Genetic and Biochemical Characterization of AXC-2 from Achromobacter ruhlandii.

Achromobacter spp. are intrinsically resistant to multiple antibiotics and can also acquire resistance to those commonly used for the treatment of respiratory infections, especially in patients with cystic fibrosis. The aim of this study was to perform the genetic and biochemical characterization of AXC-2 from A. ruhlandii and to analyze all available AXC variants. Steady-state kinetic parameters were determined on a purified AXC-2 enzyme. It exhibited higher catalytic efficiencies towards amino-penicillins and older cephalosporins, while carbapenems behaved as poor substrates. Phylogenetic analysis of all blaAXC variants available in the NCBI was conducted. AXC was encoded in almost all A. ruhlandii genomes, whereas it was only found in 30% of A. xylosoxidans. AXC-1 was prevalent among A. xylosoxidans. AXC variants were clustered in two main groups, correlating with the Achromobacter species. No association could be established between the presence of blaAXC variants and a specific lineage of A. xylosoxidans; however, a proportion of AXC-1-producing isolates corresponded to ST 182 and ST 447. In conclusion, this study provides valuable insights into the genetic context and kinetic properties of AXC-2, identified in A. ruhlandii. It also provides a thorough description of all AXC variants and their association with Achromobacter species and various lineages.

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.

Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M ß-Lactamases: Biochemical and Structural Analyses.

Design of novel ß-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two α-amido-ß-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum ß-lactamase [ESBL]) ß-lactamases were evaluated. The 50% inhibitory concentrations (IC50s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k2/K for CTX-M-96 (24,000 M-1 s-1) was twice the reported value for KPC-2 (12,000 M-1 s-1); for MB_076, the k2/K values ranged from 1,200 M-1 s-1 (KPC-2) to 3,900 M-1 s-1 (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-Å resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to ß-lactamase inhibitor design.

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.

Redefining the Origin and Evolution of Chromosomally Encoded blaCTX-M/KLU in the Context of a Revised Taxonomy of Genus Kluyvera.

Changes in Kluyvera taxonomy may clarify each species contribution for recruitment and dissemination of their relevant ß-lactamases. The CTX-M-2 subgroup is linked to Kluyvera ascorbata, KLUC to Kluyvera cryocrescens, and CTX-M-25 to Kluyvera georgiana. The CTX-M-8 subgroup can be linked to Kluyvera genomospecies 3 and CTX-M-9 to Kluyvera genomospecies 2. Kluyvera sichuanensis and Kluyvera genomospecies 1 harbor new subgroups. The CTX-M-1 subgroup has a direct counterpart in an isolate proposed as a new genomospecies 5.

Diversity of ß-lactamase-encoding genes in wastewater: bacteriophages as reporters.

A reservoir of antibiotic resistance genes (ARGs) is present in pathogenic, commensal, and environmental bacteria as well as in mobile genetic elements, including bacteriophages. Wastewater treatment plants (WWTPs) are considered hotspots for the spread of ARGs. The aim of this work was to analyze the diversity of the highly prevalent ARGs blaCTX-M and blaTEM in bacterial and bacteriophage fractions associated with human and animal environments through the study of urban waste and animal residues discharged into WWTPs to provide information about the composition and maintenance of the current resistome in Buenos Aires, Argentina. The results showed that a putative extended-spectrum variant of the blaTEM gene was the most frequently detected, with blaTEM-116 being the most prevalent, while a recently described type, blaTEM-229, was also found. In the bacteriophage fraction, we detected blaCTX-M genes from four out of the five clusters described. The detection of blaCTX- M-9-like and blaCTX-M-25-like genes was unexpected based on surveys of the ARGs from clinical pathogens circulating regionally. The finding of divergent blaCTX-M sequences associated with previously reported environmental genes argues in favor of the natural environment as a reservoir of resistance genes. ARGs were detected in bacteriophages as frequently as in bacterial communities, and furthermore, the blaCTX-M genes were more diverse in the bacteriophage fraction. Bacteriophages might therefore play a role in the spread of ARGs in the environment, but they might also be used as "reporters" for monitoring circulating ARGs.

