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.

Neonatal sepsis due to NDM-1 and VIM-2 co-producing Pseudomonas aeruginosa in Morocco.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa are being increasingly described worldwide. Here, we investigated the molecular mechanisms underlying carbapenem resistance in an extremely drug-resistant P. aeruginosa isolate from a neonatal intensive care unit in Morocco. MATERIALS AND METHODS: P. aeruginosa strain O82J1 was identified using MALDI-TOF-MS. Carba NP, immunochromatographic assay NG Carba5 and antimicrobial susceptibility testing using disc diffusion and microbroth were performed. Whole-genome sequencing using the Illumina and MinION technologies and different software packages available at the Center of Genomic Epidemiology were used to predict the resistome, sequence type and plasmid types. RESULTS: P. aeruginosa O82J1 co-expressed two metallo-ß-lactamases, blaNDM-1 and blaVIM-2, and was susceptible to colistin and apramycin only. It belonged to ST773 that is frequently reported worldwide as a high-risk P. aeruginosa clone. The blaVIM-2 gene was integron-borne on a IncP-2 465-kb plasmid, whereas the blaNDM-1 gene was chromosomally encoded and embedded in an integrative conjugative element, probably at the origin of its acquisition. A total of 23 antimicrobial resistance genes were detected including a blaPER-1 ESBL gene, and an 16S-rRNA methyltransferase gene rmtB. CONCLUSIONS: The isolation of XDR P. aeruginosa isolates expressing several carbapenemases in a neonatal intensive care unit is of great concern due to the reduced treatment options, relying only on colistin, but not recommended in neonates, and apramycin, not yet approved for human therapy. Concerns were further elevated due to the resistance to cefiderocol and ATM/AVI, two novel and last-resort antibiotics recommended to treat infections caused by Gram-negative bacteria, particularly XDR P. aeruginosa in adults.

Using artificial intelligence to improve COVID-19 rapid diagnostic test result interpretation.

Serological rapid diagnostic tests (RDTs) are widely used across pathologies, often providing users a simple, binary result (positive or negative) in as little as 5 to 20 min. Since the beginning of the COVID-19 pandemic, new RDTs for identifying SARS-CoV-2 have rapidly proliferated. However, these seemingly easy-to-read tests can be highly subjective, and interpretations of the visible "bands" of color that appear (or not) in a test window may vary between users, test models, and brands. We developed and evaluated the accuracy/performance of a smartphone application (xRCovid) that uses machine learning to classify SARS-CoV-2 serological RDT results and reduce reading ambiguities. Across 11 COVID-19 RDT models, the app yielded 99.3% precision compared to reading by eye. Using the app replaces the uncertainty from visual RDT interpretation with a smaller uncertainty of the image classifier, thereby increasing confidence of clinicians and laboratory staff when using RDTs, and creating opportunities for patient self-testing.

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.

Structural and Biochemical Features of OXA-517: a Carbapenem and Expanded-Spectrum Cephalosporin Hydrolyzing OXA-48 Variant.

OXA-48-producing Enterobacterales have now widely disseminated throughout the world. Several variants have now been reported, differing by just a few amino-acid substitutions or deletions, mostly in the region of the loop ß5-ß6. As OXA-48 hydrolyzes carbapenems but lacks significant expanded-spectrum cephalosporin (ESC) hydrolytic activity, ESCs were suggested as a therapeutic option. Here, we have characterized OXA-517, a natural variant of OXA-48- with an Arg214Lys substitution and a deletion of Ile215 and Glu216 in the ß5-ß6 loop, capable of hydrolyzing at the same time ESC and carbapenems. MICs values of E. coli expressing blaOXA-517 gene revealed reduced susceptibility to carbapenems (similarly to OXA-48) and resistance to ESCs. Steady-state kinetic parameters revealed high catalytic efficiencies for ESCs and carbapenems. The blaOXA-517 gene was located on a ca. 31-kb plasmid identical to the prototypical IncL blaOXA-48-carrying plasmid except for an IS1R-mediated deletion of 30.7-kb in the tra operon. The crystal structure of OXA-517, determined to 1.86 Å resolution, revealed an expanded active site compared to that of OXA-48, which allows for accommodation of the bulky ceftazidime substrate. Our work illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutation/deletion in the ß5-ß6 loop to extend its hydrolysis profile to encompass most ß-lactam substrates.

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.

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.

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.

Emergence and Polyclonal Dissemination of OXA-244-Producing Escherichia coli, France.

Since 2016, OXA-244-producing Escherichia coli has been increasingly isolated in France. We sequenced 97 OXA-244-producing E. coli isolates and found a wide diversity of sequence types and a high prevalence of sequence type 38. Long-read sequencing demonstrated the chromosomal location of blaOXA-244 inside the entire or truncated Tn51098.

