Klebsiella pneumoniae Carbapenemase Variants Resistant to Ceftazidime-Avibactam: an Evolutionary Overview.

First variants of the Klebsiella pneumoniae carbapenemase (KPC), KPC-2 and KPC-3, have encountered a worldwide success, particularly in K. pneumoniae isolates. These beta-lactamases conferred resistance to most beta-lactams including carbapenems but remained susceptible to new beta-lactam/beta-lactamase inhibitors, such as ceftazidime-avibactam. After the marketing of ceftazidime-avibactam, numerous variants of KPC resistant to this association have been described among isolates recovered from clinical samples or derived from experimental studies. In KPC variants resistant to ceftazidime-avibactam, point mutations, insertions and/or deletions have been described in various hot spots. Deciphering the impact of these mutations is crucial, not only from a therapeutic point of view, but also to follow the evolution in time and space of KPC variants resistant to ceftazidime-avibactam. In this review, we describe the mutational landscape of the KPC beta-lactamase toward ceftazidime-avibactam resistance based on a multidisciplinary approach including epidemiology, microbiology, enzymology, and thermodynamics. We show that resistance is associated with three hot spots, with a high representation of insertions and deletions compared with other class A beta-lactamases. Moreover, extension of resistance to ceftazidime-avibactam is associated with a trade-off in the resistance to other beta-lactams and a decrease in enzyme stability. Nevertheless, the high natural stability of KPC could underlay the propensity of this enzyme to acquire in vivo mutations conferring resistance to ceftazidime-avibactam (CAZavi), particularly via insertions and deletions.

Genetic and Phenotypic Study of the Pectobacterium versatile Beta-Lactamase, the Enzyme Most Similar to the Plasmid-Encoded TEM-1.

Genus Pectobacterium bacteria include important agricultural pathogens. Pectobacterium versatile isolates contain a chromosome-borne beta-lactamase, PEC-1. This enzyme is the closest relative of TEM-1, a plasmid-borne beta-lactamase widespread in the Enterobacterales. We performed bioinformatics and phenotypic analyses to investigate the genetic and phenotypic features of PEC-1 and its frequency and ability to spread within genus Pectobacterium. We also compared the characteristics of PEC-1 and TEM-1 and evaluated the likelihood of transfer. We found that blaPEC-1 was present principally in a small number of genetic environments in P. versatile. Identical blaPEC-1 genetic environments were present in closely related species, consistent with the high frequency of genetic exchange within the genus Pectobacterium. Despite the similarities between PEC-1 and TEM-1, their genetic environments displayed no significant identity, suggesting an absence of recent transfer. Phenotypic analyses on clonal constructs revealed similar hydrolysis spectra. Our results suggest that P. versatile is the main reservoir of PEC-1, which seems to transfer to closely related species. The genetic distance between PEC-1 and TEM-1, and the lack of conserved elements in their genetic environments, suggest that any transfer that may have occurred must have taken place well before the antibiotic era. IMPORTANCE This study aimed to compare the chromosomal beta-lactamase from Pectobacterium versatile, PEC-1, with the well-known and globally distributed TEM-1 in terms of genetic and functional properties. Despite the similarities between the enzymes, we obtained no definitive proof of gene transfer for the emergence of blaPEC-1 from blaTEM-1. Indeed, given the limited degree of sequence identity and the absence of a common genetic environment, it seems unlikely that any transfer of this gene has occurred recently. However, although blaPEC-1 was found mostly in one specific clade of the P. versatile species, certain isolates from other closely related species, such as Pectobacterium brasiliense and Pectobacterium polaris, may also carry this gene inserted into common genetic environments. This observation suggests that genetic exchanges are frequent, accounting for the diffusion of blaPEC-1 between isolates from different Pectobacterium species and, potentially, to exogenous mobile genetic elements.

[Electrophoresis of serum lipoproteins (lipoproteinogram) with the Hydragel Lipo + Lp(a)® (Sebia) kit: evaluation of Fat Red 7B staining]. / Électrophorèse des lipoprotéines sériques (lipoprotéinogramme) par le kit Hydragel Lipo + Lp(a)® (Sebia) : évaluation de la coloration au Fat Red 7B.

The lipoproteinogram (or lipidogram) consists in an electrophoretic separation of the main classes of serum lipoproteins. Separation was done in agarose gel using the Sebia Hydragel Lipo + Lp(a)® kit. A repeatability study (n=6) was conducted on 3 sera (1 normolipidemic, 1 hypertriglyceridemic and 1 with a high Lp(a) concentration). The reproducibility was studied on these 3 sera and on an ascites liquid containing chylomicrons, upon 6 days (n=6). A quantitative approach was made by studying areas under the curve and percentages of fractions. In both cases (repeatability and reproducibility), the revelation of the lipoproteins in the gel after electrophoretic migration was made either by staining with Sudan Black (procedure recommended by Sebia), or with Fat Red 7B. Regardless of staining, both repeatability and reproducibility studies show that all lipoprotein fractions were correctly detected at their respective positions, leading to satisfactory interpretations of lipoproteinograms. Our reproducibility study also confirmed a good stability of the fractions over 6 days (storage at +5 ± 3̊C). In addition, the Fat Red 7B staining leads to a shorter technical time (about 40 min) for the gel drying and staining/destaining phases, which allows us to respond more quickly to certain urgent requests such as chylothorax diagnosis.