Carbapenemases In Enterobacterales Research Articles

OXA-48-like carbapenemases in Proteus mirabilis - novel genetic environments and a challenge for detection

OXA-48-like enzymes represent the most frequently detected carbapenemases in Enterobacterales in Western Europe, North Africa and the Middle East. In contrast to other species, the presence of OXA-48-like in Proteus mirabilis leads to an unusually susceptible phenotype with low MICs for carbapenems and piperacillin-tazobactam, which is easily missed in the diagnostic laboratory. So far, there is little data available on the genetic environments of the corresponding genes, bla OXA-48-like, in P. mirabilis. In this study susceptibility phenotypes and genomic data of 13 OXA-48-like-producing P. mirabilis were investigated (OXA-48, n=9; OXA-181, n=3; OXA-162, n=1). Ten isolates were susceptible to meropenem and ertapenem and three isolates were susceptible to piperacillin-tazobactam. The gene bla OXA-48 was chromosomally located in 7/9 isolates. Thereof, in three isolates bla OXA-48 was inserted into a P. mirabilis genomic island. Of the three isolates harbouring bla OXA-181 one was located on an IncX3 plasmid and two were located on a novel MOBF plasmid, pOXA-P12, within the new transposon Tn7713. In 5/6 isolates with plasmidic location of bla OXA-48-like, the plasmids could conjugate to E. coli recipients in vitro. Vice versa, bla OXA-48-carrying plasmids could conjugate from other Enterobacterales into a P. mirabilis recipient. These data show a high diversity of bla OXA-48-like genetic environments compared to other Enterobacterales, where genetic environments are quite homogenous. Given the difficult-to-detect phenotype of OXA-48-like-producing P. mirabilis and the location of bla OXA-48-like on mobile genetic elements, it is likely that OXA-48-like-producing P. mirabilis can disseminate, escape most surveillance systems, and contribute to a hidden spread of OXA-48-like.

Carbapenem resistance determinants and their transmissibility among clinically isolated Enterobacterales in Lebanon

BackgroundThe occurrence of carbapenem-resistant bacterial infections has increased significantly over the years with Gram-negative bacteria exhibiting the broadest resistance range. In this study we aimed to investigate the genomic characteristics of clinical carbapenem-resistant Enterobacterales (CRE). MethodsSeventeen representative multi-drug resistant (MDR) isolates from a hospital setting showing high level of resistance to carbapenems (ertapenem, meropenem and imipenem) were chosen for further characterization through whole-genome sequencing. Resistance mechanisms and transferability of plasmids carrying carbapenemase-encoding genes were also determined in silico and through conjugative mating assays. ResultsWe detected 18 different β-lactamases, including four carbapenemases (blaNDM-1, blaNDM-5, blaNDM-7, blaOXA-48) on plasmids with different Inc groups. The combined results from PBRT and in silico replicon typing revealed 20 different replicons linked to plasmids ranging in size between 80 and 200 kb. The most prevalent Inc groups were IncFIB(K) and IncM. OXA-48, detected on 76-kb IncM1 conjugable plasmid, was the most common carbapenemase. We also detected other conjugative plasmids with different carbapenemases confirming the role of horizontal gene transfer in the dissemination of antimicrobial resistance genes. ConclusionOur findings verified the continuing spread of carbapenemases in Enterobacterales and revealed the types of mobile elements circulating in a hospital setting and contributing to the spread of resistance determinants. The occurrence and transmission of plasmids carrying carbapenemase-encoding genes call for strengthening active surveillance and prevention efforts to control antimicrobial resistance dissemination in healthcare settings.

Prevalence of carbapenemases in Enterobacterales from urine specimens in anuniversity hospital in Istanbul, Turkey.

