Omic insights into various ceftazidime-avibactam-resistant Klebsiella pneumoniae isolates from two southern Italian regions

Abstract

Ceftazidime-avibactam (CZA) is one of the best therapeutic options available for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. However, sporadic reports of CZA-resistant strains have been rapidly increasing in patients. Herein, we provide detailed case reports of the emergence of ceftazidime-avibactam resistance to identify their resistome and virulome using genomic molecular approaches. Sixteen isolates were collected from 13 patients at three hospitals in Catania and Catanzaro (Italy) between 2020-2021. Antimicrobial susceptibility was determined by broth microdiluition. The samples included in study were analyzed for resistome, virulome and Sequence Type (ST) using Whole Genome Sequencing (WGS). All strains were resistant to ceftazidime/avibactam, ciprofloxacin, extended-spectrum cephalosporins and aztreonam, 13/16 to meropenem, 8/16 to colistin and 7/16 to fosfomycin; 15/16 were susceptible to meropenem/vaborbactam; all strains were susceptible to cefiderocol. Molecular analysis showed circulation of three major clones: ST101, ST307 and ST512. In 10/16 strains, we found a blaKPC-3 gene; in 6/16 strains, four different blaKPC variants (blaKPC28-31-34-50) were detected. A plethora of other beta-lactam genes (blaSHV28-45-55-100-106-187-205-212, blaOXA1-9-48, blaTEM-181 and blaCTX-M-15) was observed; blaOXA-9 was found in ST307 and ST512, instead blaOXA48 in one out four ST101 strains. With regard to membrane permeability, ompK35 and ompK36 harbored frameshift mutations in 15/16 strains; analysis of ompK37 gene revealed that all strains harbored a non-functional protein and carry wild-type PBP3. There is an urgent need to characterize the mechanisms underlying carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance. Furthermore, it is becoming increasingly important to explore feasible methods for accurate detection of different KPC enzymes.

Generation and Characterization of Stable Small Colony Variants of USA300 Staphylococcus aureus in RAW 264.7 Murine Macrophages

“…Illumina sequencing was performed on the Illumina MiSeq platform (2 × 150 bp paired-end sequencing); library preparation and quantification were performed as reported, starting from 10 ng of genomic DNA [ 44 ]. The sample sheet was created using the Local Run Manager v3 software, following the instructions in the Local Run Manager v3 Software Guide provided by Illumina.…”

“…DNA Extraction S. aureus ATCC BAA-1556 wild-type and its stable SCVs isolated from the treatment with carnosine (20 mM) and erythromycin (32 mg/L) were selected for DNA isolation. For Illumina sequencing, genomic DNA was extracted and quantified as previously described [44]. For nanopore sequencing, high-molecular weight genomic DNA was obtained using a raffinose-based method as described [45,46].…”

The molecular detection of carbapenem markers with a two-levels amplification screening protocol: epidemiological and resistome insights

“…The product also includes a dUTP/UNG system To prevent contaminations. The amplification processes were used according To The manufacturer's instructions, through The Aria dx software (Agilent Technologies) (Bongiorno et al, 2023;QPCR, 2023).…”

All Roads Lead to Rome: Enhancing the Probability of Target Attainment with Different Pharmacokinetic/Pharmacodynamic Modelling Approaches

“…Ceftazidime (CAZ) is a cephalosporin that is active against P. aeruginosa ; together with avibactam (AVI), a non-ß-lactam ß-lactamase-inhibitor inhibiting enzymes belonging to Ambler classes A and C and selected class D beta-lactamases [ 58 ], it extends the spectrum against Enterobacteriaceae and several multi-drug resistant (MDR) Pseudomonas isolates [ 59 , 60 , 61 ]. In the last five years, novel beta-lactamase inhibitors, such as taniborbactam combined with a cephalosporin [ 62 , 63 ], as well as reports on the resistance of ceftazidime-avibactam (CAZ-AVI), have emerged [ 64 , 65 ]; however, we would like to highlight how the PK/PD targets were determined for AVI alone and how target attainment was achieved for the combination using PK/PD modelling. CAZ-AVI has been studied extensively in preclinical in vitro and in vivo models, as summarized elsewhere [ 66 ].…”