KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: blaKPC-80, blaKPC-81, blaKPC-96 and blaKPC-97.

Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of blaKPC-2 resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant blaKPC genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for blaKPC-80, blaKPC-96, and blaKPC-97 by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including blaTEM-1, blaOXA-18 and blaOXA-1, were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of blaKPC-2 and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies. IMPORTANCE: The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, blaKPC-2. The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

Emergence of KPC-113 and KPC-114 variants in ceftazidime-avibactam-resistant Klebsiella pneumoniae belonging to high-risk clones ST11 and ST16 in South America.

Two novel variants of Klebsiella pneumoniae carbapenemase (KPC) associated with resistance to ceftazidime-avibactam (CZA) and designated as KPC-113 and KPC-114 by NCBI were identified in 2020, in clinical isolates of Klebsiella pneumoniae in Brazil. While K. pneumoniae of ST16 harbored the blaKPC-113 variant on an IncFII-IncFIB plasmid, K. pneumoniae of ST11 carried the blaKPC-114 variant on an IncN plasmid. Both isolates displayed resistance to broad-spectrum cephalosporins, ß-lactam inhibitors, and ertapenem and doripenem, whereas K. pneumoniae producing KPC-114 showed susceptibility to imipenem and meropenem. Whole-genome sequencing and in silico analysis revealed that KPC-113 presented a Gly insertion between Ambler positions 264 and 265 (R264_A265insG), whereas KPC-114 displayed two amino acid insertions (Ser-Ser) between Ambler positions 181 and 182 (S181_P182insSS) in KPC-2, responsible for CZA resistance profiles. Our results confirm the emergence of novel KPC variants associated with resistance to CZA in international clones of K. pneumoniae circulating in South America. IMPORTANCE KPC-2 carbapenemases are endemic in Latin America. In this regard, in 2018, ceftazidime-avibactam (CZA) was authorized for clinical use in Brazil due to its significant activity against KPC-2 producers. In recent years, reports of resistance to CZA have increased in this country, limiting its clinical application. In this study, we report the emergence of two novel KPC-2 variants, named KPC-113 and KPC-114, associated with CZA resistance in Klebsiella pneumoniae strains belonging to high-risk clones ST11 and ST16. Our finding suggests that novel mutations in KPC-2 are increasing in South America, which is a critical issue deserving active surveillance.

Biochemical and Structural Characterization of CRH-1, a Carbapenemase from Chromobacterium haemolyticum Related to KPC ß-Lactamases.

KPC-2 is one of the most relevant serine-carbapenemases among the carbapenem-resistant Enterobacterales. We previously isolated from the environmental species Chromobacterium haemolyticum a class A CRH-1 ß-lactamase displaying 69% amino acid sequence identity with KPC-2. The objective of this study was to analyze the kinetic behavior and crystallographic structure of this ß-lactamase. Our results showed that CRH-1 can hydrolyze penicillins, cephalosporins (except ceftazidime), and carbapenems with similar efficacy compared to KPC-2. Inhibition kinetics showed that CRH-1 is not well inhibited by clavulanic acid, in contrast to efficient inhibition by avibactam (AVI). The high-resolution crystal of the apoenzyme showed that CRH-1 has a similar folding compared to other class A ß-lactamases. The CRH-1/AVI complex showed that AVI adopts a chair conformation, stabilized by hydrogen bonds to Ser70, Ser237, Asn132, and Thr235. Our findings highlight the biochemical and structural similarities of CRH-1 and KPC-2 and the potential clinical impact of this carbapenemase in the event of recruitment by pathogenic bacterial species.

Diversity of genetic platforms harboring the blaPER-2 gene in Enterobacterales and insights into the role of ISPa12 in its mobilization and dissemination.

The production of PER-like extended-spectrum ß-lactamases has recently been associated with reduced susceptibility to the last resort drugs aztreonam/avibactam and cefiderocol. PER-2 has been mainly confined to Argentina and neighboring countries. Until now, only three plasmids harboring blaPER-2 genes have been characterized but very little is known about the involvement of different plasmid groups in its dissemination. The diversity of genetic platforms associated with blaPER-2 genes from a collection of PER-producing Enterobacterales was analysed by describing the close environment and the plasmid backbones. Full sequences of 11 plasmids were obtained by short read (Illumina) and long read (Oxford Nanopore or PacBio) sequencing technologies. De novo assemblies, annotation and sequence analysis were performed by Unicycler, Prokka and BLAST. Plasmid analysis revealed that the blaPER-2 gene is encoded on plasmids of different incompatibility groups (A, C, FIB, HI1B, N2), indicating that this gene may have been disseminated through a variety of plasmids. Comparison with the few publicly available nucleotide sequences describing the blaPER-2 genetic environment, including those from the environmental species Pararheinheimera spp. (considered as the progenitor of blaPER genes), indicates a role of ISPa12 in blaPER-2 gene mobilization from the chromosome of Pararheinheimera spp. Also, the blaPER-2 gene was carried by a novel ISPa12-composite transposon, Tn7390. In addition, its association with ISKox2-like elements in the close genetic environment in all plasmids analysed suggests a role of these insertion sequence elements in further dissemination of blaPER-2 genes.

