Comparison of testing methods assessing the in vitro efficacy of the combination of aztreonam with avibactam on multidrug-resistant Gram-negative bacilli.

BACKGROUND: Aztreonam-avibactam (ATM-AVI) combination shows promising effectiveness on most carbapenemase-producing Gram-negatives, yet standardized antibiotic susceptibility testing (AST) methods for evaluating the combination in clinical laboratories is lacking. We aimed to evaluate different ATM-AVI AST approaches. METHODS: 96 characterized carbapenem-resistant clinical isolates belonging to 9 Enterobacterales (EB; n = 80) and P. aeruginosa (PA; n = 16) species, including 90 carbapenemase producers and 72 strains resistant to both CAZ-AVI and ATM, were tested. Paper disk elution (DE; Bio-Rad) and E-test gradient strips stacking (SS; bioMérieux) were performed for the ATM + CAZ-AVI combination. MIC Test Strip (MTS; Liofilchem) was evaluated for ATM-AVI MIC determination. Results were interpreted applying ATM clinical breakpoints of the EUCAST guidelines and compared to the broth microdilution method (Sensititre, Thermofisher). RESULTS: According to broth microdilution method, 93% of EB and 69% of PA were tested susceptible to ATM-AVI. The synergistic effect of ATM-AVI was of 95% for EB, but of only 17% for PA. The MTS method yielded higher categorical and essential agreement (CA/EA) rates for both EB (89%/91%) and PA (94%/94%) compared to SS, where the rates were 87%/83% for EB and 81%/81% for PA. MTS and SS yielded 2 and 3 major discrepancies, respectively, while 3 very major discrepancies each were observed for both methods. Concerning the DE method, CA reached 91% for EB and 81% for PA, but high number of very major discrepancies were observed for EB (n = 6; 8%) and for PA (n = 3; 19%). CONCLUSIONS: The ATM-AVI association displayed excellent in vitro activity against highly resistant clinical Enterobacterales strains. MTS method offers accurate ATM-AVI AST results, while the SS method might serve as better alternative then DE method in assessing the efficacy of ATM + CAZ-AVI combination. However, further investigation is needed to confirm the methods' ability to detect ATM-AVI resistance.

Using host-mimicking conditions and a murine cutaneous abscess model to identify synergistic antibiotic combinations effective against Pseudomonas aeruginosa.

Antibiotic drug combination therapy is critical for the successful treatment of infections caused by multidrug resistant pathogens. We investigated the efficacy of ß-lactam and ß-lactam/ß-lactamase inhibitor combinations with other antibiotics, against the hypervirulent, ceftazidime/avibactam resistant Pseudomonas aeruginosa Liverpool epidemic strain (LES) B58. Although minimum inhibitory concentrations in vitro differed by up to eighty-fold between standard and host-mimicking media, combinatorial effects only marginally changed between conditions for some combinations. Effective combinations in vitro were further tested in a chronic, high-density murine infection model. Colistin and azithromycin demonstrated combinatorial effects with ceftazidime and ceftazidime/avibactam both in vitro and in vivo. Conversely, while tobramycin and tigecycline exhibited strong synergy in vitro, this effect was not observed in vivo. Our approach of using host-mimicking conditions and a sophisticated animal model to evaluate drug synergy against bacterial pathogens represents a promising approach. This methodology may offer insights into the prediction of combination therapy outcomes and the identification of potential treatment failures.

In vitro antibacterial activity of sulbactam-durlobactam in combination with other antimicrobial agents against Acinetobacter baumannii-calcoaceticus complex.

Combinations of the ß-lactam/ß-lactamase inhibitor sulbactam-durlobactam and seventeen antimicrobial agents were tested against strains of Acinetobacter baumannii in checkerboard assays. Most combinations resulted in indifference with no instances of antagonism. These results suggest sulbactam-durlobactam antibacterial activity against A. baumannii is unlikely to be affected if co-dosed with other antimicrobial agents.

Molecular mechanisms responsible KPC-135-mediated resistance to ceftazidime-avibactam in ST11-K47 hypervirulent Klebsiella pneumoniae.