Evaluating 10 Commercially Available SARS-CoV-2 Rapid Serological Tests by Use of the STARD (Standards for Reporting of Diagnostic Accuracy Studies) Method.

Numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid serological tests have been developed, but their accuracy has usually been assessed using very few samples, and rigorous comparisons between these tests are scarce. In this study, we evaluated and compared 10 commercially available SARS-CoV-2 rapid serological tests using the STARD (Standards for Reporting of Diagnostic Accuracy Studies) methodology. Two hundred fifty serum samples from 159 PCR-confirmed SARS-CoV-2 patients (collected 0 to 32 days after the onset of symptoms) were tested with rapid serological tests. Control serum samples (n = 254) were retrieved from pre-coronavirus disease (COVID) periods from patients with other coronavirus infections (n = 11), positivity for rheumatoid factors (n = 3), IgG/IgM hyperglobulinemia (n = 9), malaria (n = 5), or no documented viral infection (n = 226). All samples were tested using rapid lateral flow immunoassays (LFIAs) from 10 manufacturers. Only four tests achieved ≥98% specificity, with the specificities ranging from 75.7% to 99.2%. The sensitivities varied by the day of sample collection after the onset of symptoms, from 31.7% to 55.4% (days 0 to 9), 65.9% to 92.9% (days 10 to 14), and 81.0% to 95.2% (>14 days). Only three of the tests evaluated met French health authorities' thresholds for SARS-CoV-2 serological tests (≥90% sensitivity and ≥98% specificity). Overall, the performances varied greatly between tests, with only one-third meeting acceptable specificity and sensitivity thresholds. Knowledge of the analytical performances of these tests will allow clinicians and, most importantly, laboratorians to use them with more confidence; could help determine the general population's immunological status; and may help diagnose some patients with false-negative real-time reverse transcription-PCR (RT-PCR) results.

Development and validation of a lateral flow immunoassay for rapid detection of VanA-producing enterococci.

BACKGROUND: VRE are nosocomial pathogens with an increasing incidence in recent decades. Rapid detection is crucial to reduce their spread and prevent infections and outbreaks. OBJECTIVES: To evaluate a lateral flow immunoassay (LFIA) (called NG-Test VanA) for the rapid and reliable detection of VanA-producing VRE (VanA-VRE) from colonies and broth. METHODS: NG-Test VanA was validated on 135 well-characterized enterococcal isolates grown on Mueller-Hinton (MH) agar (including 40 VanA-VRE). Different agar plates and culture broths widely used in routine laboratories for culture of enterococci were tested. RESULTS: All 40 VanA-VRE clinical isolates were correctly detected in less than 15 min irrespective of the species expressing the VanA ligase and the medium used for bacterial growth. No cross-reaction was observed with any other clinically relevant ligases (VanB, C1, C2, D, E, G, L, M and N). Overall, the sensitivity and specificity of the assay were 100% for VanA-VRE grown on MH agar plates. NG-Test VanA accurately detects VanA-VRE irrespective of the culture medium (agar and broth). Band intensity was increased when using bacteria grown on vancomycin-containing culture media or on MH close to the vancomycin disc as a consequence of VanA induction. The limit of detection of the assay was 6.3 × 106 cfu per test with bacteria grown on MH plates and 4.9 × 105 cfu per test with bacteria grown on ChromID® VRE plates. CONCLUSIONS: NG-Test VanA is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of VanA-VRE.

Optimization of the rapid carbapenem inactivation method for use with AmpC hyperproducers.

OBJECTIVES: Detection of carbapenemase-producing Enterobacterales (CPEs) is sometimes difficult with AmpC-hyperproducing Enterobacterales (AHEs), as they may falsely be classified as CPEs. Here, we present a rapid Carbapenem Inactivation Method (rCIM) optimized for AmpC producers (rCIM-A) that allows rapid and easy discrimination between AHEs and CPEs. METHODS: Enterobacterales (n = 249), including natural AmpC producers, AHEs, CPEs and non-carbapenemase-producing carbapenem-resistant control strains were evaluated, using Carba NP, rCIM and rCIM-A. The rCIM-A differs from the rCIM by the addition of cloxacillin (400 µg/mL) to the initial antibiotic incubation step. RESULTS: The rCIM-A yielded a sensitivity and specificity of 84.26% (95% CI: 76.00%-90.55%) and 99.29% (95% CI: 96.11%-99.98%), respectively, while those of the rCIM were 86.11% (95% CI: 78.13%-92.01%) and 80.85% (95% CI: 73.38%-86.99%), respectively; those of Carba NP were lower at 84.04% (95% CI: 75.05%-90.78%) and 91.37% (95% CI: 85.41%-95.46%), respectively, due to indeterminate results. The rCIM-A was capable of discriminating between AHEs and true CPEs, but still failed to identify OXA-23-producing Proteus mirabilis isolates and remained only partially reliable for identifying IMI-like producers and a few MBL (2 NDM-1, 1 LMB-1, 1 TMB-1 and 1 IMP-13) producers. One chromosomally encoded AmpC variant, MIR-10, gave repeatedly positive results using all three tests and was thus considered a false positive. CONCLUSIONS: Specificity for AHEs greatly improved with the rCIM-A without altering the test performance for the other resistance mechanisms. It may replace the rCIM as a cheap, easy, rapid and accurate CPE detection test.

