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.

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.

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.

In Vitro Susceptibility of Aztreonam-Vaborbactam, Aztreonam-Relebactam and Aztreonam-Avibactam Associations against Metallo-ß-Lactamase-Producing Gram-Negative Bacteria.

BACKGROUND: Despite the availability of new options (ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam and cefiderocol), it is still very difficult to treat infections caused by metallo-ß-lactamase (MBLs)-producers resistant to aztreonam. The in vitro efficacy of aztreonam in association with avibactam, vaborbactam or relebactam was evaluated on a collection of MBL-producing Enterobacterales, MBL-producing P. aeruginosa and highly drug-resistant S. maltophilia. METHODS: A total of fifty-two non-duplicate MBL-producing Enterobacterales, five MBL-producing P. aeruginosa and five multidrug-resistant S. maltophila isolates were used in this study. The minimum inhibitory concentrations (MICs) of aztreonam, meropenem-vaborbactam and imipenem-relebactam were determined by Etest® (bioMérieux, La Balme-les-Grottes) according to EUCAST recommendations. For aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam associations, the MICs were determined using Etest® on Mueller-Hinton (MH) agar supplemented with 8 mg/L of avibactam, 8 mg/L of vaborbactam and 4 mg/L of relebactam. The MICs were interpreted according to EUCAST guidelines. RESULTS: The susceptibility rates of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam with a standard exposure of aztreonam (1g × 3, IV) were 84.6% (44/52), 55.8% and 34.6% for Enterobacterales and 0% for all combinations for P. aeruginosa and S. maltophila. The susceptibility rates of aztreonam-avibactam, aztreonam-vaborbactam and aztreonam-relebactam with a high exposure of aztreonam (2g × 4, IV) were 92.3%, 78.9% and 57.7% for Enterobacterales, 75%, 60% and 60% for P. aeruginosa and 100%, 100% and 40% for S. maltophila. CONCLUSIONS: As previously demonstrated for an aztreonam/ceftazidime-avibactam combination, aztreonam plus imipenem-relebactam and aztreonam plus meropenem-vaborbactam might be useful options, but with potentially lower efficiency, to treat infections caused by aztreonam-non-susceptible MBL-producing Gram-negative strains.

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.

In Vitro Activity of Imipenem-Relebactam, Meropenem-Vaborbactam, Ceftazidime-Avibactam and Comparators on Carbapenem-Resistant Non-Carbapenemase-Producing Enterobacterales.

Background: Avibactam, relebactam and vaborbactam are ß-lactamase inhibitors that proved their efficiency against KPC-producing Enterobacterales. Regarding their inhibitor activity towards Ambler's class A extended spectrum ß-lactamases (ESBL) and Ambler's class C cephalosporinase (AmpC), they should be active on most of the carbapenem-resistant non-carbapenemase-producing Enterobacterales (CR non-CPE). Objectives: Determine the in vitro activity of ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam and comparators against CR non-CPE. Methods: MICs to ceftazidime/avibactam, imipenem/relebactam, meropenem/vaborbactam, but also temocillin, ceftolozane/tazobactam, ertapenem, colistin, eravacycline and tigecycline were determined by broth microdilution (ThermoFisher) on a collection of 284 CR non-CPE (inhibition zone diameter < 22 mm to meropenem). Whole genome sequencing was performed on 90 isolates to assess the genetic diversity as well as resistome. Results: According to EUCAST breakpoints, susceptibility rates of ceftazidime, imipenem, meropenem and ertapenem used at standard dose were 0.7%, 45.1%, 14.8% and 2.5%, respectively. Increased exposure of ceftazidime, imipenem and meropenem led to reach 3.5%, 68.3% and 67.7% susceptibility, respectively. Using the EUCAST clinical breakpoints, susceptibility rates of ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam were 88.4%, 81.0% and 80.6%, respectively. Susceptibility rates of temocillin, ceftolozane/tazobactam, tigecycline, eravacycline, and colistin were 0%, 4.6%, 27.8%, 54.9% and 90.1%. MICs distributions with and without the presence of the inhibitor demonstrated a better ability of avibactam and relebactam compared to vaborbactam to restore susceptibility to the associated ß-lactam. Conclusions: This study demonstrated the in vitro efficacy of ceftazidime/avibactam, imipenem/relebactam and to a lesser extent meropenem/vaborbactam against CR non-CPE. Moreover, to test all ß-lactams/ß-lactamases inhibitors combinations without a priori for CRE, non-CPE is crucial since resistance to one of the ß-lactam/ß-lactamase inhibitor combinations does not predict resistance to another molecule, depending on the resistance mechanisms involved.