We aimed to investigate the prevalence of carbapenemases in Enterobacterales strains isolated from urine specimens between July 2019 and July 2020.CIM and modified CIM tests were applied as well as detection of blaOXA-48, blaNDM, blaVIM, blaKPC and blaIMP genes was performed by multiplex PCR.One hundred fifty of 3,242 Enterobacterales strains were found to be carbapenem resistant and 46 were included in the study. Forty five (98%) of the 46 strains included in the study were Klebsiella spp. and one (2%) of them was Escherichia coli. Susceptibility to ceftazidime-avibactam, amikacin and gentamicin was 97%, 11% and 9%, respectively. Forty three (94%) isolates were found positive at 2 and 4 h with CIM test. Forty four (97%) strains were found positive at 4 h and 43 (94%) strains were found positive at 2 h with modified CIM test.While blaOXA-48, blaNDM and blaOXA-48 with blaNDM association were found in Klebsiella spp. isolates in 55%, 27% and 11%, respectively, blaVIM, blaKPC, blaIMP were not found. Only blaOXA-48 and blaNDM-1 were detected in the E. coli strain.None of the investigated genes were detected in three Klebsiella strains but with whole genome analysis the combination of blaOXA-534, blaCMY-99 and blaKPC-3 was found in the first strain, blaOXA-370 in the second strain and no resistance gene was found in the third strain.Ceftazidime-avibactam was found to be active against 97% of strains, and the most common resistance genes were blaOXA-48 and blaNDM-1. Previously undetected resistance genes have been identified in our country.

Incidence of ESBLs and carbapenemases among Enterobacterales and carbapenemases in Pseudomonas aeruginosa isolates collected globally: results from ATLAS 2017-2019.

To assess the global and regional distribution of ESBLs in Enterobacterales and carbapenemases in Enterobacterales and Pseudomonas aeruginosa. Antimicrobial susceptibility of isolates collected from ATLAS (2017-2019) was determined per CLSI guidelines. Enterobacterales exhibiting meropenem MICs ≥2 mg/L and/or ceftazidime/avibactam and/or aztreonam/avibactam MICs ≥16 mg/L, Escherichia coli and Klebsiella pneumoniae with aztreonam and/or ceftazidime MICs ≥2 mg/L, and P. aeruginosa with meropenem MICs ≥4 mg/L were screened for β-lactamases by PCR and sequencing. Globally, ESBL-positive E. coli (23.7%, 4750/20047) and K. pneumoniae (35.1%, 6055/17229) carried predominantly the CTX-M-15 variant (E. coli: 53.9%; K. pneumoniae: 80.0%) with highest incidence in Africa/Middle East (AfME). Among carbapenem-resistant (CR) E. coli (1.1%, 217/20047) and Enterobacter cloacae (3.8%, 259/6866), NDMs were predominant (E. coli in AfME: 62.5%; E. cloacae in Asia Pacific: 59.7%). CR K. pneumoniae (13.3%, 2299/17 229) and P. aeruginosa (20.3%, 4187/20 643) carried predominantly KPC (30.9%) and VIM (14.7%), respectively, with highest frequency in Latin America. Among ESBL-positive Enterobacterales, susceptibility to ceftazidime/avibactam (>90.0%) and amikacin (>85.0%) was higher than to piperacillin/tazobactam (>45.0%) and ciprofloxacin (>7.4%). In CR Enterobacterales, susceptibility to amikacin (>54.0%) and ceftazidime/avibactam (>31.0%) was higher than to ciprofloxacin (>2.7%) and piperacillin/tazobactam (>0.5%). CR P. aeruginosa similarly demonstrated higher susceptibility to amikacin (63.4%) and ceftazidime/avibactam (61.9%) than to ciprofloxacin (26.2%) and piperacillin/tazobactam (25.3%). Varied distribution of resistance genotypes across regions among ESBL-positive Enterobacterales and CR Enterobacterales and P. aeruginosa provide crucial insights on major resistance mechanisms and trends observed in recent years. Continued surveillance is warranted for monitoring global dissemination and resistance.

Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city

The emergence of extended-spectrum β-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10−1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Comparison of three lateral flow immunochromatographic assays for the rapid detection of KPC, NDM, IMP, VIM and OXA-48 carbapenemases in Enterobacterales.

Comparison of three lateral flow immunochromatographic assays for the rapid detection of KPC, NDM, IMP, VIM and OXA-48 carbapenemases in Enterobacterales.