Ceftazidime-avibactam resistance attributable to the blaKPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the blaKPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The blaKPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the blaKPC-2-positive cloned strain, only the MIC of ceftazidime increased against blaKPC-135-positive K. pneumoniae and wasn't inhibited by avibactam (MIC 32 µg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

Effect of ceftazidime-avibactam combined with different antimicrobials against carbapenem-resistant Klebsiella pneumoniae.

This study aimed to assess the in vitro efficacy of ceftazidime-avibactam (CZA) in combination with various antimicrobial agents against carbapenem-resistant Klebsiella pneumoniae (CRKP). We selected 59 clinical CRKP isolates containing distinct drug resistance mechanisms. The minimum inhibitory concentrations (MICs) of meropenem (MEM), colistin (COL), eravacycline (ERA), amikacin (AK), fosfomycin (FOS), and aztreonam (ATM), both individually and in combination with CZA, were tested using the checkerboard method. The interactions of antimicrobial agent combinations were assessed by fractional inhibitory concentration index (FICI) and susceptible breakpoint index (SBPI). The time-kill curve assay was employed to dynamically evaluate the effects of these drugs alone and in combination format. In the checkerboard assay, the combination of CZA+MEM showed the highest level of synergistic effect against both KPC-producing and carbapenemase-non-producing isolates, with synergy rates of 91.3% and 100%, respectively. Following closely was the combination of FOS+CZA . For metallo-beta-lactamases (MBLs) producing strains, ATM+CZA displayed complete synergy, while the combination of MEM+CZA showed a synergy rate of only 57.14% for NDM-producing strains and 91.67% for IMP-producing strains. In the time-kill assay, MEM+CZA also demonstrated significant synergistic effects against the two KPC-2-producing isolates (Y070 and L70), the two carbapenemase-non-producing isolates (Y083 and L093), and the NDM-1-producing strain L13, with reductions in log10 CFU/mL exceeding 10 compared to the control. Against the IMP-producing strain Y047, ATM+CZA exhibited the highest synergistic effect, resulting in a log10 CFU/mL reduction of 10.43 compared to the control. The combination of CZA and MEM exhibited good synergistic effects against KPC-producing and non-enzyme-producing strains, followed by the FOS+CZA combination. Among MBL-producing strains, ATM+CZA demonstrated the most pronounced synergistic effect. However, the combinations of CZA with ERA, AK, and COL show irrelevant effects against the tested clinical isolates. IMPORTANCE: Our study confirmed the efficacy of the combination CZA+MEM against KPC-producing and non-carbapenemase-producing strains. For metalloenzyme-producing strains, CZA+ATM demonstrated the most significant synergy. Additionally, CZA exhibited a notable synergy effect when combined with FOS. These combination therapies present promising new options for the treatment of CRKP infection.

First report of KPC variants conferring ceftazidime-avibactam resistance in Colombia: introducing KPC-197.

Resistance to ceftazidime-avibactam (CZA) due to Klebsiella pneumoniae carbapenemase (KPC) variants is increasing worldwide. We characterized two CZA-resistant clinical Klebsiella pneumoniae strains by antimicrobial susceptibility test, conjugation assays, and WGS. Isolates belonged to ST258 and ST45, and produced a KPC-31 and a novel variant KPC-197, respectively. The novel KPC variant presents a deletion of two amino acids on the Ω-loop (del_168-169_EL) and an insertion of two amino acids in position 274 (Ins_274_DS). Continued surveillance of KPC variants conferring CZA resistance in Colombia is warranted. IMPORTANCE: Latin America and the Caribbean is an endemic region for carbapenemases. Increasingly high rates of Klebsiella pneumoniae carbapenemase (KPC) have established ceftazidime-avibactam (CZA) as an essential antimicrobial for the treatment of infections due to MDR Gram-negative pathogens. Although other countries in the region have reported the emergence of CZA-resistant KPC variants, this is the first description of such enzymes in Colombia. This finding warrants active surveillance, as dissemination of these variants could have devastating public health consequences.

In vitro mimicry of in vivo KPC mutations by ceftazidime-avibactam: phenotypes, mechanisms, genetic structure and kinetics of enzymatic hydrolysis.