Evaluation of ceftolozane-tazobactam susceptibility on a French nationwide collection of Enterobacterales.

OBJECTIVES: Ceftolozane-tazobactam (C/T) proved its efficacy for the treatment of infections caused by non-carbapenemase producing Pseudomonas aeruginosa and Enterobacterales. Here, we aimed to provide susceptibility data on a large series of Enterobacterales since the revision of EUCAST categorization breakpoints in 2020. METHODS: First, C/T susceptibility was determined on characterized Enterobacterales resistant to third generation cephalosporins (3GCs) (extended spectrum ß-lactamase [ESBL] production or different levels of AmpC overexpression) (n = 213) and carbapenem-resistant Enterobacterales (CRE) (n = 259), including 170 carbapenemase producers (CPE). Then, 1632 consecutive clinical Enterobacterales responsible for infection were prospectively collected in 23 French hospitals. C/T susceptibility was determined by E-test® (biomérieux) and broth microdilution (BMD) (Sensititre™, Thermo Scientific) to perform method comparison. RESULTS: Within the collection isolates, 88% of 3GC resistant strains were susceptible to C/T, with important variation depending on the resistance mechanism: 93% vs. 13% susceptibility for CTX-M and SHV-ESBL producers, respectively. Only 20% of the CRE were susceptible to C/T. Among CPE, 80% of OXA-48-like producers were susceptible to C/T, whereas all metallo-ß-lactamase producers were resistant. The prospective study revealed that 95.6% of clinical isolates were susceptible to C/T. Method comparison performed on these 1632 clinical isolates demonstrated 99% of categorization agreement between MIC to C/T determined by E-test® in comparison with the BMD (reference) and only 74% of essential agreement. CONCLUSION: Overall, C/T showed good activity against wild-type Enterobacterales, AmpC producers, and ESBL-producing Escherichia coli but is less active against ESBL-producing Klebsiella pneumoniae, and CRE. E-test® led to an underestimation of the MICs in comparison to the BMD reference.

Evaluation of a colorimetric test for the rapid detection of carbapenemase activity in Gram negative bacilli: the MAST® PAcE test.

The MAST® Carba PAcE test is a colorimetric test used to detect carbapenemase-producing Gram-negative bacilli from cultured colonies. The performances of this test were compared to ß-CARBA™, Carba NP test and RAPIDEC® CARBA NP tests using a collection of 280 characterized isolates. Sensitivity and specificity of the MAST® Carba PAcE test were 79.8% (95%IC: 73.3%-85.1%) and 98.9% (95%IC: 92.9%-99.9%). The MAST® Carba PAcE sensitivity was the lowest mainly due to interpretation difficulties (particularly OXA-48-like).

Polyclonal Dissemination of OXA-232 Carbapenemase-Producing Klebsiella pneumoniae, France, 2013-2021.

During 2013-2021, increased prevalence of oxacillinase 232-producing Enterobacterales was observed in France, mostly driven by its emergence in Klebsiella pneumoniae. Whole-genome sequencing identified that oxacillinase 232-producing K. pneumoniae belonged to 14 sequence types (STs), among which 2 polyclonal high-risk clones, ST-231 and ST-2096, were overrepresented.