Detecting KPC-2 and NDM-1 Coexpression in Klebsiella pneumoniae Complex from Human and Animal Hosts in South America.

ABSTRACTReports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for “CZA 50” (30 µg ceftazidime [CAZ] and 20 µg AVI) and “CZA 14” (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide.IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.

Detection and Characterization of Carbapenemases in Enterobacterales With a New Rapid and Simplified Carbapenemase Detection Method Called rsCDM.

ObjectiveThis study aimed to develop a new rapid and simplified carbapenemase detection method (rsCDM) for detection and characterization of carbapenemase with 3-aminophenylboronic acid (APBA), ethylenediaminetetraacetic acid (EDTA), and cloxacillin (CLO) β-lactamase inhibitors.MethodsA panel of 182 carbapenem-resistant Enterobacterales (CRE) strains with blaKPC (88), blaNDM (60), blaIMP (10), blaVIM (3), blaOXA-181 (5), blaKPC, and blaNDM (7), porin changes in combination with an extended-spectrum β-lactamase (ESBL) (3) or AmpC hyper-production (6) and 43 carbapenem-susceptible Enterobacterales isolates were used to evaluate the performance of rsCDM and EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined with specific inhibitors at 4, 6, and 18 h by rsCDM, and the difference between imipenem (IMI) and meropenem (MEM) disks was simultaneously compared.ResultsThe sensitivity of rsCDM using IMI was 97.1% at the three time points, with a specificity of 100%, independent of the culture duration. Similar to IPM, MEM disk also showed high sensitivity (97.1%) and specificity (100%) at 6 h. And the sensitivity of eCIM was 95.4% and the specificity was 100%. Based on a decision algorithm, the characterization number of IMI and MEM in KPC-producing isolates was 88 vs. 87, metallo-β-lactamases (MBLs) was 73 vs. 72, KPC and NDM carbapenemase was 7 vs. 7 at 4 h, respectively. After 6 h, the category number changed insignificantly except for isolates with combined AmpC overproduction and porin changes, showing an increase in IMI (6) and MEM (2), and there was no difference in the results between 6 and 18 h for the two tablets. OXA-181-producing strains can’t be distinguished by rsCDM. For eCIM, the characterization number in KPC-, OXA- 181-, and MBLs-producing strains was 88, 5, and 72, but it failed to detect multi-enzyme-producing isolates (KPC and NDM).ConclusionrsCDM accurately discriminated carbapenemase within 4 h and could differentiate multi-enzyme (KPC and NDM) and AmpC in conjunction with porin changes strains. Hence, rsCDM represents a rapid, simple, easy readout, and accurate tool that can be used without any specialized equipment.

1251. Comparative Evaluation of Phenotypic Synergy Tests vs. Resist-4 O.K.N.V® and NG Test Carba 5® Lateral Flow Immunoassays for the Detection and Differentiation of Carbapenemases in Enterobacterales and Pseudomonas aeruginosa

