In vitro activity of cefepime-enmetazobactam on carbapenem resistant Gram negatives.

OBJECTIVES: Cefepime-enmetazobactam is a new ß-lactam-ß-lactamase inhibitor (BL/BLI) combination with broad-spectrum activity against multidrug-resistant Enterobacterales including ESBL producers. This study evaluated the in vitro activity of cefepime-enmetazobactam towards a collection of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa and Acinetobacter baumannii compared to the other BL/BLI combinations. METHODS: MIC of cefepime, cefepime-enmetazobactam, ceftazidime, ceftazidime-avibactam, meropenem, meropenem-vaborbactam, imipenem, imipenem-relebactam and ertapenem were determined by broth microdilution on 2,212 CRE including 2,089 carbapenemase producers (1000 OXA-48-like, 49 KPC, 697 NDM, 180 VIM, 1 IMP, 9 IMI, 158 multiple carbapenemases) and 123 non-carbapenemase producers (CRE non-CPE) received at the French National Reference Center (1st March to 31th August 2023), 50 P. aeruginosa and 30 A. baumannii. All strains were fully sequenced. RESULTS: We confirmed the absence of inhibitory activity of enmetazobactam towards metallo-ß-lactamases. Cefepime-enmetazobactam and ceftazidime-avibactam exhibited a similar susceptibility (96.7% vs 99.5%, respectively) on OXA-48-producers. Cefepime-enmetazobactam exhibited 66.9% and 63.3% susceptibility for CRE non-EPC and KPC while those rates rose to 96.7%/95.9%, 93.4%/95.9%, 95.9%/98.0% for ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam, respectively. Low MICs (≤0.25 mg/L) were obtained for ceftazidime-avibactam resistant KPC variants. Cefepime-enmetazobactam did not display a significant added value compared to cefepime alone on P. aeruginosa and A. baumannii. CONCLUSION: OXA-48 producers displayed high susceptibility to cefepime-enmetazobactam which is similar to ceftazidime-avibactam, including for OXA-48 producers that co-produce a ceftazidime hydrolyzing enzyme (ESBL or AmpC). In vivo experiments have to be implemented to confim if cefepime-enmetazobactam might be a relevant alternative to ceftazidime-avibactam for the treatment of infections caused by OXA-48 producers.

Genetic and biochemical characterization of OXA-1186, a novel OXA-198-type carbapenemase hydrolysing cephalosporins in Citrobacter freundii.

OBJECTIVES: This study described OXA-1186, a novel carbapenemase related to OXA-198 carbapenemase and produced by a clinical isolate of Citrobacter freundii. METHODS: WGS was used to characterize the resistome, virulome and plasmid types of the C. freundii 315C8 isolate and to reconstruct the blaOXA-1186-carrying plasmid. Disc diffusion and broth microdilution assays were used to determine MICs. The blaOXA-1186 gene was cloned into plasmid pTOPO and then transformed into Escherichia coli TOP10 or HB4. It was also cloned in pET41b and transformed into E. coli BL21 DE3 for protein purification. Steady-state kinetic parameters were determined on purified OXA-1186. RESULTS: C. freundii 315C8, belonging to ST8, was resistant to penicillins including temocillin and broad-spectrum cephalosporins and displayed reduced susceptibility to carbapenems. It was negative for one of the five main carbapenemases. WGS revealed that the blaOXA-1186 gene encoded a novel carbapenemase that shared 83% amino acid identity with OXA-198. The blaOXA-1186 gene was carried on an IncP6-type plasmid and was embedded within a class 1 integron. Cloning and expression in E. coli revealed that expression of the blaOXA-1186 gene conferred resistance to penicillins, cephalosporins and carbapenems, where it was associated with impaired outer membrane permeability. Kinetic parameters confirmed the hydrolysis of ceftazidime, cefepime and aztreonam, in addition to imipenem and meropenem. CONCLUSIONS: Here, we described a novel carbapenemase, OXA-1186, identified in C. freundii. Unlike OXA-198, OXA-1186 is able to hydrolyse broad-spectrum cephalosporins. This carbapenemase was carried on a broad-spectrum IncP6 plasmid identified in other Citrobacter species and non-fermenters.