Characterization of Emerging Pathogens Carrying blaKPC-2 Gene in IncP-6 Plasmids Isolated From Urban Sewage in Argentina.

Untreated wastewater is a reservoir for multidrug-resistant bacteria, but its role in the spread of antibiotic resistance in the human population remains poorly investigated. In this study, we isolated a KPC-2-producing ST2787 Klebsiella quasipneumoniae subsp. quasipneumoniae (WW14A), recovered from raw sewage at a wastewater treatment plant in Argentina in 2018 and determined its complete genome sequence. Strain WW14A was resistant to all ß-lactams, ciprofloxacin and amikacin. A core genome phylogenetic analysis indicated that WW14A was closely related to a GES-5-producing Taiwanese strain isolated from hospital wastewater in 2015 and it was clearly distinct from strains isolated recently in Argentina and Brazil. Interestingly, blaKPC-2 was harbored by a recently described IncP-6 broad-spectrum plasmid which was sporadically reported worldwide and had never been reported before in Argentina. We investigated the presence of the IncP-6 replicon in isolates obtained from the same sampling and found a novel non-typable/IncP-6 hybrid plasmid in a newly assigned ST1407 Enterobacter asburiae (WW19C) also harboring blaKPC-2. Nanopore sequencing and hybrid assembly of strains WW14A and WW19C revealed that both IncP-6 plasmids shared 72% of coverage (~20 kb), with 99.99% of sequence similarity and each one also presented uniquely combined regions that were derived from other plasmids recently reported in different countries of South America, Asia, and Europe. The region harboring the carbapenem resistance gene (~11 kb) in both plasmids contained a Tn3 transposon disrupted by a Tn3-ISApu-flanked element and the core sequence was composed by ΔISKpn6/blaKPC-2/ΔblaTEM-1/ISKpn27. Both strains also carried genes conferring resistance to heavy metals (e.g., arsenic, mercury, lead, cadmium, copper), pesticides (e.g., glyphosate), disinfectants, and several virulence-related genes, posing a potential pathogenic risk in the case of infections. This is the first study documenting blaKPC-2 associated with IncP-6 plasmids in K. quasipneumoniae and Enterobacter cloacae complex from wastewater in Argentina and highlights the circulation of IncP-6 plasmids as potential reservoirs of blaKPC-2 in the environment.

Structural and Biochemical Characterization of the Novel CTX-M-151 Extended-Spectrum ß-Lactamase and Its Inhibition by Avibactam.

The diazabicyclooctane (DBO) inhibitor avibactam (AVI) reversibly inactivates most serine ß-lactamases, including the CTX-M ß-lactamases. Currently, more than 230 unique CTX-M members distributed in five clusters with less than 5% amino acid sequence divergence within each group have been described. Recently, a variant named CTX-M-151 was isolated from a Salmonella enterica subsp. enterica serovar Choleraesuis strain in Japan. This variant possesses a low degree of amino acid identity with the other CTX-Ms (63.2% to 69.7% with respect to the mature proteins), and thus it may represent a new subgroup within the family. CTX-M-151 hydrolyzes ceftriaxone better than ceftazidime (kcat/Km values 6,000-fold higher), as observed with CTX-Ms. CTX-M-151 is well inhibited by mechanism-based inhibitors like clavulanic acid (inactivation rate [kinact]/inhibition constant [Ki ] = 0.15 µM-1 · s-1). For AVI, the apparent inhibition constant (Kiapp), 0.4 µM, was comparable to that of KPC-2; the acylation rate (k2/K) (37,000 M-1 · s-1) was lower than that for CTX-M-15, while the deacylation rate (koff) (0.0015 s-1) was 2- to 14-fold higher than those of other class A ß-lactamases. The structure of the CTX-M-151/AVI complex (1.32 Å) reveals that AVI adopts a chair conformation with hydrogen bonds between the AVI carbamate and Ser70 and Ser237 at the oxyanion hole. Upon acylation, the side chain of Lys73 points toward Ser130, which is associated with the protonation of Glu166, supporting the role of Lys73 in the proton relay pathway and Glu166 as the general base in deacylation. To our knowledge, this is the first chromosomally encoded CTX-M in Salmonella Choleraesuis that shows similar hydrolytic preference toward cefotaxime (CTX) and ceftriaxone (CRO) to that toward ceftazidime (CAZ).