BackgroundThe spread of carbapenem resistant Pseudomonas aeruginosa and carbapenemase-producing Enterobacterales (CPE) has had a great impact on morbidity and mortality. COVID-19 pandemic has favoured the selection of these microorganisms because of the excessive and prolonged use of broad-spectrum antibiotics and the outbreaks related to patient transfer between hospitals and inadequate use of personal protective equipment. Therefore, detection is considered essential for their control. Our aim was to compare conventional phenotypic synergy tests and two lateral flow immunoassays for detecting carbapenemases in Enterobacterales and P. aeruginosa.MethodsWe analysed 100 carbapenem-resistant Gram-negative bacilli isolates, 80 Enterobacterales and 20 Pseudomonas aeruginosa, (86 isolates producing KPC, NDM, OXA-48, IMP and VIM carbapenemases and 14 non-carbapenemase-producing isolates). We performed a modified Hodge test, boronic acid and ethylenediaminetetraacetic acid (EDTA) synergy tests, and two lateral flow immunoassays: RESIST-4 O.K.N.V (Coris Bioconcept®) and NG Test Carba 5® (NG Biotech®).ResultsIn total, 76 KPC, 7 VIM, 1 NDM, 1 OXA-48 and 1 isolate coproducing KPC + NDM enzymes were included. The concordance of different methods estimated by Kappa index was 0.432 (Standard error: 0.117), thus showing a high variability with the synergy tests with boronic acid and EDTA and reporting 16 false negatives that were detected by the two immunochromatographic methods. Co-production was only detected using immunoassays. Figure 1. Errors in synergy tests. 1) KPC-producing K. pneumoniae not detected with boronic acid and EDTA (1a). Positive result adjusting distance to 5 mm (1b) and positive result by NG Test Carba 5® and RESIST-4 O.K.N.V® immunoassays (1c). 2) VIM -producing P. aeruginosa with inconclusive EDTA result (2a), Positive result adjusting distance to 5 mm (2b), confirmation of VIM by immunoassay (2c). 3) K. pneumoniae with KPC and NDM co-production without synergistic effect with boronic acid or EDTA (3a), positivity for both enzymes by immunoassay (3b). 4) Impact of distance between disks: False negative for serine-carbapenemases with 15 mm (4a) positive result at 10 mm (4b). Differences in boronic acid tests for serin-carbapenemases at 10 mm, 5 mm and 0 mm distance (4c, 4d). Differences in synergy between imipenem, meropenem and ertapenem discs at 0 mm distance (4e).Table 2. Results of synergy methods and immunoassay tests for Enterobacterales and Pseudomonas aeruginosa MHTModified Hodge Test, APB: boronic acid synergy; EDTA: EDTA synergy; Pos: positive; Neg: negative. KPC (Klebsiella pneumoniae Carbapenemase), VIM (Verona integron-mediated metallo- β-lactamase), NDM (New Delhi metallo-β-lactamase), OXA (oxacillinase-48-like carbapenemase (OXA-48))ConclusionConventional phenotypic synergy tests with boronic acid and EDTA used for detecting carbapenemases are suboptimal and their routine use should be reconsidered. They depend on the degree of enzyme expression and the distance between disks. Lateral flow immunoassay tests are a rapid and cost-effective tool to detect and differentiate carbapenemases, improving clinical outcomes through targeted therapy and promoting infection prevention measures. Disclosures Diego Josa, Msc, ALIFAX (Speaker’s Bureau) German Esparza, n/a, Biomerieux (Consultant)Pfizer (Speaker’s Bureau) Luis Reyes, n/a, MSD (Speaker’s Bureau)

Evaluation of the rCIM for carbapenemase detection in Enterobacterales and Pseudomonas aeruginosa and description of the TSBrCIM, an optimized variant

ObjectivesThe rapid Carbapenem Inactivation Method (rCIM) was evaluated with a strain collection of 164 and 69 carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa, respectively, that produced various carbapenemases. For an improved carbapenemase detection in Enterobacterales, an optimized variant of the rCIM named TSBrCIM was developed. MethodsBacterial isolates were incubated with two meropenem disks in distilled water (rCIM) or tryptic soy broth (TSBrCIM). After centrifugation, the supernatant was incubated with a susceptible E. coli indicator strain in tryptic soy broth. Growth of the indicator strain implied carbapenemase activity in the test strain. ResultsThe rCIM detected 100/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 88.5% and a specificity of 94.1%. For P. aeruginosa, sensitivity and specificity were 96.0% and 100%, respectively. The TSBrCIM was able to detect 105/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 92.9% and a specificity of 96.1%. ConclusionThis study shows that the TSBrCIM can be valuable tool for detection of carbapenemases in Enterobacterales in the clinical laboratory, while the rCIM showed the best results for carbapenemase detection in P. aeruginosa.

