[Application of Rapid Detection Method Based on NG-Test® CARBA 5 in Bloodstream Infections Associated With Carbapenem-Resistant Enterobacterales].

Objective: To compare the consistency and accuracy of a rapid test method and a traditional test method for pathogen identification, antimicrobial susceptibility and carbapenemase type identification of positive blood culture samples. Methods: A total of 51 positive blood culture samples of bloodstream infection (BSI) were collected between March 2022 and May 2022. All samples were found to be "positive for Gram-negative bacilli" according to the blood smear results. The rapid method was adopted to perform rapid antimicrobial susceptibility test (RAST) and analysis of the positive blood culture samples. According to the RAST result interpretation standards, NG-Test® CARBA 5 was used for rapid carbapenemase detection of the imipenem-resistant strains and the results were confirmed by PCR. In addition, mass spectrometry, VITEK 2 Compact drug sensitivity analysis, and carbapenemase type identification were performed with the colonies cultured with positive samples according to the traditional method. Results: In the identification of bacteria, the rapid method and the traditional method had 100% consistency rate in the identification results of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In the antimicrobial susceptibility test, the consistency rate between the results of the two methods was high and the consistency rate for results for susceptibility to imipenem was 100%. In the identification of carbapenemase type, 18 serinase-producing strains and 3 metal-ß-lactamase-producing strains of Enterobacterales were detected by the traditional method. With the rapid method, 18 Klebsiella pneumoniae carbapenemase (KPC)-producing strains, 2 New Delhi metallo-betalactamase (NDM)-producing strains, and 1 imipenem enzyme (IMP)-producing strain were identified in the blood culture samples by using a testing kit. Compared with the PCR results, the sensitivity and specificity of the rapid test for determining carbapenemase types were 100%. In this study, we investigated a rapid method for bacteria and carbapenemase type identification of positive blood culture specimens and found that the turnaround time (TAT) of the rapid method was reduced by 1.94 days on average in comparison with the TAT of the traditional method. Conclusion: The rapid method established in the study can effectively shorten the TAT for pathogenic microorganism identification and antimicrobial susceptibility test of blood culture samples, and the joint report of colloidal gold carbapenemase type identification results can provide a reference for clinicians to use antibiotics appropriately and accurately manage multi-drug resistant bacterial infections.

Xpert Carba-R Assay on Flagged Blood Culture Samples: Clinical Utility in Intensive Care Unit Patients with Bacteremia Caused by Enterobacteriaceae.

Introduction and background: Rapid molecular diagnostics to predict carbapenem resistance well before the availability of routine drug sensitivity testing (DST) can serve as an antimicrobial stewardship tool in the context of high rates of Carbapenem-resistant Enterobacteriaceae (CRE). Materials and methods: A retrospective observational study of patients more than 18 years of age on whom Xpert Carba-R (FDA approved for rectal swab specimen) was done on gram-negative bacteria (GNB) flagged blood culture samples, in an Indian intensive care unit between January 2015 and November 2018. We analyzed the performance of Xpert Carba-R in comparison with routine DST. Results: A total of 164 GNBs were isolated from 160 patients. Klebsiella pneumoniae and Escherichia coli were the predominant isolates. Carba-R was positive in 35.36% of samples and 45.34% were carbapenem-resistant (CR) on routine DST. The distribution of the CR gene was: Oxacillinase (OXA) (50%), NDM (32.7%) followed by OXA and NDM co-expression (15.51%). The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value, and negative predictive value of Carba-R were 90.74, 93.15, 13.25, 0.10, 83.58 and 96.31% for Enterobacteriaceae. The median time to obtain the Carba-R report was 30 hours 34 minutes vs 74 hours and 20 minutes for routine DST. Based on the Carba-R report, 9.72% of patients had escalation and 27.08% had de-escalation of antibiotics. Conclusion: Xpert Carba-R serves as a rapid diagnostic tool for predicting carbapenem resistance in intensive care unit patients with bacteremia caused by Enterobacteriaceae. How to cite this article: Rajendran S, Gopalakrishnan R, Tarigopula A, Kumar DS, Nambi PS, Sethuraman N, et al. Xpert Carba-R Assay on Flagged Blood Culture Samples: Clinical Utility in Intensive Care Unit Patients with Bacteremia Caused by Enterobacteriaceae. Indian J Crit Care Med 2023;27(9):655-662.