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).

FONA-7, a Novel Extended-Spectrum ß-Lactamase Variant of the FONA Family Identified in Serratia fonticola.

Serratia fonticola is a human pathogen widely found in the environment, with birds being reported as possible natural hosts. During an epidemiological and genomic surveillance study conducted to monitor the occurrence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in South American wild birds, we identified an ESBL-positive S. fonticola in a fecal sample collected from a Hudsonian Whimbrel, during its non-breeding range on the Pacific Coast of Chile. Whole genome sequencing analysis and "in silico" modeling revealed a novel variant of the class A ESBLs FONA family, designated FONA-7, which shows 96.28% amino acid identity with FONA-6; with amino acid substitutions occurring in the signal peptide sequence (Thr22→Ser), and in the mature protein (Ser39→Asn and Thr227→Ile). This finding denotes that migratory birds can be potential vectors for the transboundary spread of ESBL-producing bacteria, creating a further theoretical risk for the origin of novel plasmid-encoded ß-lactamases.

Comparative Kinetic Analysis of OXA-438 with Related OXA-48-Type Carbapenem-Hydrolyzing Class D ß-Lactamases.

Novel variants of OXA-48-type enzymes with the ability to hydrolyze oxyimino-cephalosporins and carbapenems are increasingly reported. Since its first report in 2011, OXA-163 is now extensively spread throughout Argentina, and several variants like OXA-247 have emerged. Here, we characterized a new blaOXA-48-like variant, OXA-438, and we performed a comparative kinetic analysis with the local variants OXA-247 and OXA-163 and the internationally disseminated OXA-48. blaOXA-163, blaOXA-247, and blaOXA-438 were located in a 70 kb IncN2 conjugative plasmid. OXA-438 presented mutations in the vicinity of conserved KTG (214-216), with a 2-aa deletion (R220-I221) and a D224E shift (as in OXA-163) compared to OXA-48. Despite Kpn163 (OXA-163), Kpn247 (OXA-247) and Eco438 (OXA-438) were resistant to meropenem and ertapenem, and the transconjugants (TC) remained susceptible (however, the carbapenems minimum inhibitory concentrations were ≥3 times 2-fold dilutions higher than the acceptor strain). TC163 and Eco48 were resistant to oxyimino-cephalosporins, unlike TC247 and TC438. kcat/Km values for cefotaxime in OXA-163 were slightly higher than the rest of the variants that were accompanied by a lower Km for carbapenems. For OXA-163, OXA-247, and OXA-438, the addition of NaHCO3 improved kcat values for both cefotaxime and ceftazidime; carbapenems kcat/Km values were higher than for oxyimino-cephalosporins. Mutations occurring near the conserved KTG in OXA-247 and OXA-438 are probably responsible for the improved carbapenems hydrolysis and decreased inactivation of oxyimino-cephalosporins compared to OXA-163. Dichroism results suggest that deletions at the ß5-ß6 loop seem to impact the structural stability of OXA-48 variants. Finally, additional mechanisms are probably involved in the resistance pattern observed in the clinical isolates.

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.

Whole Genome Sequence Analysis of Burkholderia contaminans FFH2055 Strain Reveals the Presence of Putative ß-Lactamases.