Comparison of Etest and broth microdilution for evaluating the susceptibility of Staphylococcus aureus and Streptococcus pneumoniae to ceftaroline and of carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa to ceftazidime/avibactam

Decreased susceptibility to ceftazidime/avibactam (CZA) and ceftaroline (CPT) has been reported during antimicrobial resistance surveillance and therapy. Conventional laboratories are unable to provide timely susceptibility testing for CZA and CPT because these antimicrobial agents are not incorporated in automated susceptibility testing systems. We evaluated Etest and the Sensititre broth microdilution (BMD) method for testing CZA against carbapenem-resistant Gram-negative bacilli and CPT against important Gram-positive cocci bloodstream isolates. Genotypes of carbapenemases in Enterobacterales were also determined using the Xpert® Carba-R assay. Etest showed ≥90% agreement with Sensititre BMD for carbapenem-resistant Klebsiella pneumoniae (CRKP) (n=187), carbapenem-resistant Escherichia coli (CREC) (n=28) and Streptococcus pneumoniae (n=35); however, the very major error rate exceeded 3%. Agreement between Etest and Sensititre BMD was <90% for carbapenem-resistant Pseudomonas aeruginosa (CRPA) (n=81), methicillin-susceptible Staphylococcus aureus (MSSA) (n=92) and methicillin-resistant S. aureus (MRSA) (n=170). Both agents remained potent with a high susceptibility rate by Sensititre BMD as follows: CZA against CRKP (95.0%), CREC (89.3%) and CRPA (84.5%); and CPT against MSSA (100.0%), MRSA (95.3%) and S. pneumoniae (94.3%). CZA was active against blaKPC-carrying CRKP (98.5% susceptible), and resistance in the majority of CZA-resistant Enterobacterales isolates (6 of 10 CRKP and 2 of 3 CREC) was due to the presence of a metallo-β-lactamase gene. Our results suggest that interpretation of susceptibility results obtained by Etest for both agents should be undertaken cautiously and remains challenging.

Evaluation of NitroSpeed-Carba NP test for rapid identification among different classes of carbapenemases in Enterobacterales and Pseudomonas aeruginosa

ObjectivesThis study aimed to evaluate the performance of the NitroSpeed-Carba NP test for detecting carbapenemases in the clinical strains of Enterobacterales and Pseudomonas aeruginosa (P. aeruginosa), and analyze its advantages and limitations. MethodsThe antimicrobial susceptibility tests were performed according to the agar dilution method. Using the modified carbapenemase inactivation method (mCIM), polymerase chain reaction (PCR), and sequencing, the production of carbapenemase and the prevalence of genes were studied. The NitroSpeed-Carba NP test was performed to detect different types of carbapenemases in Enterobacterales and P. aeruginosa. The results of PCR and sequencing were used as the gold standard. ResultsAmong 144 carbapenemase-producing and 54 carbapenemase-negative strains of Enterobacterales and P. aeruginosa, the NitroSpeed-Carba NP test correctly detected 143 of 144 carbapenemase producers and 51 of 54 non-carbapenemase producers. Moreover, the sensitivity and specificity of all tested isolates were 99.31% and 94.44%, respectively (99.28% and 92.86% for Enterobacterales; 100% and 100% for P. aeruginosa). The sensitivity was 100% for class A (56 of 56), 100% for class B (60 of 60), and 100% for class D (27 of 27). ConclusionsThe results suggest that NitroSpeed-Carba NP test is a simple and valuable assay that could be used as a rapid and stable detection method to identify the carbapenemases in Enterobacterales and P. aeruginosa strains.

Genetic Diversity, Biochemical Properties, and Detection Methods of Minor Carbapenemases in Enterobacterales.