Success and Challenges Associated with Large-Scale Collaborative Surveillance for Carbapenemase Genes in Gram-Negative Bacteria.

The emergence and spread of antimicrobial resistance, especially in Gram-negative bacteria, has led to significant morbidity and increased cost of health care. Large surveillance studies such as the one performed by the Antibiotic Resistance Laboratory Network are immensely valuable in understanding the scope of resistance mechanisms, especially among carbapenemase-producing Gram-negative bacteria. However, the routine laboratory detection of carbapenemases in these bacteria remains challenging and requires further optimization.

Implementation of Chromatic Super CAZ/AVI® medium for active surveillance of ceftazidime-avibactam resistance: preventing the loop from becoming a spiral.

Acquired resistance towards ceftazidime-avibactam (CAZ-AVI) is increasingly reported. Several mechanisms can be involved, but mutations in the Ω-loop region of ß-lactamases are the most described. Herein, we assessed the implementation of Chromatic Super CAZ/AVI® medium in rectal swab surveillance cultures in a geographic area with endemic distribution of KPC-producing Klebsiella pneumoniae. Routine rectal swabs collected from the intensive care unit (ICU) and non-ICU patients were screened for carbapenemase-producing Enterobacterales (CPE), carbapenem-resistant Gram-negative organisms (CR-GN) and CAZ-AVI-resistant organisms by Chromatic CRE and Super CAZ/AVI® media. Among the 1839 patients screened, 146 (7.9%) were found to be colonized by one or more CPE and/or CR-GN isolates during hospitalization. Overall, among colonized patients the most common bacteria encountered were KPC-producing Enterobacterales (n = 60; 41.1%), carbapenem-resistant Pseudomonas aeruginosa (n = 41; 28.1%) and carbapenem-resistant A. baumannii (n = 34; 23.3%). Among patients colonized by KPC-producing Enterobacterales, thirty-five (58.3%) had CAZ-AVI-resistant strains. A 30.5% rate of faecal carriage of CAZ-AVI-resistant KPC-producing K. pneumoniae, substantially higher than that of susceptible isolates (2.8%), was observed in the COVID-19 ICU. Prevalence of faecal carriage of metallo-ß-lactamase-producing organisms was low (0.5% and 0.2% for Enterobacterales and P. aeruginosa, respectively). Chromatic Super CAZ/AVI® medium showed 100% sensitivity in detecting CPE or CR-GN isolates resistant to CAZ-AVI regardless of both MIC values and carbapenemase content. Specificity was 86.8%. The Chromatic Super CAZ/AVI® medium might be implemented in rectal swab surveillance cultures for identification of patients carrying CAZ-AVI-resistant organisms to contain the spread of these difficult-to-treat pathogens.

Distribution of carbapenemase genes in clinical isolates of Acinetobacter baumannii & a comparison of MALDI-TOF mass spectrometry-based detection of carbapenemase production with other phenotypic methods.

