-resistant isolates (92.4% and 92.9% susceptible, respectively). Although activity was slightly reduced against MBL-negative subsets of ceftazidime-non-susceptible (79.6% susceptible), meropenem-non-susceptible (85.1% susceptible) and MDR (81.6% susceptible) P. aeruginosa, ceftazidime/avibactam remained the second most active entity, after colistin, compared with all other comparator agents tested. At the country level, susceptibility to ceftazidime/avibactam ranged from 74.6% to 99.6%, with decreased susceptibilities only observed in countries where MBLs are more frequently encountered, such as the Czech Republic, Greece, Romania and Russia. Ceftazidime/avibactam was also active in vitro against 87.6% of meropenem-non-susceptible isolates in which no acquired ß-lactamases were detected by molecular methods; these isolates were assumed to hyperproduce the chromosomally encoded AmpC in combination with alterations in OprD or drug efflux. As expected, ceftazidime/avibactam was not active against isolates carrying MBLs. Conclusions: The data show that ceftazidime/avibactam is highly potent in vitro against clinical isolates of P. aeruginosa collected in European countries, including isolates that exhibit resistance to ceftazidime, meropenem and colistin and combined resistance to agents from multiple drug classes.

In Vitro Activity of Ceftazidime-Avibactam against Clinical Isolates of Enterobacteriaceae and Pseudomonas aeruginosa Collected in Asia-Pacific Countries: Results from the INFORM Global Surveillance Program, 2012 to 2015.

The in vitro activities of ceftazidime-avibactam and comparators against 9,149 isolates of Enterobacteriaceae and 2,038 isolates of Pseudomonas aeruginosa collected by 42 medical centers in nine countries in the Asia-Pacific region from 2012 to 2015 were determined as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance program. Antimicrobial susceptibility testing was conducted by Clinical and Laboratory Standards Institute (CLSI) broth microdilution, and isolate subset analysis was performed on the basis of the resistant phenotypes and ß-lactamase content. Ceftazidime-avibactam demonstrated potent in vitro activity (MIC, ≤8 µg/ml) against all Enterobacteriaceae tested (99.0% susceptible) and was the most active against isolates that were metallo-ß-lactamase (MBL) negative (99.8% susceptible). Against P. aeruginosa, 92.6% of all isolates and 96.1% of MBL-negative isolates were susceptible to ceftazidime-avibactam (MIC, ≤8 µg/ml). The rates of susceptibility to ceftazidime-avibactam ranged from 97.0% (Philippines) to 100% (Hong Kong, South Korea) for Enterobacteriaceae and from 83.1% (Thailand) to 100% (Hong Kong) among P. aeruginosa isolates, with lower susceptibilities being observed in countries where MBLs were more frequently encountered (Philippines, Thailand). Ceftazidime-avibactam inhibited 97.2 to 100% of Enterobacteriaceae isolates, per country, that carried serine ß-lactamases, including extended-spectrum ß-lactamases, AmpC cephalosporinases, and carbapenemases (KPC, GES, OXA-48-like). It also inhibited 91.3% of P. aeruginosa isolates that were carbapenem nonsusceptible in which no acquired ß-lactamase was detected. Among MBL-negative Enterobacteriaceae isolates that were ceftazidime nonsusceptible, meropenem nonsusceptible, colistin resistant, and multidrug resistant, ceftazidime-avibactam inhibited 96.1, 87.7, 100, and 98.8% of isolates, respectively, and among MBL-negative P. aeruginosa isolates that were ceftazidime nonsusceptible, meropenem nonsusceptible, colistin resistant, and multidrug resistant, ceftazidime-avibactam inhibited 79.6, 83.6, 83.3, and 68.2% of isolates, respectively. Overall, clinical isolates of Enterobacteriaceae and P. aeruginosa collected in nine Asia-Pacific countries from 2012 to 2015 were highly susceptible to ceftazidime-avibactam.