Evaluation of the Reveal® AST (SPECIFIC) for Antimicrobial Susceptibility Testing from Positive Blood Culture Spiked with Carbapenem-Resistant Isolates.

As bloodstream infections and associated septic shock are common causes of mortality in hospitals, rapid antibiotic susceptibility testing (AST) performed directly on positive blood cultures is needed to implement an efficient therapy in clinical settings. We evaluated the Reveal® rapid AST system on a collection of 197 fully characterized carbapenem-resistant Enterobacterales, including 177 carbapenemase producers (CPE) spiked in blood culture bottles. The clinical categorization based on the Minimal Inhibitory Concentration (MIC) determination of eighteen antimicrobial molecules was compared to the clinical categorization based on the disk diffusion assay as a reference. The Reveal AST system provided results within a mean time to result of 5 h. Overall, the categorical agreement (CA) between the two techniques was 94.1%. The rates of very major errors (VMEs), major errors (MEs) and minor errors (mEs) were 3.8%, 3.7% and 5.6%, respectively. Imipenem was the antimicrobial with the lowest CA rate (78.7%), with rates of 15% VMEs and 10.7% MEs, but the performances were better when considering only the non-CPE category (CA of 89%). On this resistant collection of Enterobacterales with numerous acquired ß-lactamases, the Specific Reveal assay proved to be useful for a rapid determination of AST compatible with a quick adaptation of the patient's antimicrobial treatment.

Oxacillinase-484-Producing Enterobacterales, France, 2018-2023.

We examined the emergence and characteristics of oxacillinase-484-producing Enterobacterales in France during 2012-2023. Genomic analysis identified 2 predominant sequence types in Escherichia coli: ST410 and ST1722. Plasmid analysis revealed that blaOXA-484 genes were carried mostly on an IncX3-type plasmid associated with genetic elements including insertion sequences IS3000 and ISKpn19.

Molecular, Genetic, and Biochemical Characterization of OXA-484 Carbapenemase, a Difficult-to-Detect R214G Variant of OXA-181.

OXA-244, an R214G variant of OXA-48, is silently spreading worldwide likely because of difficulties in detection using classical screening media. Here, we characterized two clinical isolates of Escherichia coli and Citrobacter youngae that displayed reduced susceptibility to carbapenems but were lacking significant carbapenemase activity as revealed by negative Carba NP test results. However, positive test results were seen for OXA-48-like enzymes by lateral flow immunoassays. WGS revealed the presence of a blaOXA-181-like gene that codes for OXA-484, an R214G variant of OXA-181. BlaOXA-484 gene was located on a 58.4-kb IncP1-like plasmid (pN-OXA-484), that upon transfer into E. coli HB4 with impaired permeability, conferred carbapenem and temocillin resistance (MICs > 32 mg/L). E. coli TOP10 (pTOPO-OXA-484) revealed reduced MICs in most substrates as compared to E. coli TOP10 (pTOPO-OXA-181), especially for imipenem (0.25 mg/L versus 0.75 mg/L) and temocillin (16 mg/L versus 1028 mg/L). Catalytic efficiencies of OXA-484 were reduced as compared to OXA-181 for most ß-lactams including imipenem and temocillin with 27.5- and 21.7-fold reduction, respectively. Molecular modeling confirmed that the salt bridges between R214, D159, and the R1 substituent's carboxylate group of temocillin were not possible with G214 in OXA-484, explaining the reduced affinity for temocillin. In addition, changes in active site's water network may explain the decrease in hydrolysis rate of carbapenems. OXA-484 has weak imipenem and temocillin hydrolytic activities, which may lead to silent spread due to underdetection using selective screening media or biochemical imipenem hydrolysis confirmatory tests.

IMI-Type Carbapenemase-Producing Enterobacter cloacae Complex, France and Overseas Regions, 2012-2022.

We characterized a collection of IMI-like-producing Enterobacter spp. isolates (n = 112) in France. The main clone corresponded to IMI-1-producing sequence type 820 E. cloacae subspecies cloacae that was involved in an outbreak. Clinicians should be aware of potential antimicrobial resistance among these bacteria.

Spread of carbapenemase-producing Morganella spp from 2013 to 2021: a comparative genomic study.