First Occurrence of the OXA-198 Carbapenemase in Enterobacterales.

A carbapenem-resistant Citrobacter sp. was recovered from routine screening of multidrug-resistant bacteria. This isolate coproduced OXA-48 and OXA-198. OXA-48 was carried by the prototypical IncL plasmid, whereas OXA-198 was carried by a peculiar IncHI-type plasmid. This carbapenemase gene was inserted within a class 1 integron located on a conjugative plasmid. This report describes the first occurrence of OXA-198 in Enterobacterales.

Evaluation of the Revogene Carba C Assay for Detection and Differentiation of Carbapenemase-Producing Gram-Negative Bacteria.

The Revogene Carba C assay (formerly GenePOC Carba assay) is a multiplex nucleic acid-based in vitro diagnostic test intended for the detection of carbapenemase-producing Enterobacterales (CPE) from cultured colonies. This assay was evaluated directly on colonies of 118 well-characterized Enterobacterales with reduced susceptibility to carbapenems and on 49 multidrug-resistant (MDR) Pseudomonas aeruginosa and 40 MDR Acinetobacter baumannii isolates. The Revogene Carba C assay's performance was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). In Enterobacterales, sensitivity and specificity were 100%. When extrapolating the results to the French CPE epidemiology between 2012 and 2018, this assay would have detected 99.28% of the 9,624 CPE isolates sent to the French NRC, missing 69 CPE isolates (2 GES-5, 10 OXA-23, 2 TMB-1, 1 SME-4, 53 IMI, and 1 FRI). The overall sensitivity and specificity for CP P. aeruginosa were 93.7 and 100%, respectively, as two rare IMP variants (IMP-31 and -46) were not detected. Extrapolating these results to the French epidemiology of CP P. aeruginosa in 2017, 93.3% would have been identified, missing only 1 DIM and 10 GES variants. The Revogene Carba C assay accurately identified the targeted carbapenemase genes in A. baumannii, but when extrapolating these results to the French CP A. baumannii epidemiology of 2017, only 12.50% of them could be detected, as OXA-23 is the most prevalent carbapenemase in CP A. baumannii The Revogene Carba C assay showed excellent sensitivity and specificity for the five most common carbapenemases regardless of the bacterial host. It is well adapted to the CPE and CP P. aeruginosa epidemiology of many countries worldwide, which makes it suitable for use in the routine microbiology laboratory, with a time to result of ca. 85 min for eight isolates simultaneously.

Screening of OXA-244 producers, a difficult-to-detect and emerging OXA-48 variant?

BACKGROUND: OXA-244, a single amino acid variant of OXA-48, demonstrates weaker hydrolytic activity towards carbapenems and temocillin compared with OXA-48. Of note, these antimicrobials are present in high concentrations in several carbapenemase-producing Enterobacterales (CPE) screening media. As a result, some screening media fail to grow OXA-244-producing isolates, while the prevalence of OXA-244 producers is constantly increasing in France. METHODS: Here, we evaluate the performance of three commercially available CPE screening media [ChromID® CARBA SMART (bioMérieux), Brilliance™ CRE (Thermo Fisher) and mSuperCARBA™ (MAST Diagnostic)] for their ability to detect OXA-244 producers (n = 101). As OXA-244 producers may also express an ESBL, two additional ESBL screening media were tested (Brilliance™ ESBL and ChromID® BLSE). MICs of temocillin and imipenem were determined by broth microdilution. The clonality of OXA-244-producing Escherichia coli isolates (n = 97) was assessed by MLST. RESULTS: Overall, the sensitivity of the ChromID® CARBA SMART, Brilliance™ CRE and mSuperCARBA™ media were 14% (95% CI = 8.1%-22.5%), 54% (95% CI = 43.3%-63.4%) and 99% (95% CI = 93.8%-100%), respectively, for the detection of OXA-244 producers. Among the 101 OXA-244-producing isolates, 96% were E. coli and 77%-78% grew on ESBL screening media. MLST analysis identified five main STs among OXA-244-producing E. coli isolates: ST38 (n = 37), ST361 (n = 17), ST69 (n = 12), ST167 (n = 11) and ST10 (n = 8). CONCLUSIONS: Our results demonstrated that the mSuperCARBA™ medium is very efficient in the detection of OXA-244 producers, unlike the ChromID® CARBA SMART medium. The high prevalence of ESBLs among OXA-244 producers allowed detection of 77%-78% of them using ESBL-specific screening media.