Frequency and Mechanism of Spontaneous Resistance to Sulbactam Combined with the Novel ß-Lactamase Inhibitor ETX2514 in Clinical Isolates of Acinetobacter baumannii.

The novel diazabicyclooctenone ETX2514 is a potent, broad-spectrum serine ß-lactamase inhibitor that restores sulbactam activity against resistant Acinetobacter baumannii The frequency of spontaneous resistance to sulbactam-ETX2514 in clinical isolates was found to be 7.6 × 10-10 to <9.0 × 10-10 at 4× MIC and mapped to residues near the active site of penicillin binding protein 3 (PBP3). Purified mutant PBP3 proteins demonstrated reduced affinity for sulbactam. In a sulbactam-sensitive isolate, resistance also mapped to stringent response genes associated with resistance to PBP2 inhibitors, suggesting that in addition to ß-lactamase inhibition, ETX2514 may enhance sulbactam activity in A. baumannii via inhibition of PBP2.

In Vitro Activity of Aztreonam-Avibactam against Enterobacteriaceae and Pseudomonas aeruginosa Isolated by Clinical Laboratories in 40 Countries from 2012 to 2015.

The combination of the monobactam aztreonam and the non-ß-lactam ß-lactamase inhibitor avibactam is currently in clinical development for the treatment of serious infections caused by metallo-ß-lactamase (MBL)-producing Enterobacteriaceae, a difficult-to-treat subtype of carbapenem-resistant Enterobacteriaceae for which therapeutic options are currently very limited. The present study tested clinically significant isolates of Enterobacteriaceae (n = 51,352) and Pseudomonas aeruginosa (n = 11,842) collected from hospitalized patients in 208 medical center laboratories from 40 countries from 2012 to 2015 for in vitro susceptibility to aztreonam-avibactam, aztreonam, and comparator antimicrobial agents using a standard broth microdilution methodology. Avibactam was tested at a fixed concentration of 4 µg/ml in combination with 2-fold dilutions of aztreonam. The MIC90s of aztreonam-avibactam and aztreonam were 0.12 and 64 µg/ml, respectively, for all Enterobacteriaceae isolates; >99.9% of all isolates and 99.8% of meropenem-nonsusceptible isolates (n = 1,498) were inhibited by aztreonam-avibactam at a concentration of ≤8 µg/ml. PCR and DNA sequencing identified 267 Enterobacteriaceae isolates positive for MBL genes (NDM, VIM, IMP); all Enterobacteriaceae carrying MBLs demonstrated aztreonam-avibactam MICs of ≤8 µg/ml and a MIC90 of 1 µg/ml. Against all P. aeruginosa isolates tested, the MIC90 of both aztreonam-avibactam and aztreonam was 32 µg/ml; against MBL-positive P. aeruginosa isolates (n = 452), MIC90 values for aztreonam-avibactam and aztreonam were 32 and 64 µg/ml, respectively. The current study demonstrated that aztreonam-avibactam possesses potent in vitro activity against a recent, sizeable global collection of Enterobacteriaceae clinical isolates, including isolates that were meropenem nonsusceptible, and against MBL-positive isolates of Enterobacteriaceae, for which there are few treatment options.

Global Dissemination of blaKPC into Bacterial Species beyond Klebsiella pneumoniae and In Vitro Susceptibility to Ceftazidime-Avibactam and Aztreonam-Avibactam.