BACKGROUND: Morganella spp are opportunistic pathogens involved in various infections. Intrinsic resistance to multiple antibiotics (including colistin) combined with the emergence of carbapenemase producers reduces the number of active antimicrobials. The aim of this study was to characterise genetic features related to the spread of carbapenem-resistant Morganella spp. METHODS: This comparative genomic study included extensively drug-resistant Morganella spp isolates collected between Jan 1, 2013, and March 1, 2021, by the French National Reference Center (NRC; n=68) and European antimicrobial resistance reference centres in seven European countries (n=104), as well as one isolate from Canada, two reference strains from the Pasteur Institute collection (Paris, France), and two colistin-susceptible isolates from Bicêtre Hospital (Kremlin-Bicêtre, France). The isolates were characterised by whole-genome sequencing, antimicrobial susceptibility testing, and biochemical tests. Complete genomes from GenBank (n=103) were also included for genomic analysis, including phylogeny and determination of core genomes and resistomes. Genetic distance between different species or subspecies was performed using average nucleotide identity (ANI). Intrinsic resistance mechanisms to polymyxins were investigated by combining genetic analysis with mass spectrometry on lipid A. FINDINGS: Distance analysis by ANI of 275 isolates identified three groups: Morganella psychrotolerans, Morganella morganii subspecies sibonii, and M morganii subspecies morganii, and a core genome maximum likelihood phylogenetic tree showed that the M morganii isolates can be separated into four subpopulations. On the basis of these findings and of phenotypic divergences between isolates, we propose a modified taxonomy for the Morganella genus including four species, Morganella psychrotolerans, Morganella sibonii, Morganella morganii, and a new species represented by a unique environmental isolate. We propose that M morganii include two subspecies: M morganii subspecies morganii (the most prevalent) and M morganii subspecies intermedius. This modified taxonomy was supported by a difference in intrinsic resistance to tetracycline and conservation of metabolic pathways such as trehalose assimilation, both only present in M sibonii. Carbapenemase producers were mostly identified among five high-risk clones of M morganii subspecies morganii. The most prevalent carbapenemase corresponded to NDM-1, followed by KPC-2, and OXA-48. A cefepime-zidebactam combination was the most potent antimicrobial against the 172 extensively drug-resistant Morganella spp isolates in our collection from different European countries, which includes metallo-ß-lactamase producers. Lipid A analysis showed that the intrinsic resistance to colistin was associated with the presence of L-ARA4N on lipid A. INTERPRETATION: This global characterisation of, to our knowledge, the widest collection of extensively drug-resistant Morganella spp highlights the need to clarify the taxonomy and decipher intrinsic resistance mechanisms, and paves the way for further genomic comparisons. FUNDING: None.

Role of amino acid 159 in carbapenem and temocillin hydrolysis of OXA-933, a novel OXA-48 variant.

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The blaOXA-933 gene was borne on Tn2208, a 2,696-bp composite transposon made of two IS1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the blaOXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the blaOXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the ß5-ß6 loop, but interacting with key residues of it.

Regional dissemination of NDM-1 producing Enterobacter hormaechei ST1740, with a subset of strains co-producing VIM-4 or IMP-13, France, 2019 to 2022.

BackgroundFrom 2019 to 2022, the French National Reference Centre for Antibiotic Resistance (NRC) received a total of 25 isolates of Enterobacter hormaechei subsp. hoffmannii sequence type (ST)1740. All produced metallo-ß-lactamase(s) and were from the Lyon area.AimTo understand these strains' spread and evolution, more extended microbiological and molecular analyses were conducted.MethodsPatients' demographics and specimen type related to isolates were retrieved. All strains underwent short-read whole genome sequencing, and for 15, long-read sequencing to understand carbapenemase-gene acquisition. Clonal relationships were inferred from core-genome single nt polymorphisms (SNPs). Plasmids and the close genetic environment of each carbapenemase-encoding gene were analysed.ResultsPatients (10 female/15 male) were on average 56.6 years old. Seven isolates were recovered from infections and 18 through screening. With ≤ 27 SNPs difference between each other's genome sequences, the 25 strains represented a clone dissemination. All possessed a chromosome-encoded bla NDM-1 gene inside a composite transposon flanked by two IS3000. While spreading, the clone independently acquired a bla VIM-4-carrying plasmid of IncHI2 type (n = 12 isolates), or a bla IMP-13-carrying plasmid of IncP-1 type (n = 1 isolate). Of the 12 isolates co-producing NDM-1 and VIM-4, seven harboured the colistin resistance gene mcr9.2; the remaining five likely lost this gene through excision.ConclusionThis long-term outbreak was caused by a chromosome-encoded NDM-1-producing ST1740 E. hormaechei subsp. hoffmannii clone, which, during its dissemination, acquired plasmids encoding VIM-4 or IMP-13 metallo-ß-lactamases. To our knowledge, IMP-13 has not prior been reported in Enterobacterales in France. Epidemiological and environmental investigations should be considered alongside microbiological and molecular ones.