Ceftazidime-avibactam (CZA) is employed for the treatment of infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP). Resistance to CZA is frequently linked to point mutations in the blaKPC. We conducted in vitro simulations of in vivo blaKPC mutations using CZA. Four pre-therapy KPC-KP isolates (K1, K2, K3, and K4) were evaluated, all initially exhibited susceptibility to CZA and produced KPC-2. The crucial distinction was that following CZA treatment, the blaKPC-2 mutated in K1, K2, and K3, rendering them resistant to CZA, while K4 achieved microbiological clearance, and blaKPC-2 remained unaltered. The induction assay identified various blaKPC-2 variants, including blaKPC-25, blaKPC-127, blaKPC-100, blaKPC-128, blaKPC-137, blaKPC-138, blaKPC-144 and blaKPC-180. Our findings suggest that the resistance of KPC-KP to CZA primarily results from the emergence of KPC variants, complemented by increased blaKPC expression. A close correlation exists between avibactam concentration and the rate of increased CZA minimum Inhibitory concentration, as well as blaKPC mutation. Inadequate avibactam concentration is more likely to induce resistance in strains against CZA, there is also a higher likelihood of mutation in the blaKPC-2 and the optimal avibactam ratio remains to be determined. Simultaneously, we selected a blaKPC-33-producing K. pneumoniae strain (mutated from blaKPC-2) and induced it with imipenem and meropenem, respectively. The blaKPC-2 was detected during the process, indicating that the mutation is reversible. Clinical use of carbapenems to treat KPC variant strains increases the risk of infection, as the gene can mutate back to blaKPC-2, rendering the strain even more cross-resistant to carbapenems and CZA.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Evaluation of a simple method for testing aztreonam and ceftazidime-avibactam synergy in New Delhi metallo-beta-lactamase producing Enterobacterales.

NDM-producing carbapenem-resistant bacterial infections became a challenge for clinicians. Combination therapy of aztreonam and ceftazidime-avibactam is a prudent choice for these infections. However, there is still no recommendation of a practically feasible method for testing aztreonam and ceftazidime-avibactam synergy. We proposed a simple method for testing aztreonam and ceftazidime-avibactam synergy and compared it with reference broth micro-dilution and other methods. Carbapenem-resistant Enterobacterales clinical isolates were screened for the presence of the NDM gene by the Carba R test. NDM harbouring isolates were tested for aztreonam and ceftazidime-avibactam synergy by broth microdilution (reference method), E strip-disc diffusion, double disc diffusion, and disc replacement methods. In the newly proposed method, the MHA medium was supplemented with ceftazidime-avibactam (corresponding to an aztreonam concentration of 4µg/ml). The MHA medium was then inoculated with the standard inoculum (0.5 McFarland) of the test organism. An AZT disc (30 µg) was placed on the supplemented MHA medium, and the medium was incubated overnight at 37°C. Aztreonam zone diameter on the supplemented MHA medium (in the presence of ceftazidime-avibactam) was compared with that from a standard disc diffusion plate (without ceftazidime-avibactam), performed in parallel. Interpretation of synergy was based on the restoration of aztreonam zone diameter (in the presence of ceftazidime-avibactam) crossing the CLSI susceptibility breakpoint, i.e., ≥ 21 mm. Of 37 carbapenem-resistant NDM-producing isolates, 35 (94.6%) were resistant to aztreonam and tested synergy positive by the proposed method. Its sensitivity and specificity were 97.14% and 100%, respectively. Cohen's kappa value showed substantial agreement of the reference method with the proposed method (κ = 0.78) but no other methods. The proposed method is simple, easily interpretable, and showed excellent sensitivity, specificity, and agreement with the reference method. Therefore, the new method is feasible and reliable for testing aztreonam synergy with avibactam in NDM-producing Enterobacterales.

Emergence of ceftazidime-avibactam resistance in blaKPC-33-harbouring ST11 Klebsiella pneumoniae in a paediatric patient.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially children. This study reports the infection caused by CRKP in a paediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that 12 strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC >64 mg/L) and restored the carbapenem susceptibility (Imipenem, MIC =0.25 mg/L; Meropenem, MIC =2 mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the blaKPC-33-positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient Escherichia coli DH5α and blaKPC-2-positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3-26 single nucleotide polymorphisms [SNPs]). A single base mutation (G532T) of blaKPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into blaKPC-33 during the treatment of blaKPC-2-positive CRKP in paediatric-infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP.