Structural Insights into the Inhibition of the Extended-Spectrum ß-Lactamase PER-2 by Avibactam.

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-ß-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D ß-lactamases (i.e., k2/K of ≈103 M-1 s-1) but lower than other class A ß-lactamases (i.e., k2/K = 104 to 105 M-1 s-1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum ß-lactamases (ESBLs). Comparatively, relebactam combined with a ß-lactam is more potent against Escherichia coli producing PER-2 variants than ß-lactam-AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

Defining Substrate Specificity in the CTX-M Family: the Role of Asp240 in Ceftazidime Hydrolysis.

The natural diversification of CTX-M ß-lactamases led to the emergence of Asp240Gly variants in the clinic that confer reduced susceptibility to ceftazidime (CAZ). In this study, we compared the impact of this substitution on CAZ and ceftazidime-avibactam (CZA) MICs against isogenic Escherichia coli strains with different porin deficiencies. Our results show a noticeable increase in CAZ resistance in clones expressing Asp240Gly-harboring CTX-M when combined with OmpF porin deficiency. Kinetic analysis revealed that the kcat/Km for CAZ was 5- to 15-fold higher for all Asp240Gly variants but remained 200- to 725-fold lower than that for cefotaxime (CTX). In vitro selection of CAZ-resistant clones yielded nonsusceptible CTX-M producers (MIC of >16 µg/ml) only after overnight incubation; the addition of avibactam (AVI) decreased MICs to a susceptible range against these variants. In contrast, the use of CZA as a selective agent did not yield resistant clones. AVI inactivated both CTX-M-12 and CTX-M-96, with an apparent inhibition constant comparable to that of SHV-2 and 1,000-fold greater than that of PER-2 and CMY-2, and k2/K for CTX-M-12 was 24- and 35-fold higher than that for CTX-M-96 and CTX-M-15, respectively. Molecular modeling suggests that AVI interacts similarly with CTX-M-96 and CTX-M-15. We conclude that the impact of Asp240Gly in resistance may arise when other mechanisms are also present (i.e., OmpF deficiency). Additionally, CAZ selection could favor the emergence of CAZ-resistant subpopulations. These results define the role of Asp240 and the impact of the -Gly substitution and allow us to hypothesize that the use of CZA is an effective preventive strategy to delay the development of resistance in this family of extended-spectrum ß-lactamases.

Impact of Mutations at Arg220 and Thr237 in PER-2 ß-Lactamase on Conformation, Activity, and Susceptibility to Inhibitors.

PER-2 accounts for up to 10% of oxyimino-cephalosporin resistance in Klebsiella pneumoniae and Escherichia coli in Argentina and hydrolyzes both cefotaxime and ceftazidime with high catalytic efficiencies (kcat/Km ). Through crystallographic analyses, we recently proposed the existence of a hydrogen bond network connecting Ser70-Gln69-oxyanion water-Thr237-Arg220 that might be important for the activity and inhibition of the enzyme. Mutations at Arg244 in most class A ß-lactamases (such as TEM and SHV) reduce susceptibility to mechanism-based inactivators, and Arg220 in PER ß-lactamases is equivalent to Arg244. Alterations in the hydrogen bond network of the active site in PER-2, through modifications in key residues such as Arg220 and (to a much lesser extent) Thr237, dramatically impact the overall susceptibility to inactivation, with up to ∼300- and 500-fold reductions in the rate constant of inactivation (kinact)/Ki values for clavulanic acid and tazobactam, respectively. Hydrolysis on cephalosporins and aztreonam was also affected, although to different extents compared to with wild-type PER-2; for cefepime, only an Arg220Gly mutation resulted in a strong reduction in the catalytic efficiency. Mutations at Arg220 entail modifications in the catalytic activity of PER-2 and probably local perturbations in the protein, but not global conformational changes. Therefore, the apparent structural stability of the mutants suggests that these enzymes could be possibly selected in vivo.