The Klebsiella pneumoniae carbapenemase (KPC), first described in the United States in 1996, is now a widespread global problem in several Gram-negative species. A worldwide surveillance study collected Gram-negative pathogens from 202 global sites in 40 countries during 2012 to 2014 and determined susceptibility to ß-lactams and other class agents by broth microdilution testing. Molecular mechanisms of ß-lactam resistance among carbapenem-nonsusceptible Enterobacteriaceae and Pseudomonas aeruginosa were determined using PCR and sequencing. Genes encoding KPC enzymes were found in 586 isolates from 22 countries (76 medical centers), including countries in the Asia-Pacific region (32 isolates), Europe (264 isolates), Latin America (210 isolates), and the Middle East (19 isolates, Israel only) and the United States (61 isolates). The majority of isolates were K. pneumoniae (83.4%); however, KPC was detected in 13 additional species. KPC-2 (69.6%) was more common than KPC-3 (29.5%), with regional variation observed. A novel KPC variant, KPC-18 (KPC-3[V8I]), was identified during the study. Few antimicrobial agents tested remained effective in vitro against KPC-producing isolates, with ceftazidime-avibactam (MIC90, 4 µg/ml), aztreonam-avibactam (MIC90, 0.5 µg/ml), and tigecycline (MIC90, 2 µg/ml) retaining the greatest activity against Enterobacteriaceae cocarrying KPC and other ß-lactamases, whereas colistin (MIC90, 2 µg/ml) demonstrated the greatest in vitro activity against KPC-positive P. aeruginosa This analysis of surveillance data demonstrated that KPC is widely disseminated. KPC was found in multiple species of Enterobacteriaceae and P. aeruginosa and has now become a global problem.

Assessment of the In Vitro Activity of Ceftazidime-Avibactam against Multidrug-Resistant Klebsiella spp. Collected in the INFORM Global Surveillance Study, 2012 to 2014.

Increasing resistance in Gram-negative bacilli, including Klebsiella spp., has reduced the utility of broad-spectrum cephalosporins. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, protects ß-lactams from hydrolysis by Gram-negative bacteria that produce extended-spectrum ß-lactamases (ESBLs) and serine carbapenemases, including Ambler class A and/or class C and some class D enzymes. In this analysis, we report the in vitro activity of ceftazidime-avibactam and comparators against multidrug-resistant (MDR) Klebsiella spp. from the 2012-2014 INFORM surveillance study. Isolates collected from 176 sites were sent to a central laboratory for confirmatory identification and tested for susceptibility to ceftazidime-avibactam and comparator agents, including ceftazidime alone. A total of 2,821 of 10,998 (25.7%) Klebsiella species isolates were classified as MDR, based on resistance to three or more classes of antimicrobials. Among the MDR isolates, 99.4% had an ESBL screen-positive phenotype, and 27.4% were not susceptible to meropenem as an example of a carbapenem. Ceftazidime-avibactam was highly active against MDR isolates, including ESBL-positive and serine carbapenemase-producing isolates, with MIC50/90 values of 0.5/2 µg/ml and 96.6% of all MDR isolates and ESBL-positive MDR isolates inhibited at the FDA breakpoint (MIC value of ≤8 µg/ml). Ceftazidime-avibactam did not inhibit isolates producing class B enzymes (metallo-ß-lactamases) either alone or in combination with other enzymes. These in vitro results support the continued investigation of ceftazidime-avibactam for the treatment of MDR Klebsiella species infections.

In Vitro Susceptibility of Global Surveillance Isolates of Pseudomonas aeruginosa to Ceftazidime-Avibactam (INFORM 2012 to 2014).