Susceptibility of OXA-48-producing Enterobacterales to imipenem/relebactam, meropenem/vaborbactam and ceftazidime/avibactam.

Relebactam and vaborbactam are among the newest ß-lactamase inhibitors marketed. They were originally designed to inhibit the Ambler class A carbapenemase KPC. In this study, susceptibility to imipenem/relebactam and meropenem/vaborbactam was determined against a collection of OXA-48-like-producing Enterobacterales (n = 407). The clonality and resistomes of the isolates were determined by whole-genome sequencing. Comparison was performed with other relevant antibiotics such as carbapenems alone, ceftazidime/avibactam and ceftolozane/tazobactam. Addition of relebactam and vaborbactam did not significantly modify the MIC50 and MIC90 values obtained for imipenem and meropenem alone. In contrast, addition of avibactam strongly restored ceftazidime susceptibility. According to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, MIC50/MIC90 values were at 2/4, 2/4, 2/8, 2/8, 32/>32 and 0.5/2 mg/L for imipenem, imipenem/relebactam, meropenem, meropenem/vaborbactam, ceftazidime and ceftazidime/avibactam, respectively. No differences were observed depending on the species. This study highlights the lack of benefit in vitro for carbapenem/inhibitor combination compared with carbapenem alone against OXA-48-producing isolates as well as the difficulties in comparing molecules since carbapenem/inhibitor combinations were not developed with the same dosage of carbapenem.

Activity of mecillinam against carbapenem-resistant Enterobacterales.

BACKGROUND: Despite the fact that carbapenem-resistant Enterobacterales (CRE) mostly cause urinary tract infections (UTIs), only few studies have focused on the efficacity of mecillinam against these CRE. OBJECTIVES: To evaluate the mecillinam susceptibility of a huge collection of CRE, including carbapenemase-producing Enterobacterales (CPE) and non-CPE (ESBL and AmpC producers with decreased permeability of the outer membrane). METHODS: A total of 8310 non-duplicate clinical CRE, including 4042 OXA-48-like producers, 1094 NDM producers, 411 VIM producers, 174 KPC producers, 42 IMI producers, 153 multiple-carbapenemase producers and 45 isolates producing other types of carbapenemases (such as IMP-like enzymes or GES-5), were included in the study. WGS was performed on all CPE using Illumina technology. Categorization of susceptibility to mecillinam was performed using disc diffusion (mecillinam discs at 10 µg; I2A, France) according to EUCAST recommendations. The results were interpreted according to EUCAST guidelines (S ≥15 mm). RESULTS: Significantly higher susceptibility rates were observed for carbapenem-resistant Proteus spp. (85%) and carbapenem-resistant Escherichia coli (84%), which are the two most common species responsible for UTIs, than for Klebsiella pneumoniae (67%), Enterobacter cloacae complex (75%), Citrobacter spp. (65%), Serratia spp. (34%) and Morganella morganii (12%). Susceptibility rates were 84%, 71% and 91% for OXA-48-like, NDM and IMI producers and 70% for non-CPE CRE. Mecillinam was less active against VIM and KPC producers (14% and 0%, respectively). CONCLUSIONS: Mecillinam might be an alternative for the treatment of infections due to CRE, particularly UTIs, except for VIM and KPC producers and for M. morganii and Serratia spp species.

Comparison of disk diffusion, MIC test strip and broth microdilution methods for cefiderocol susceptibility testing on carbapenem-resistant enterobacterales.