Gram-negative bacteria, especially Enterobacterales, have emerged as major players in antimicrobial resistance worldwide. Resistance may affect all major classes of anti-gram-negative agents, becoming multidrug resistant or even pan-drug resistant. Currently, β-lactamase-mediated resistance does not spare even the most powerful β-lactams (carbapenems), whose activity is challenged by carbapenemases. The dissemination of carbapenemases-encoding genes among Enterobacterales is a matter of concern, given the importance of carbapenems to treat nosocomial infections. Based on their amino acid sequences, carbapenemases are grouped into three major classes. Classes A and D use an active-site serine to catalyze hydrolysis, while class B (MBLs) require one or two zinc ions for their activity. The most important and clinically relevant carbapenemases are KPC, IMP/VIM/NDM, and OXA-48. However, several carbapenemases belonging to the different classes are less frequently detected. They correspond to class A (SME-, Nmc-A/IMI-, SFC-, GES-, BIC-like…), to class B (GIM, TMB, LMB…), class C (CMY-10 and ACT-28), and to class D (OXA-372). This review will address the genetic diversity, biochemical properties, and detection methods of minor acquired carbapenemases in Enterobacterales.

Correction for Nordmann et al., "NitroSpeed-Carba NP Test for Rapid Detection and Differentiation between Different Classes of Carbapenemases in Enterobacterales".

Volume 58, no. 9, e00932-20, 2020, https://doi.org/10.1128/JCM.00932-20. This article was published on 24 August 2020 with a CC BY 4.0 copyright line (“Copyright © 2020 Nordmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.”). The authors elected to remove open access for the article after publication, necessitating replacement of the original copyright line, and this change was made on 29 September 2020.

Multicenter Evaluation of the BD Phoenix CPO Detect Test for Detection and Classification of Carbapenemase-Producing Organisms in Clinical Isolates

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

Development of a MALDI-TOF MS-based screening panel for accelerated differential detection of carbapenemases in Enterobacterales using the direct-on-target microdroplet growth assay

Carbapenemase-producing bacteria are a growing issue worldwide. Most phenotypic detection methods are culture-based, requiring long incubation times. We present a phenotypic screening panel for detection of carbapenem non-susceptibility and differentiation of carbapenemase classes and AmpC, the MALDI-TOF MS-based direct-on-target microdroplet growth assay (DOT-MGA). It was validated on 7 reference strains and 20 challenge Enterobacterales isolates. Broth microdilution (BMD) and combination disk test (CDT) were also performed, as well as PCR as reference method. The panel based on the synergy between meropenem and carbapenemase inhibitors, determined by incubating these substances with bacterial suspension on a MALDI-TOF MS target and subsequently assessing bacterial growth on the target’s spots by MS. After 4 hours of incubation, DOT-MGA correctly identified KPC, MBL and OXA (100% agreement with PCR). Detection of AmpC coincided with BMD and CDT but agreement with PCR was low, not ruling out false negative PCR results. DOT-MGA delivered more accurate results than BMD and CDT in a significantly shorter time, allowing for detection of carbapenem non-susceptibility, MIC determination and carbapenemase differentiation in one step.

Comparison of two multiplex immunochromatographic assays for the rapid detection of major carbapenemases in Enterobacterales.

ObjectivesTwo commercially available lateral flow immunochromatographic assays (ICAs) for detection of the major carbapenemases were prospectively assessed for the detection of carbapenemases in Enterobacterales: RESIST-4 O.K.N.V. (Coris BioConcept) and NG-Test CARBA 5 (NG Biotech).MethodsThese two assays were performed prospectively on consecutive Enterobacterales suspected of producing a carbapenemase that were referred to the Belgian National Reference Center for Monitoring Antimicrobial Resistance in Gram-Negative Bacteria between March and June 2018. The intensity of the band corresponding to a carbapenemase for each test was compared using ImageJ software.ResultsOf the 161 isolates tested, a carbapenemase was detected in 91 (60 OXA-48-like, 15 VIM, 9 KPC, 5 NDM, 1 IMP and 1 IMP + OXA-48); in the remaining 70, no carbapenemases were detected. For both tests, the results were 100% concordant with the results of the PCR-sequencing reference method. Two IMP producers were only detected by NG-Test CARBA 5 as IMP is not targeted by RESIST-4 O.K.N.V. The mean intensity of the OXA-48, VIM and NDM bands displayed by NG-Test CARBA 5 was 3 to 3.7 times higher than for RESIST-4 O.K.N.V., while the KPC band was on average 1.7 times more intense with RESIST-4 O.K.N.V.ConclusionsRESIST-4 O.K.N.V. and NG-Test CARBA 5 are two efficient assays for identification of the major carbapenemases. NG-Test CARBA 5 offers the advantage of detecting IMP, which remains rare in Western countries.