Broth microdilution antimicrobial susceptibility testing was performed for ceftazidime-avibactam and comparator agents against 7,062 clinical isolates of Pseudomonas aeruginosa collected from 2012 to 2014 in four geographic regions (Europe, Asia/South Pacific, Latin America, Middle East/Africa) as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance program. The majority of isolates were susceptible to ceftazidime-avibactam, with the proportions susceptible differing marginally across the four regions (MIC90, 8 to 16 µg/ml; 88.7 to 93.2% susceptible), in contrast to lower susceptibilities to the following comparator ß-lactam agents: ceftazidime (MIC90, 32 to 64 µg/ml; 71.5 to 80.8% susceptible), meropenem (MIC90, >8 µg/ml; 64.9 to 77.4% susceptible), and piperacillin-tazobactam (MIC90, >128 µg/ml; 62.3 to 71.3% susceptible). Compared to the overall population, susceptibility to ceftazidime-avibactam of isolates that were nonsusceptible to ceftazidime (n = 1,627) was reduced to between 56.8% (Middle East/Africa; MIC90, 64 µg/ml) and 68.9% (Asia/South Pacific; MIC90, 128 µg/ml), but these percentages were higher than susceptibilities to other ß-lactam agents (0 to 44% susceptible, depending on region and agent; meropenem MIC90, >8 µg/ml; 26.5 to 43.9% susceptible). For this subset of isolates, susceptibilities to amikacin (MIC90, >32 µg/ml; 53.2 to 80.0% susceptible) and colistin (MIC90, 1 µg/ml; 98.5 to 99.5% susceptible) were comparable to or higher than that of ceftazidime-avibactam. A similar observation was made with isolates that were nonsusceptible to meropenem (n = 1,926), with susceptibility to ceftazidime-avibactam between 67.8% (Middle East/Africa; MIC90, 64 µg/ml) and 74.2% (Europe; MIC90, 32 µg/ml) but again with reduced susceptibility to comparators except for amikacin (MIC90, >32 µg/ml; 56.8 to 78.7% susceptible) and colistin (MIC90, 1 µg/ml; 98.9 to 99.3% susceptible). Of the 8% of isolates not susceptible to ceftazidime-avibactam, the nonsusceptibility of half could be explained by their possession of genes encoding metallo-ß-lactamases. The data reported here are consistent with results from other country-specific and regional surveillance studies and show that ceftazidime-avibactam demonstrates in vitro activity against globally collected clinical isolates of P. aeruginosa, including isolates that are resistant to ceftazidime and meropenem.

In Vitro Susceptibility to Ceftazidime-Avibactam of Carbapenem-Nonsusceptible Enterobacteriaceae Isolates Collected during the INFORM Global Surveillance Study (2012 to 2014).

The activity of ceftazidime-avibactam was assessed against 961 isolates of meropenem-nonsusceptible Enterobacteriaceae Most meropenem-nonsusceptible metallo-ß-lactamase (MBL)-negative isolates (97.7%) were susceptible to ceftazidime-avibactam. Isolates that carried KPC or OXA-48-like ß-lactamases, both alone and in combination with extended-spectrum ß-lactamases (ESBLs) and/or AmpC ß-lactamases, were 98.7% and 98.5% susceptible to ceftazidime-avibactam, respectively. Meropenem-nonsusceptible, carbapenemase-negative isolates demonstrated 94.7% susceptibility to ceftazidime-avibactam. Ceftazidime-avibactam activity was compromised only in isolates for which carbapenem resistance was mediated through metallo-ß-lactamases.

In Vitro Activity of AZD0914, a Novel Bacterial DNA Gyrase/Topoisomerase IV Inhibitor, against Clinically Relevant Gram-Positive and Fastidious Gram-Negative Pathogens.

AZD0914, a new spiropyrimidinetrione bacterial DNA gyrase inhibitor with a novel mode of inhibition, has activity against bacterial species commonly cultured from patient infection specimens, including fluoroquinolone-resistant isolates. This study assessed the in vitro activity of AZD0914 against key Gram-positive and fastidious Gram-negative clinical isolates collected globally in 2013. AZD0914 demonstrated potent activity, with MIC90s for AZD0914 of 0.25 mg/liter against Staphylococcus aureus (n = 11,680), coagulase-negative staphylococci (n = 1,923), streptococci (n = 4,380), and Moraxella catarrhalis (n = 145), 0.5 mg/liter against Staphylococcus lugdunensis (n = 120) and Haemophilus influenzae (n = 352), 1 mg/liter against Enterococcus faecalis (n = 1,241), and 2 mg/liter against Haemophilus parainfluenzae (n = 70). The activity against Enterococcus faecium was more limited (MIC90, 8 mg/liter). The spectrum and potency of AZD0914 included fluoroquinolone-resistant isolates in each species group, including methicillin-resistant staphylococci, penicillin-resistant streptococci, vancomycin-resistant enterococci, ß-lactamase-producing Haemophilus spp., and M. catarrhalis. Based on these in vitro findings, AZD0914 warrants further investigation for its utility against a variety of Gram-positive and fastidious Gram-negative bacterial species.