Concomitant carriage of KPC-producing and non-KPC-producing Klebsiella pneumoniae ST512 within a single patient.

BACKGROUND: KPC-producing Klebsiella pneumoniae of clonal group 258 are prominent in healthcare settings in many regions of the world. The blaKPC gene is mostly carried by a multireplicon IncFIIk-IncFI plasmid suspected to be highly compatible and stable in this genetic background. Here, we analysed the genetic diversity of an ST512 K. pneumoniae population in a single patient. METHODS: Twelve K. pneumoniae isolates (n = 5 from urine samples and n = 7 from rectal swabs) were recovered from one patient over a 2 month period. Antimicrobial susceptibility testing, plasmid extraction and WGS were performed on all isolates. The first K. pneumoniae isolate, D1, was used as a reference for phylogenetic analysis. RESULTS: Antimicrobial susceptibility testing, plasmid analysis and WGS revealed concomitant carriage of carbapenem-resistant and carbapenem-susceptible K. pneumoniae isolates of ST512, with the absence of the entire blaKPC-carrying plasmid in the susceptible population. Furthermore, 14 other genetic events occurred within the genome, including 3 chromosomal deletions (of 71 kb, 33 kb and 11 bp), 2 different insertions of ISKpn26 and 9 SNPs. Interestingly, most of the events occurred in the same chromosomal region that has been deleted independently several times, probably after homologous recombination involving 259 bp repeated sequences. CONCLUSIONS: Our study revealed (to the best of our knowledge) the first case of in vivo blaKPC-carrying plasmid curing and a wide within-patient genetic diversity of a single K. pneumoniae ST512 clone over a short period of carriage. This within-patient diversity must be taken into account when characterizing transmission chains using WGS during nosocomial outbreaks.

Comparison of the Superpolymyxin and ChromID Colistin R Screening Media for the Detection of Colistin-Resistant Enterobacteriaceae from Spiked Rectal Swabs.

The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) has led to the increased use of colistin, which has resulted in the emergence of colistin-resistant Enterobacteriaceae worldwide. One of the most threatening scenarios is the dissemination of colistin resistance in CPE, particularly the plasmid-encoded resistance element MCR. Thus, it has now become mandatory to possess reliable media to screen for colistin-resistant Gram-negative bacterial isolates, especially Enterobacteriaceae In this study, we evaluated the performances of the Superpolymyxin medium (ELITechGroup) and the ChromID Colistin R medium (bioMérieux) to screen for colistin-resistant Enterobacteriaceae from spiked rectal swabs. Stool samples were spiked with a total of 94 enterobacterial isolates (Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Enterobacter cloacae), including 53 colistin-resistant isolates. ESwabs (Copan Diagnostics) were then inoculated with those spiked fecal suspensions, and culture proceeded as recommended by both manufacturers. The sensitivity of detection of colistin-resistant Enterobacteriaceae was 86.8% (95% confidence interval [95% CI] = 74.0% to 94.0%) using both the Superpolymyxin medium and the ChromID Colistin R plates. Surprisingly, the isolates that were not detected were not the same for both media. The specificities were high for both media, at 97.9% (95% CI = 87.3% to 99.9%) for the Superpolymyxin medium and 100% (95% CI = 90.4% to 100%) for the ChromID Colistin R medium. Both commercially available media, ChromID Colistin R and Superpolymyxin, provide useful tools to screen for colistin-resistant Enterobacteriaceae from patient samples (rectal swabs) regardless of the level and mechanism of colistin resistance.

Improvement of the Immunochromatographic NG-Test Carba 5 Assay for the Detection of IMP Variants Previously Undetected.

Here, we evaluated the immunochromatographic assay NG-Test Carba 5v2 (NG-Biotech), with improved IMP variant detection on 31 IMP producers, representing the different branches of the IMP phylogeny, including 32 OXA-48, 19 KPC, 12 VIM, 14 NDM, and 13 multiple carbapenemase producers (CPs), 13 CPs that were not targeted, and 13 carbapenemase-negative isolates. All tested IMP variants were accurately detected without impairing detection of the other carbapenemases. Thus, NG-Test Carba 5v2 is now well adapted to countries with high IMP prevalence and to the epidemiology of CP-Pseudomonas aeruginosa, where IMPs are most frequently detected.