Susceptibility profile and ß-lactamase content of global Pseudomonas aeruginosa isolates resistant to ceftolozane/tazobactam and/or imipenem/relebactam-SMART 2016-21.

Objectives: To determine susceptibility profiles and ß-lactamase content for ceftolozane/tazobactam-resistant and imipenem/relebactam-resistant Pseudomonas aeruginosa isolates collected in eight global regions during 2016-21. Methods: Broth microdilution MICs were interpreted using CLSI breakpoints. PCR to identify ß-lactamase genes or WGS was performed on selected isolate subsets. Results: Ceftolozane/tazobactam-resistant [from 0.6% (Australia/New Zealand) to 16.7% (Eastern Europe)] and imipenem/relebactam-resistant [from 1.3% (Australia/New Zealand) to 13.6% (Latin America)] P. aeruginosa varied by geographical region. Globally, 5.9% of isolates were both ceftolozane/tazobactam resistant and imipenem/relebactam resistant; 76% of these isolates carried MBLs. Most ceftolozane/tazobactam-resistant/imipenem/relebactam-susceptible isolates carried ESBLs (44%) or did not carry non-intrinsic (acquired) ß-lactamases (49%); 95% of imipenem/relebactam-resistant/ceftolozane/tazobactam-susceptible isolates did not carry non-intrinsic ß-lactamases. Isolates that carried indicators of strong PDC (Pseudomonas-derived cephalosporinase) up-regulation without a mutation known to expand the spectrum of PDC, or non-intrinsic ß-lactamases, showed an 8-fold increase in ceftolozane/tazobactam modal MIC; however, this rarely (3%) resulted in ceftolozane/tazobactam resistance. Isolates with a PDC mutation and an indicator for PDC upregulation were ceftolozane/tazobactam non-susceptible (MIC,  ≥ 8 mg/L). MICs ranged widely (1 to >32 mg/L) for isolates with a PDC mutation and no positively identified indicator for PDC up-regulation. Imipenem/relebactam-resistant/ceftolozane/tazobactam-susceptible isolates without non-intrinsic ß-lactamases frequently (91%) harboured genetic lesions implying OprD loss of function; however, this finding alone did not account for this phenotype. Among imipenem-non-susceptible isolates without non-intrinsic ß-lactamases, implied OprD loss only shifted the distribution of imipenem/relebactam MICs up by 1-2 doubling dilutions, resulting in ∼10% imipenem/relebactam-resistant isolates. Conclusions: P. aeruginosa with ceftolozane/tazobactam-resistant/imipenem/relebactam-susceptible and imipenem/relebactam-resistant/ceftolozane/tazobactam-susceptible phenotypes were uncommon and harboured diverse resistance determinants.

Epidemiology of Resistance Determinants Identified in Meropenem-Nonsusceptible Enterobacterales Collected as Part of a Global Surveillance Study, 2018 to 2019.

The objective of this study was to describe the frequency of resistance determinants in meropenem-nonsusceptible (MEM-NS) Enterobacterales isolates collected in 2018 and 2019 as a part of the ATLAS global surveillance program. Among a total of 39,368 Enterobacterales isolates collected in 2018 and 2019, 5.7% were MEM-NS (MIC ≥2 µg/mL). Among the different regions, the proportion of MEM-NS isolates ranged from 1.9% (North America) to 8.4% (Asia/Pacific). The majority of MEM-NS isolates collected were of the species Klebsiella pneumoniae (71.5%). Among the MEM-NS Enterobacterales isolates collected, metallo-ß-lactamases (MBL) were identified in 36.7%, KPC in 25.5%, and OXA-48-like in 24.1%. The predominance of resistance mechanisms among MEM-NS isolates varied by region: MBLs were dominant in isolates collected in Africa and Middle East (AfME, 49%) and Asia/Pacific (59.4%), OXA-48-like carbapenemases were predominant in Europe (30%), and KPC in Latin America (51.9%) and North America (53.6%). NDM ß-lactamases accounted for the majority of MBLs identified (88.4%). Of the 38 carbapenemase variants identified, NDM-1 (68.7%), KPC-2 (54.6%), OXA-48 (54.3%), and VIM-1 (76.1%) were the common variants within their respective families. Among the MEM-NS isolates, 7.9% co-carried two carbapenemases. Notably, the proportion of MEM-NS Enterobacterales increased from 4.9% in 2018 to 6.4% in 2019. The results of this study show a continuation of the trend of increasing carbapenem-resistance within clinical Enterobacterales with mechanisms of resistance varying across different regions. The existential threat to public health posed by the continued spread of nearly untreatable pathogens requires a multifaceted approach to prevent the collapse of modern medicine.