OBJECTIVES: Cefiderocol is a catechol-substituted cephalosporin dedicated to the treatment of infections caused by multidrug resistant gram-negative rods. Cefiderocol susceptibility testing might be complex. We compared cefiderocol susceptibility testing methods on a relevant collection of carbapenem-resistant Enterobacterales. METHODS: CE-IVD (European CE marking required for all in vitro diagnostic (IVD)) broth microdilution (BMD) plate (ThermoFisher, Waltham, MA, USA) using regular Mueller-Hinton broth, MIC test strip (Liofilchem, Teramo, Italy), and disk diffusion (Liofilchem) were compared to a frozen BMD plate prepared with iron depleted Mueller-Hinton broth. First, a collection of 100 entirely sequenced carbapenem-resistant Enterobacterales was used to compare these methods. Then, a prospective comparison of disk diffusion and CE-IVD BMD was performed on 827 consecutive carbapenem non-susceptible Enterobacterales including 634 carbapenemase-producers. RESULTS: Compared to reference method, CE-IVD BMD plate gave 95.0% (95% CI, 88.8-97.9) categorisation agreement (CA), 2.8% (95% CI, 0.4-14.2) very major errors (VME), and 1.6% (95% CI, 0.3-8.7) major errors (ME) with high reproducibility. MIC strip gave only 63% (95% CI, 53.2-71.8) of CA and 94.9% (95% CI, 83.1-98.6) of VME due to critical underestimation of the MICs. Disk diffusion gave 77% (95% CI, 67.9-84.2) CA with additional 8% of the isolates within the area of technical uncertainty of 18-22 mm. Prospectively, disk diffusion gave 81.7% (95% CI, 79.0-84.2) CA, 23.3% (95% CI, 15.1-34.2%)VME, and 4.9% (95% CI, 3.6-6.7) ME. Additionally, 21.3% (95% CI, 18.6-24.2) of CRE were within the area of technical uncertainty. DISCUSSION: Commercial CE-IVD BMD (ThermoFisher) is accurate for cefiderocol MIC determination in difficult-to-treat Enterobacterales whereas MIC test strip (Liofilchem), that was formulated for Pseudomonas aeruginosa only, should be avoided. Disk diffusion might be useful for screening, but many of these CRE have to be re-tested using BMD to assess definitive categorization.

Emergence of VIM-producing Enterobacter cloacae complex in France between 2015 and 2018.

OBJECTIVES: To genetically characterize VIM-producing Enterobacter cloacae complex (ECC) isolates recovered in France from 2015 to 2018. METHODS: WGS, species determination, MLST, clonal relationship and genetic characterization were performed on 149 VIM-producing ECC isolates. RESULTS: Among VIM-producing Enterobacterales, the prevalence of ECC increased drastically from 6% in 2012 to 52% in 2018. The most prevalent species were Enterobacter hormaechei subsp. hoffmannii (40.9%), E. hormaechei subsp. steigerwaltii (21.5%), E. hormaechei subsp. xiangfangensis (14.8%) and ECC clade S (17.4%). Major STs were ST-873 (17.5%), ST-66 (12.1%), ST-78 (9.4%), ST-419 (8.1%), ST-145 (4.7%), ST-50 (4.0%), ST-118 (4.0%) and ST-168 (4.0%). Finally, six different integrons were identified, with some being specific to a given blaVIM variant (In916 with blaVIM-1-aacA4'-aphA15-aadA1-catB2 and In416 with blaVIM-4-aacA7-dfrA1b-aadA1b-smr2 genes). CONCLUSIONS: This study demonstrated the genetic diversity among VIM-producing ECC isolates, indicating that their spread is not linked to a single clone.

Outbreak of CTX-M-15 Extended-Spectrum ß-Lactamase-Producing Klebsiella pneumoniae ST394 in a French Intensive Care Unit Dedicated to COVID-19.

Infections caused by extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (ESBL-KP) are constantly rising worldwide and are often reported as causative agent of outbreaks in intensive care units (ICUs). During the first wave of the COVID-19 pandemic, bacterial cross-transmission was thought unlikely to occur due to the reinforcement of hygiene measures and prevention control. However, we report here an ESBL-producing K. pneumoniae (ST394) isolate responsible for a nosocomial outbreak in an ICU dedicated to COVID-19 patients.