Capsules and their traits shape phage susceptibility and plasmid conjugation efficiency.

Bacterial evolution is affected by mobile genetic elements like phages and conjugative plasmids, offering new adaptive traits while incurring fitness costs. Their infection is affected by the bacterial capsule. Yet, its importance has been difficult to quantify because of the high diversity of confounding mechanisms in bacterial genomes such as anti-viral systems and surface receptor modifications. Swapping capsule loci between Klebsiella pneumoniae strains allowed us to quantify their impact on plasmid and phage infection independently of genetic background. Capsule swaps systematically invert phage susceptibility, revealing serotypes as key determinants of phage infection. Capsule types also influence conjugation efficiency in both donor and recipient cells, a mechanism shaped by capsule volume and conjugative pilus structure. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The least capsule-sensitive pili (F-like) are the most frequent in the species' plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer.

Evaluation of the French novel disc diffusion-based algorithm for the phenotypic screening of carbapenemase-producing Enterobacterales.

OBJECTIVES: The early identification of carbapenemase-producing Enterobacterales (CPE) is required to prevent their spread and initiate proper therapy. Accordingly, it is crucial to develop efficient algorithms using susceptibility testing results to discriminate non-carbapenemase producers (non-CPE) from those that require complementary tests. In 2022, to adapt its recommendations to the evolution of CPE epidemiology (increased prevalence of OXA-244 producers), the Antibiogram Committee of the French Society of Microbiology (CA-SFM) proposed a new algorithm for the screening of CPE. We compared this algorithm to the former algorithm (2015-2021). METHODS: From July 2022 to January 2023, all nonduplicate enterobacterial isolates referred to French National Reference Centre for carbapenemase detection (n = 518) were subjected to the former CA-SFM algorithm (2015 to 2021) using inhibition diameters of ertapenem, ticarcillin-clavulanate, temocillin and meropenem or imipenem, and the novel CA-SFM algorithm (since 2022) using inhibition diameters of ceftazidime-avibactam, temocillin, and meropenem or imipenem. RESULTS: Sensitivity, specificity, negative predictive value, and positive predictive value were of 80.8% (CI95 76.3%-84.6%), 66.2% (58.1%-73.5%), 59.3% (51.5%-66.6%), and 85.0% (80.7% - 88.5%) for the old CA-SFM algorithm and 97.8% (95.5%-99.0%), 45.5% (37.5%-53.7%), 89.7% (80.3%-95.2%), and 80.9% (76.9%-84.4%) for the novel CA-SFM algorithm. DISCUSSION: The novel CA-SFM algorithm possesses the best performance for the screening of CPE particularly in countries with a high prevalence of OXA-48-like producers.

Emergence and rapid dissemination of highly resistant NDM-14-producing Klebsiella pneumoniae ST147, France, 2022.