Background & objectives: Carbapenemase-producing Acinetobacter baumannii (CRAB) poses a continuous threat to the current antimicrobial era with its alarming spread in critical care settings. The present study was conducted to evaluate the diagnostic potential of phenotypic methods for carbapenemase [carbapenem-hydrolyzing class D ß-lactamases (CHDLs) and metallo-ß-lactamases (MBLs)] production, by comparing with molecular detection of genes. Methods: One hundred and fifty clinical CRAB isolates collected between August 2013 and January 2014 were studied. Multiplex PCR was performed to identify the carbapenemases produced (class D blaOXA-51, blaOXA-23, blaOXA-48, blaOXA-58; class B blaVIM, blaNDM-1, blaIMP; class A blaKPC). Each isolate was evaluated for carbapenemase production by studying the pattern of imipenem hydrolysis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results: The most commonly encountered carbapenemase genes were blaOXA-51(100%), blaOXA-23(98%), blaVIM(49.3%), blaNDM-1(18.7%) and blaOXA-58(2%). MALDI-TOF MS was able to detect 30.6 per cent carbapenemases within three hours (P=0.001 for MBL and P>0.05 for CHDL) and 65.3 per cent within six hours (P=0.001 for MBL and P>0.05 for CHDL). Interpretation & conclusions: MALDI-TOF MS reliably detected carbapenemase activity within a short span of time, thus helping in tailoring patient therapy. MALDI-TOF MS, once optimized, can prove to be a useful tool for timely detection of carbapenemase production by A. baumannii and consequently in directing appropriate antimicrobial therapy.

Direct ß-Lactam Inactivation Method: a New Low-Cost Assay for Rapid Detection of Carbapenemase- or Extended-Spectrum-ß-Lactamase-Producing Enterobacterales Directly from Positive Blood Culture Bottles.

We validate and evaluate a new phenotypic assay, named the direct ß-lactam inactivation method (dBLIM), for the rapid and simultaneous detection of carbapenemase or extended-spectrum-cephalosporinase activity directly from Enterobacterales (EB)-positive blood cultures (BCs). It originates from the carbapenem inactivation method (CIM), an inexpensive and highly sensitive assay for carbapenemase activity detection. dBLIM cutoff values to detect extended-spectrum ß-lactamase (ESBL) and carbapenemase activities resulted in diameters of ≤12 mm for a 5-µg-cefotaxime disk and for a 10-µg-meropenem disk. dBLIM assessment was determined with both aerobic and anaerobic BC bottles spiked with 422 characterized EB strains, classifiable into the following 4 phenotypic groups: (i) ESBL/AmpC-type ß-lactamase (ACBL)/carbapenemase (CARB)-nonproducing (np-ESBL/ACBL/CARB) EB (n = 116), (ii) ESBL-producing EB (n = 111), (iii) AmpC-ß-lactamase-producing EB (n = 33), and (iv) carbapenemase-producing EB (n = 162). No false-positive results were obtained in any of the np-ESBL/ACBL/CARB EB, ESBL, and AmpC groups, demonstrating an overall assay specificity of 100%. There were no significant discrepancies in dBLIM performance between aerobic and anaerobic BCs across all groups, except with VIM-type carbapenemase-expressing EB. Interestingly, among BCs spiked with blaVIM-harboring EB, the sensitivity rates of the assay in anaerobic and aerobic bottles were 53.6% and 100%, respectively. In contrast, dBLIM performance was deemed excellent for the KPC, OXA-48, and NDM carbapenemase producers regardless of the type of bottle being tested, with a sensitivity rate ranging between 99% and 100%. Concerning the detection of the extended-spectrum cephalosporinases of the ESBL-producing and AmpC types, dBLIM sensitivities was 100% and 84 to 87%, respectively. dBLIM may be a cost-effective and highly robust phenotypic screening method for the reliable detection of carbapenemases or extended-spectrum cephalosporinases directly from BCs on the same day of bottle positivity detection.

Laboratory validation of a KPC-producing strain identification method based on the detection of a specific 11,109 Da peak via Maldi-Tof-Vitek MS in an endemic area.

Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Vitek MS system is a useful technique to identify bacteria strains isolated in clinical samples. In this paper, we applied this method to KPC-producing Enterobacteriaceae detection through the determination of a specific 11,109 (±8) Da peak. We assayed the presence, specificity and reliability of this peak on routine workflow through the analysis of 183 Enterobacteriaceae strains isolated from clinical samples and characterized by classical approaches. The peak was detected in 95.5% (129/135) of carbapenemase-producing strains spectra compared with the 48 extended spectrum beta-lactamase producing controls strains, which all lacked this peak. Hence, this 11,109 Da peak determination showed a Positive Predictive Value (PPV) of 100% and a Negative Predictive Value (NPV) of 94.4%. The characterization of this specific peak in a MALDI-TOF Vitek MS system might be considered a valuable tool to reveal KPC-producing Enterobacteriaceae especially in KPC endemic region.