Performance of disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales.

OBJECTIVES: To evaluate the performance of an in-house developed disk diffusion method for aztreonam in combination with avibactam against Enterobacteriales. METHODS: The in vitro antibacterial activity of aztreonam with avibactam against 204 carbapenemase-producing Enterobacteriales was determined by a disk diffusion method, with a broth microdilution method as a reference. RESULTS: The optimal S/R breakpoints for disk diffusion tests of 30/20 and 10/4 µg disks, calculated by the dBETs software using the model-based approaches, were ≥22/≤21 and ≥12/≤11 mm, respectively. On the basis of the estimated breakpoints, the CAs for disk diffusion tests of 30/20 and 10/4 µg aztreonam/avibactam disks were both 98.0%, with 0.5% major error and 37.5% very major error. CONCLUSIONS: The home-made disk diffusion method is an economical and practical method for clinical microbiology laboratories to determine the antibacterial susceptibility of aztreonam with avibactam against Enterobacteriales.

Durlobactam, a Diazabicyclooctane ß-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum ß-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb ß-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KI app 9.2 ± 0.9 µM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KI app 3.3 ± 0.6 µM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KI app values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 µg/mL, similar to the ranges for meropenem (1-32 µg/mL) and imipenem (0.5-64 µg/mL). In ß-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel ß-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

Antimicrobial activity of ceftazidime-avibactam against KPC-2-producing Enterobacterales: a cross-combination and dose-escalation titration study with relebactam and vaborbactam.

With the introduction of ceftazidime-avibactam worldwide, the antimicrobial activity of new ß-lactam/ß-lactamase inhibitors (BL/BLIs) needs to be investigated. From January 2020 to June 2023, Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were collected. With a broth microdilution test of new BL/BLIs, cross-activity test with nine combinations of BLs and new BLIs and dose-escalation titration test for non-susceptible isolates were conducted to investigate inhibitory activities of new BLIs. A total of 188 isolates was collected and most isolates (186/188, 98.9%) carried the KPC-2 gene exclusively, while two isolates (1.1%) co-harbored NDM-1. Among the 186 KPC-2-producing isolates, 184 (98.9%) were susceptible to ceftazidime-avibactam, 173 (93.0%) to imipenem-relebactam, and 184 (98.9%) to meropenem-vaborbactam. All isolates non-susceptible to imipenem-relebactam or meropenem-vaborbactam became susceptible when avibactam replaced relebactam or vaborbactam, with 7 of 11 (63.6%) imipenem-relebactam non-susceptible isolates and both (100.0%) of the meropenem-vaborbactam non-susceptible isolates. When the minimum inhibitory concentrations (MICs) of BLs were compared using log2 scales, combinations with avibactam showed statistically significant efficacy in lowering MICs compared to relebactam and vaborbactam (all P < 0.05). In the dose-escalation test of new BLIs, increasing dose of all new BLIs corresponded to increased susceptibility to BLs. Ceftazidime-avibactam exhibited excellent susceptibility against KPC-2-producing Enterobacterales unless co-harboring metallo-ß-lactamase. The cross-combination test against non-susceptible isolates suggests that the inhibitory activity of avibactam was superior to those of relebactam or vaborbactam. Increasing the dose of new BLIs produced increased susceptibility to BLs, suggesting that high-concentration regimen need to be developed. IMPORTANCE: This study investigated 188 Klebsiella pneumoniae carbapenemase (KPC)-2-producing Enterobacterales collected from January 2020 to June 2023 in a tertiary care hospital of Korea. Most isolates were susceptible to ceftazidime-avibactam (98.9%) and meropenem-vaborbactam (98.9%), while susceptibility to imipenem-relebactam was lower (93.0%). The cross-combination test using nine combinations of the individual ß-lactams (BLs) and new ß-lactamase inhibitors (BLIs) showed that the inhibitory activity of avibactam was significantly superior to relebactam or vaborbactam when the Log2 MIC of BLs were compared for each combination with BLIs (all P < 0.05). The dose-escalation test of new BLIs demonstrated that increasing doses of new BLIs corresponded to increased susceptibility to BLs. Taken together, this study illustrates the excellent activity of ceftazidime-avibactam against KPC-2-producing Enterobacterales and suggests further investigation into high-concentration regimens for potentially non-susceptible clinical isolates.