BackgroundSince 2021, an emergence of New Delhi metallo-ß-lactamase (NDM)-14-producing Klebsiella pneumoniae has been identified in France. This variant with increased carbapenemase activity was not previously detected in Enterobacterales.AimWe investigated the rapid dissemination of NDM-14 producers among patients in hospitals in France.MethodsAll NDM-14-producing non-duplicate clinical isolates identified in France until June 2022 (n = 37) were analysed by whole genome sequencing. The phylogeny of NDM-14-producers among all K. pneumoniae sequence type (ST) 147 reported in France since 2014 (n = 431) was performed. Antimicrobial susceptibility testing, conjugation experiments, clonal relationship and molecular clock analysis were performed.ResultsThe 37 NDM-14 producers recovered in France until 2022 belonged to K. pneumoniae ST147. The dissemination of NDM-14-producing K. pneumoniae was linked to a single clone, likely imported from Morocco and responsible for several outbreaks in France. The gene bla NDM-14 was harboured on a 54 kilobase non-conjugative IncFIB plasmid that shared high homology with a known bla NDM-1-carrying plasmid. Using Bayesian analysis, we estimated that the NDM-14-producing K. pneumoniae ST147 clone appeared in 2020. The evolutionary rate of this clone was estimated to 5.61 single nucleotide polymorphisms per genome per year. The NDM-14 producers were highly resistant to all antimicrobials tested except to colistin, cefiderocol (minimum inhibitory concentration 2 mg/L) and the combination of aztreonam/avibactam.ConclusionHighly resistant NDM-14 producing K. pneumoniae can rapidly spread in healthcare settings. Surveillance and thorough investigations of hospital outbreaks are critical to evaluate and limit the dissemination of this clone.

Nationwide molecular epidemiology of carbapenemase-producing Citrobacter spp. in France in 2019 and 2020.

IMPORTANCE: The emergence of carbapenemase producers in Enterobacterales mostly involves Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex species. However, in France, we observed the emergence and the rapid dissemination of carbapenemase in Citrobacter spp. In this study, we demonstrated that a wide variety of carbapenemases is produced by many different species of Citrobacter spp. However, we clearly identify three high-risk clones of Citrobacter freundii, ST8, ST22, and ST91 that drive the spread of carbapenemase in France. This epidemiological study paves the way of further analysis that would aim to identify the virulence factors involved in this pellicular ability of these three clones to disseminate at the hospital.

Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9.

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to ß-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Km = 53 µM) than for NDM-1 (Km = 161 µM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 µM-1.s-1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol.

Population Analysis of Escherichia coli Sequence Type 361 and Reduced Cefiderocol Susceptibility, France.

Cefiderocol resistance is increasingly reported in New Delhi metallo-ß-lactamase-producing Enterobacterales. Genomic and phenotypic analysis of Escherichia coli sequence type 361, a primary clone causing carbapenemase spread in France, revealed mutations leading to cefiderocol resistance. Continued genomic surveillance of carbapenem-resistant Enterobacterales could clarify prevalence of cefiderocol-resistant E. coli in Europe.

Role of blaTEM and OmpC in the piperacillin-tazobactam resistance evolution by E. coli in patients with complicated intra-abdominal infection.

Piperacillin-tazobactam resistance (P/T-R) is increasingly reported among Escherichia coli isolates. Although in vitro experiments have suggested that blaTEM gene plays a key role in the P/T-R acquisition, no clinical in vivo study has yet confirmed the role of blaTEM or other genes. Therefore, we aimed to identify the mechanisms underlying P/T-R by following up patients with E. coli complicated intra-abdominal infections (cIAI) who experienced P/T treatment failure. Four pairs of strains, clonally related from four patients, were isolated both before and after treatment with P/T dosed at 4 g/0.5 g intravenously. The P/T MIC was tested using broth microdilution, and ß-lactamase activity was determined in these isolates. Whole-genome sequencing (WGS) was performed to decipher the role of blaTEM and other genes associated with P/T-R. Changes in the outer membrane protein (OMP) profile were analyzed using SDS-PAGE, and blaTEM and ompC transcription levels were measured by RT-qPCR. In addition, in vitro competition fitness was performed between each pairs of strains (P/T-susceptible vs. P/T-resistant). We found a higher copy number of blaTEM gene in P/T-R isolates, generated by three different genetic events: (1) IS26-mediated duplication of the blaTEM gene, (2) generation of a small multicopy plasmid (ColE-like) carrying blaTEM, and (3) adaptive evolution via reduction of plasmid size, leading to a higher plasmid copy number. Moreover, two P/T-R strains showed reduced expression of OmpC. This study describes the mechanisms involved in the acquisition of P/T-R by E. coli in patients with cIAI. The understanding of P/T-R evolution is crucial for effectively treating infected patients and preventing the spread of resistant microorganisms.