Activity of ceftolozane/tazobactam against clinical isolates of Pseudomonas aeruginosa from patients in the Middle East and Africa - Study for Monitoring Antimicrobial Resistance Trends (SMART) 2017-2020.

OBJECTIVES: We evaluated the activity of ceftolozane/tazobactam (C/T), and comparators against clinical Pseudomonas aeruginosa isolates collected for the global Study for Monitoring Antimicrobial Resistance Trends (SMART) surveillance program in ten countries in the Middle East and Africa to augment scarce standardized surveillance data in this region. METHODS: Minimum inhibitory concentrations (MICs) were determined using Clinical and Laboratory Standards Institute broth microdilution and interpreted with European Committee on Antimicrobial Susceptibility Testing breakpoints. P. aeruginosa isolates testing with C/T MIC >4 mg/l or imipenem MIC >2 mg/l were screened for ß-lactamase genes. RESULTS: C/T was active against 91.4% and 87.0% of P. aeruginosa isolates from the Middle East and Africa, respectively (14-21 and 7-16 percentage points higher than most ß-lactam comparators, respectively). Considerable variation in susceptibility was seen across countries, which largely correlated with the observed prevalence of carbapenemases and/or extended-spectrum ß-lactamases. Differences across countries were smaller for C/T than for the ß-lactam comparators, ranging from 81% C/T-susceptible among isolates from Jordan to 95% for Qatar. Among subsets resistant to meropenem, ceftazidime, or piperacillin/tazobactam, C/T maintained activity against 51-73% of isolates from the Middle East and against 27-54% from Africa (where metallo-ß-lactamase and GES carbapenemase rates were higher). CONCLUSION: Given the desirability of ß-lactam use among clinicians, C/T represents an important option in the treatment of infections caused by P. aeruginosa.

Molecular characterization of clinical isolates of Enterobacterales with elevated MIC values for aztreonam-avibactam from the INFORM global surveillance study, 2012-2017.

OBJECTIVES: While aztreonam-avibactam is a potent ß-lactam-ß-lactamase-inhibitor combination, reduced in vitro activity against some Enterobacterales isolates has been reported. In this study, globally collected clinical isolates of Enterobacterales with elevated minimum inhibitory concentrations (MICs) for aztreonam-avibactam were examined for potential resistance mechanisms. METHODS: Isolates with aztreonam-avibactam MICs ≥8 µg/mL (n = 55: Escherichia coli, n = 38; Enterobacter cloacae, n = 10; Klebsiella pneumoniae, n = 3; others, n = 4) and <8 µg/mL (n = 18) collected for the INFORM global surveillance programme were characterized by short read whole-genome sequencing. Sequences were inspected for the presence of ß-lactamase genes, penicillin-binding protein (PBP) mutations, and disruptions in the coding sequences of porin genes. RESULTS: All isolates of E. coli testing with aztreonam-avibactam MIC values ≥8 µg/mL carried a previously documented four-amino-acid insertion in PBP3 at position 333 of YRI(K/N/P). Such mutations were absent in isolates with MICs <2 µg/mL (n = 6). Among other species, carriage of PER- or VEB-type ß-lactamases was identified in 10/17 (58.8%) of isolates testing with aztreonam-avibactam MICs ≥8 µg/mL, but no isolates with lower MIC values (n = 11). CONCLUSIONS: PBP3 mutations are known to confer resistance to aztreonam in E. coli, providing a rationale for the elevated MIC values for aztreonam-avibactam in these isolates. Elevated MICs in other isolates were associated with the carriage of PER-type ß-lactamases, which have been previously shown to be inhibited less effectively by avibactam than other Class A ß-lactamases and may contribute to this phenotype. Other resistance mechanisms contributing to poor in vitro activity for aztreonam-avibactam in some of these isolates are not yet elucidated.

Prevalence of mcr-type genes among colistin-resistant Enterobacteriaceae collected in 2014-2016 as part of the INFORM global surveillance program.

A set of 908 clinically derived colistin-resistant Enterobacteriaeae isolates collected worldwide in 2014-2016 were screened for the presence of the plasmid-borne mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 genes. In total 3.2% (29/908) of the collection were positive for mcr, including 27 Escherichia coli, 1 Klebsiella pneumoniae and 1 Enterobacter cloacae. Twenty-four isolates possessed genes from the mcr-1 family, including the original mcr-1 (n = 22), as well as mcr-1.2 (n = 1) and mcr-1.5 (n = 1), which each differ from mcr-1 by encoding single amino acid variations. Genes from the mcr-3 family were found in isolates from Thailand, including mcr-3.1 (n = 3) and mcr-3.2 (n = 1). An E. coli isolated from a patient with a urinary tract infection in Colombia contained the recently discovered mcr-5. The full colistin-resistant collection was tested against a panel of antimicrobial agents with ceftazidime-avibactam and tigecycline exhibiting the highest activity.