Burkholderia contaminans is a member of the Burkholderia cepacia complex (Bcc), a pathogen with increasing prevalence among cystic fibrosis (CF) patients and the cause of numerous outbreaks due to the use of contaminated commercial products. The antibiotic resistance determinants, particularly ß-lactamases, have been poorly studied in this species. In this work, we explored the whole genome sequence (WGS) of a B. contaminans isolate (FFH 2055) and detected four putative ß-lactamase-encoding genes. In general, these genes have more than 93% identity with ß-lactamase genes found in other Bcc species. Two ß-lactamases, a class A (Pen-like, suggested name PenO) and a class D (OXA-like), were further analyzed and characterized. Amino acid sequence comparison showed that Pen-like has 82% and 67% identity with B. multivorans PenA and B. pseudomallei PenI, respectively, while OXA-like displayed strong homology with class D enzymes within the Bcc, but only 22-44% identity with available structures from the OXA family. PCR reactions designed to study the presence of these two genes revealed a heterogeneous distribution among clinical and industrial B. contaminans isolates. Lastly, blaPenO gene was cloned and expressed into E. coli to investigate the antibiotic resistance profile and confers an extended-spectrum ß-lactamase (ESBL) phenotype. These results provide insight into the presence of ß-lactamases in B. contaminans, suggesting they play a role in antibiotic resistance of these bacteria.

Proposing Kluyvera georgiana as the Origin of the Plasmid-Mediated Resistance Gene fosA4.

A putative fosA gene in Kluyvera georgiana 14751 showed 99% nucleotide identity with plasmid-encoded fosA4 Due to a single-nucleotide insertion translating to a truncated protein, K. georgiana 14751 fosA does not confer fosfomycin resistance. However, analysis of another genome deposit (Kluyvera ascorbata WCH1410) that could be recategorized as K. georgiana after phylogenetic analysis revealed a fosA gene 100% identical to the plasmid-borne fosA4 gene. We suggest that Kluyvera georgiana represents the most probable origin of fosA4.

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.

Complete Sequence of the IncA/C1 Plasmid pCf587 Carrying blaPER-2 from Citrobacter freundii.

The blaPER-2-harboring plasmid pCf587 (191,541 bp) belongs to lineage IncA/C1 and is closely related to pRA1. It contains a large resistance island including the blaPER-2 gene between two copies of ISKox2-like elements, the toxin-antitoxin module pemK-pemI, several other resistance genes inserted within a Tn2 transposon, a Tn21-like structure, and a class 1 integron. pCf587 belongs to sequence type 13 (ST13), a new plasmid multilocus sequence typing (pMLST) ST.

Crystal structure and kinetic analysis of the class B3 di-zinc metallo-ß-lactamase LRA-12 from an Alaskan soil metagenome.

We analyzed the kinetic properties of the metagenomic class B3 ß-lactamase LRA-12, and determined its crystallographic structure in order to compare it with prevalent metallo-ß-lactamases (MBLs) associated with clinical pathogens. We showed that LRA-12 confers extended-spectrum resistance on E. coli when expressed from recombinant clones, and the MIC values for carbapenems were similar to those observed in enterobacteria expressing plasmid-borne MBLs such as VIM, IMP or NDM. This was in agreement with the strong carbapenemase activity displayed by LRA-12, similar to GOB ß-lactamases. Among the chelating agents evaluated, dipicolinic acid inhibited the enzyme more strongly than EDTA, which required pre-incubation with the enzyme to achieve measurable inhibition. Structurally, LRA-12 contains the conserved main structural features of di-zinc class B ß-lactamases, and presents unique structural signatures that differentiate this enzyme from others within the family: (i) two loops (α3-ß7 and ß11-α5) that could influence antibiotic entrance and remodeling of the active site cavity; (ii) a voluminous catalytic cavity probably responsible for the high hydrolytic efficiency of the enzyme; (iii) the absence of disulfide bridges; (iv) a unique Gln116 at metal-binding site 1; (v) a methionine residue at position 221that replaces Cys/Ser found in other B3 ß-lactamases in a predominantly hydrophobic environment, likely playing a role in protein stability. The structure of LRA-12 indicates that MBLs exist in wild microbial populations in extreme environments, or environments with low anthropic impact, and under the appropriate antibiotic selective pressure could be captured and disseminated to pathogens.