Longitudinal analysis of ESBL and carbapenemase carriage among Enterobacterales and Pseudomonas aeruginosa isolates collected in Europe as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme, 2013-17.

To determine the spread of ESBLs and carbapenemases in Enterobacterales and Pseudomonas aeruginosa in Europe. 45 335 Gram-negative bacilli were collected in 18 European countries as part of the International Network for Optimal Resistance Monitoring (INFORM) global surveillance programme from 2013 to 2017. Antimicrobial susceptibility was determined using broth microdilution, and 9546 isolates were screened for β-lactamase genes by PCR and sequencing. ESBLs were identified in 35.5% of Klebsiella pneumoniae and 18.5% of Escherichia coli. ESBL carriage was lowest among isolates in Northern/Western Europe and highest in Eastern Europe. CTX-M-15 was the dominant ESBL in all countries except Greece, where SHV-type ESBLs were more common. Carbapenemases (KPC, OXA-48-like, GES, NDM and VIM) were found in 3.4% of Enterobacterales and were most common among K. pneumoniae (10.5% of those collected). Carbapenemase carriage was lowest in Northern/Western and highest in Southern Europe. KPC-positive Enterobacterales were most abundant but the percentages of OXA-48-like-, NDM- and VIM-positive isolates increased over time and were correlated with an increase in meropenem non-susceptibility. Carbapenemases (VIM, IMP, NDM and GES) were also identified in 5.1% of P. aeruginosa and were commonly found in Eastern Europe. Carbapenemase carriage and meropenem non-susceptibility among P. aeruginosa fluctuated over the 5 years studied and were not well correlated. ESBL and carbapenemase carriage varied by species and European subregion. Meropenem non-susceptibility in European isolates of Enterobacterales can be attributed to carbapenemase carriage and is increasingly caused by MBLs and OXA-48-like carbapenemases. Carbapenemases or other β-lactamases are not a common cause of meropenem non-susceptibility in P. aeruginosa in Europe.

Lateral flow assay for the rapid detection of carbapenemases in Enterobacterales

With the development of carbapenemase inhibitors such as avibactam, relebactam and vaborbactam, it has become important to detect the type of carbapenemase produced for guiding antibiotic therapy. Among the several methods available for the same, we chose lateral flow assay (Resist-3 O. K. N., Coris BioConcepts, Gembloux, Belgium) and compared it against a commercially available molecular test (Xpert Carba R assay, version 2, Cepheid, Sunnyvale, CA, USA). Sixteen clinical isolates, which were resistant to carbapenems on phenotypic testing, were selected. Fourteen of these were Klebsiella pneumoniae and two were Escherichia coli a total of 22 carbapenemases. (OXA-48-14, New Delhi metallo-beta-lactamase [NDM]-7 and K. pneumoniae carbapenemase [KPC]-1) were detected by the Carba-R assay. Six isolates (K. pneumoniae-5 and E. coli-1) had co-production of OXA-48 and NDM. Resist-3 assay detected all twenty two enzymes. The distribution of enzymes in these 16 isolates was identical to that shown by the Carba-R (100% agreement). Hence, we conclude that lateral flow immunochromatography assay is a simple, rapid and cost-effective method for the detection of carbapenemases. This would help the clinician select the appropriate antibiotic and support antibiotic stewardship in the Indian settings.

Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm

ObjectivesThe aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories. MethodsThe immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric β-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2). ResultsThe overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6–89.2%)/100% (CI 91.8–100%) for RESIST-4, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for CARBA-5, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for Xpert Carba-R, 73.7% (CI 66.2–80.0%)/100% (CI 93.4–99.0%) for β-CARBA, and 97.4% (CI 87.9–99.6%)/97.7% (CI 87.9–99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for β-CARBA (76.6% (CI 68.4–83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3–98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed. ConclusionsExcept for β-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.