A novel KPC-166 in ceftazidime/avibactam resistant ST307 Klebsiella pneumoniae causing an outbreak in intensive care COVID Unit, Italy.

INTRODUCTION: Aim of the study was the molecular characterization of 21 ceftazidime/avibactam resistant (CZA-R) Klebsiella pneumoniae strains, collected in the period October 2021-March 2022 from an Intensive Care COVID Unit in a Northern Italian Hospital. METHODS: After growth on selective/chromogenic culture media and susceptibility tests assessment, resistance genes content was ascertained for all the isolates by the HybriSpot 12 multiplexing, PCR and Whole-Genome Sequencing (WGS). Clonality was assessed by PFGE and MLST according to the Pasteur scheme. A SNPs-based phylogenetic tree was obtained comparing representative isolates and global genomes. The blaKPC gene horizontal transmission was evaluated by conjugation experiments. blaKPC-166 was cloned in a pCR2.1 vector and transformed in chemically competent TOP10 cells. RESULTS: Sixteen inpatients resulted positive for colonization and/or infection by KPC-producing K. pneumoniae (KPC-Kp) strains. The 21 CZA-R KPC-Kp isolates obtained showed MDR phenotype; susceptibility to meropenem was always retained. All the CZA-R KPC-Kp presented a novel blaKPC variant, named blaKPC-166, showing a single nucleotide substitution (T811C) compared to the blaKPC-94; but related to blaKPC-2. TWO DIFFERENT PULSOTYPES WERE DETECTED: A in 18/21 and B in 1/21 cases, two strains from the same patient being untypable by PFGE. Interestingly, the outbreak was sustained by the high-risk clone ST307, although the ST22, ST6342, ST6418 and ST6811 have also been identified and associated to KPC-166. Worryingly, blaKPC-166 could be transferred horizontally and, after cloning, it conferred resistance to CZA. DISCUSSION: This novel variant confers CZA-resistance and carbapenems susceptibility restoration. As KPC-166 was found expressed by multiple Kp clones, greater efforts should be made to prevent the further dissemination of such strains in Italian clinical settings.

In vitro activity of ceftazidime/avibactam, cefiderocol, meropenem/vaborbactam and imipenem/relebactam against clinical strains of the Stenotrophomonas maltophilia complex.

BACKGROUND: Infections caused by Stenotrophomonas maltophilia and related species are increasing worldwide. Unfortunately, treatment options are limited, whereas the antimicrobial resistance is increasing. METHODS: We included clinical isolates identified as S. maltophilia by VITEK 2 Compact. Ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam, cefiderocol, quinolones, and tetracycline family members were evaluated by broth microdilution method and compared with first-line treatment drugs. Minimum inhibitory concentrations (MICs) were reported for all antibiotics. We sequenced the Whole Genome of cefiderocol resistant strains (CRSs) and annotated their genes associated with cefiderocol resistance (GACR). Presumptive phylogenetic identification employing the 16S marker was performed. RESULTS: One hundred and one clinical strains were evaluated, sulfamethoxazole and trimethoprim, levofloxacin and minocycline showed susceptibilities of 99.01%, 95.04% and 100% respectively. Ceftazidime was the antibiotic with the highest percentage of resistance in all samples (77.22%). Five strains were resistant to cefiderocol exhibiting MIC values ≥ 2 µg/mL (4.95%). The ß-lactamase inhibitors meropenem/vaborbactam and imipenem/relebactam, failed to inhibit S. maltophilia, preserving both MIC50 and MIC90 ≥64 µg/mL. Ceftazidime/avibactam restored the activity of ceftazidime decreasing the MIC range. Tigecycline had the lowest MIC range, MIC50 and MIC90. Phylogeny based on 16S rRNA allowed to identify to cefiderocol resistant strains as putative species clustered into Stenotrophomonas maltophilia complex (Smc). In these strains, we detected GARCs such as Mutiple Drug Resistance (MDR) efflux pumps, L1-type ß-lactamases, iron transporters and type-1 fimbriae. CONCLUSION: Antimicrobial resistance to first-line treatment is low. The in vitro activity of new ß-lactamase inhibitors against S. maltophilia is poor, but avibactam may be a potential option. Cefiderocol could be considered as a potential new option for multidrug resistant infections. Tetracyclines had the best in vitro activity of all antibiotics evaluated.