In vitro susceptibility of characterized ß-lactamase-producing strains tested with avibactam combinations.

Avibactam, a broad-spectrum ß-lactamase inhibitor, was tested with ceftazidime, ceftaroline, or aztreonam against 57 well-characterized Gram-negative strains producing ß-lactamases from all molecular classes. Most strains were nonsusceptible to the ß-lactams alone. Against AmpC-, extended-spectrum ß-lactamase (ESBL)-, and KPC-producing Enterobacteriaceae or Pseudomonas aeruginosa, avibactam lowered ceftazidime, ceftaroline, or aztreonam MICs up to 2,048-fold, to ≤4 µg/ml. Aztreonam-avibactam MICs against a VIM-1 metallo-ß-lactamase-producing Enterobacter cloacae and a VIM-1/KPC-3-producing Escherichia coli isolate were 0.12 and 8 µg/ml, respectively.

In vitro activity of ceftolozane-tazobactam as determined by broth dilution and agar diffusion assays against recent U.S. Escherichia coli isolates from 2010 to 2011 carrying CTX-M-type extended-spectrum ß-lactamases.

Ceftolozane MIC(50)/MIC(90)s were 4/8 µg/ml when tested against 26 CTX-M-14-type-producing isolates and 64/>64 µg/ml against 219 CTX-M-15-type-producing isolates. The addition of 4 µg/ml tazobactam lowered the ceftolozane MIC50/MIC9(0)s to ≤ 0.25/0.5 µg/ml by broth microdilution and Etest. The zone diameters for the ceftolozane-tazobactam disks were 23 to 29 mm for 92.2% of the isolates.

Generation of mice with a conditional allele for the transforming growth factor beta3 gene.

The transforming growth factor beta (TGFß) pathway is involved in embryonic development and several inherited and acquired human diseases. The gene for TGFß3 (Tgfb3) encodes one of the three ligands for TGFß receptors. It is widely expressed in the embryo and its mutation or misexpression is found in human diseases. Tgfb3-/- mice die at birth from cleft palate, precluding functional studies in adults. Here, we generated mice in which exon 6 of Tgfb3 was flanked with LoxP sites (Tgfb3flox/flox). The adult mice were normal and fertile. EIIa-Cre-mediated deletion of exon 6 in Tgfb3flox/flox mice efficiently generated Tgfb3 conditional knockout (Tgfb3cko/cko) mice which died at birth from the same cleft palate defect as Tgfb3-/- mice, indicating that the conditional and knockout alleles are functionally equivalent. This Tgfb3cko allele will now enable studies of TGFß3 function in different cell or tissue types in embryonic development and during adulthood.

Altered tissue behavior of a non-aneurysmal descending thoracic aorta in the mouse model of Marfan syndrome.

Aortic aneurysm is predominantly found in the ascending aorta in patients with Marfan syndrome (MFS). However, descending aortic disease has emerged as a problem since people are living longer because of improved medical and surgical management of the ascending aorta. Diagnostic procedures before disease onset and the mechanisms involved in the transition of normal aortic tissue to aneurysm remain unclear. We determined signs of descending aortic disease before disease onset in mice with a mutation in the fibrillin 1 gene (Fbn1(+/C1039G)), a validated mouse model of disease susceptibility and progression of aortic aneurysm of MFS. We analyzed a tubular unfixed non-aneurysmal descending thoracic aorta from 8-month-old wild-type and Fbn1(+/C1039G) mice by a tubular biaxial tester that works in conjunction with a two-photon nonlinear microscope. Fbn1(+/C1039G) mouse aorta was more compliant in the circumferential direction. Two-photon imaging showed defective organization of adventitial collagen fibers in the pressurized aortas of Fbn1(+/C1039G) mice. Moreover, disruption in the elastic lamina was noted in the absence of aneurysms in pressurized aortas but not unpressurized aortas of Fbn1(+/C1039G) mice. At the molecular level, this altered tissue behavior in non-aneurysmal descending aortas of Fbn1(+/C1039G) mice was accompanied by an increasing trend of canonical but not noncanonical, transforming growth factor-ß (TGFß) signaling. Finally, assays of in vitro collagen lattice formation in mouse wild-type and TGFß1-deficient embryonic fibroblasts indicate that TGFß1 can regulate collagen organization. The ability to reveal the presence of altered biomechanics and microstructure coupled with subtle changes in TGFß signaling provides a novel surrogate measure of tissue susceptibility to aneurysm before disease onset.