Pyridodiazepine amines are selective therapeutic agents for helicobacter pylori by suppressing growth through inhibition of glutamate racemase but are predicted to require continuous elevated levels in plasma to achieve clinical efficacy.

A pyridodiazepine amine inhibitor of Helicobacter pylori glutamate racemase (MurI) was characterized. The compound was selectively active against H. pylori, and growth suppression was shown to be mediated through the inhibition of MurI by several methods. In killing kinetics experiments, the compound showed concentration-independent activity, with about a 2-log loss of viability in 24 h. A demonstration of efficacy in a mouse infection model was attempted but not achieved, and this was attributed to the failure to attain extended exposure levels above the MIC for >95% of the time. This index and magnitude were derived from pharmacokinetic-pharmacodynamic (PK-PD) studies with amoxicillin, another inhibitor of peptidoglycan biosynthesis that showed slow killing kinetics similar to those of the pyridodiazepine amines. These studies indicate that MurI and other enzymes involved in peptidoglycan biosynthesis may be less desirable targets for monotherapy directed against H. pylori if once-a-day dosing is required.

A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria.

Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.

Pyrazolopyrimidines establish MurC as a vulnerable target in Pseudomonas aeruginosa and Escherichia coli.

The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials, as proven by the clinical success of ß-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, N-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates l-alanine to UDP-N-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore, inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with subnanomolar potency against both Escherichia coli and Pseudomonas aeruginosa MurC enzymes, which demonstrates a concomitant bactericidal activity against efflux-deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis, and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers such as colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery.

Discovery of inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) to validate PPAT as a target for antibacterial therapy.

Inhibitors of 4'-phosphopantetheine adenylyltransferase (PPAT) were identified through high-throughput screening of the AstraZeneca compound library. One series, cycloalkyl pyrimidines, showed inhibition of PPAT isozymes from several species, with the most potent inhibition of enzymes from Gram-positive species. Mode-of-inhibition studies with Streptococcus pneumoniae and Staphylococcus aureus PPAT demonstrated representatives of this series to be reversible inhibitors competitive with phosphopantetheine and uncompetitive with ATP, binding to the enzyme-ATP complex. The potency of this series was optimized using structure-based design, and inhibition of cell growth of Gram-positive species was achieved. Mode-of-action studies, using generation of resistant mutants with targeted sequencing as well as constructs that overexpress PPAT, demonstrated that growth suppression was due to inhibition of PPAT. An effect on bacterial burden was demonstrated in mouse lung and thigh infection models, but further optimization of dosing requirements and compound properties is needed before these compounds can be considered for progress into clinical development. These studies validated PPAT as a novel target for antibacterial therapy.

Exploitation of structural and regulatory diversity in glutamate racemases.

Glutamate racemase is an enzyme essential to the bacterial cell wall biosynthesis pathway, and has therefore been considered as a target for antibacterial drug discovery. We characterized the glutamate racemases of several pathogenic bacteria using structural and biochemical approaches. Here we describe three distinct mechanisms of regulation for the family of glutamate racemases: allosteric activation by metabolic precursors, kinetic regulation through substrate inhibition, and D-glutamate recycling using a d-amino acid transaminase. In a search for selective inhibitors, we identified a series of uncompetitive inhibitors specifically targeting Helicobacter pylori glutamate racemase that bind to a cryptic allosteric site, and used these inhibitors to probe the mechanistic and dynamic features of the enzyme. These structural, kinetic and mutational studies provide insight into the physiological regulation of these essential enzymes and provide a basis for designing narrow-spectrum antimicrobial agents.

Compound efflux in Helicobacter pylori.

Susceptibility testing with a variety of structurally unrelated compounds showed that hefC in Helicobacter pylori is involved in multidrug efflux. This efflux was shown to depend on the proton motive force, as demonstrated by ethidium bromide accumulation experiments. Thus, H. pylori contains an active multidrug efflux mechanism.