Characterization of Acinetobacter baumannii-calcoaceticus complex isolates and microbiological outcome for patients treated with sulbactam-durlobactam in a phase 3 trial (ATTACK).

Acinetobacter baumannii-calcoaceticus complex (ABC) causes severe, difficult-to-treat infections that are frequently antibiotic resistant. Sulbactam-durlobactam (SUL-DUR) is a targeted ß-lactam/ß-lactamase inhibitor combination antibiotic designed to treat ABC infections, including those caused by multidrug-resistant strains. In a global, pathogen-specific, randomized, controlled phase 3 trial (ATTACK), the efficacy and safety of SUL-DUR were compared to colistin, both dosed with imipenem-cilastatin as background therapy, in patients with serious infections caused by carbapenem-resistant ABC. Results from ATTACK showed that SUL-DUR met the criteria for non-inferiority to colistin for the primary efficacy endpoint of 28-day all-cause mortality with improved clinical and microbiological outcomes compared to colistin. This report describes the characterization of the baseline ABC isolates from patients enrolled in ATTACK, including an analysis of the correlation of microbiological outcomes with SUL-DUR MIC values and the molecular drivers of SUL-DUR resistance.

In vitro activity of ceftazidime-avibactam against gram-negative bacteria in patients with bacteremia and skin and soft-tissue infections in Colombia 2019-2021.

OBJECTIVES: Ceftazidime-avibactam (CAZ-AVI) is an option for infections caused by MDR gram-negative bacilli. In this study, we aimed to analyze the in vitro antimicrobial activity of CAZ-AVI and other antimicrobial agents against gram-negative bacilli that were collected in Colombia between 2019 and 2021 from patients with bacteremia and skin and soft-tissue infections (SSTIs). METHODS: A total of 600 Enterobacterales and 259 P. aeruginosa strains were analyzed. The phenotypic resistance of isolates, particularly non-susceptibility to meropenem, multidrug-resistant (MDR) isolates, and difficult-to-treat (DTR) P. aeruginosa, was evaluated according to CLSI breakpoints. RESULTS: Enterobacterales had the most susceptibility to CAZ-AVI (96.5 %) and tigecycline (95 %). Tigecycline and CAZ-AVI were the antimicrobial agents with the most in vitro activity against carbapenem-resistant Enterobacterales (CRE). CAZ-AVI was the antimicrobial treatment with the most activity against P. aeruginosa. CONCLUSIONS: Tigecycline and CAZ-AVI were the antimicrobial agents with the most activity against CRE and MDR Enterobacterales. For P. aeruginosa, CAZ-AVI was the antimicrobial treatment with the most in vitro activity.

Determination of aztreonam/ceftazidime-avibactam synergism and proposal of a new methodology for the evaluation of susceptibility in vitro.

We proposed a new methodology, the microelution ATM/CZA (mATM/CZA), based on the antibiotic disc elution and the use of resazurin, for rapid (<4h) determination of in vitro susceptibility to aztreonam combined with ceftazidime-avibactam among Enterobacterales. The mATM/CZA presented excellent accuracy with 1.9 %, 98.1 % and 100 % of major error, specificity and sensitivity, respectively. Furthermore, we assessed synergism between aztreonam and ceftazidime-avibactam in Enterobacterales and Pseudomonas aeruginosa, which was observed in 37/55 Enterobacterales and 31/56 P. aeruginosa. As reference methodologies (checkerboard, time-kill curve) are not compatible with the routine of the clinical microbiology laboratories, mATM/CZA is an important alternative to evaluate susceptibility of the combination in a scenario where its clinical use is increasingly important.