Detection of Carbapenemase-Producing Enterobacteriaceae in Positive Blood Culture Using an Immunochromatographic RESIST-4 O.K.N.V. Assay.

We evaluated the performance of the RESIST-4 O.K.N.V. assay (Coris) with 98 isolates to detect OXA-48-like and KPC-, NDM-, and VIM-type carbapenemases directly on positive human blood cultures. OXA-48-like and KPC-type isolates were correctly detected, but the detection of NDM- and VIM-type carbapenemases was weak and variable. We show that repeating the test on a 4-h subculture improves the detection of NDM- and VIM-type carbapenemases to 100%.

Multicenter Evaluation of the Xpert Carba-R Assay for Detection of Carbapenemase Genes in Gram-Negative Isolates.

This multicenter study evaluated the performance of the Cepheid Xpert Carba-R assay, a qualitative PCR test designed for the rapid detection of blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48 carbapenem resistance genes from bacterial isolates grown on blood agar or MacConkey agar. The results were compared to those obtained from bidirectional DNA sequence analysis of nucleic acid extracted from pure colonies. Isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii that tested as either intermediate or resistant to a carbapenem antibiotic were analyzed. A total of 467 isolates were evaluated, including prospectively collected clinical isolates, frozen isolates, and a group of contrived broth specimens sent by a central reference laboratory. The assay was run on the GeneXpert platform and took 48 min, with less than 1 min of hands-on time. Compared to the results of the reference methods, the overall sensitivity of the assay was 100% (95% confidence interval [CI], 99.0 to 100%) for isolates grown on both blood and MacConkey agars. Overall specificity was 98.1% (95% CI, 93.1 to 99.8%) and 97.1% (95% CI, 91.7 to 99.4%) for blood and MacConkey agars, respectively. This platform, previously demonstrated to be effective for the detection of carbapenemase genes in rectal swabs, is also adequate for the detection of these genes in bacterial colonies isolated from blood and MacConkey agars.

Complete Genome Sequence of a Klebsiella pneumoniae Strain Carrying Novel Variant blaKPC-203, Cross-Resistant to Ceftazidime/Avibactam and Cefiderocol, but Susceptible to Carbapenems, Isolated in Italy, 2023.

BACKGROUND: Klebsiella pneumoniae is a concerning pathogen, responsible for hospital-associated outbreaks. Multi drug resistant (MDR) strains are especially hard to treat. We conducted whole-genome sequencing on a MDR K. pneumoniae strain in order to identify genomic features potentially linked to its phenotype. METHODS: DNA sequencing was performed on the Illumina iSeq 100 platform. Genome assembly was carried out with SPAdes. The genome was annotated with RASTtk. Typing was performed with MLST and Kaptive. Antibiotic resistance genes were detected with AMRFinderPlus and Abricate, and further verified with BLAST. RESULTS: The strain exhibited resistance to ceftazidime/avibactam and cefiderocol, but remained susceptible to carbapenems. The strain belonged to sequence type ST101, serotype O1:K17. The analysis of antibiotic resistance genes indicated that the strain carried a novel KPC variant, designated as KPC-203, featuring a EL deletion at amino acid position 166-167, within the Ω-loop, and a nine-amino-acid insertion (LAVYTRAPM) at position 259. Sequence alterations were found in porin genes ompK35 and ompK36. Unlike molecular testing, which was able to detect the KPC-203 variant, all phenotypic carbapenemase detection methods achieved negative results. CONCLUSIONS: KPC-203, a novel KPC variant, showed a sequence modification in a cephalosporin resistance-associated hotspot. Interestingly, such alterations typically correlate with the restoration of carbapenem susceptibility. We hypothesize that KPC-203 likely led to resistance to ceftazidime/avibactam and cefiderocol, while maintaining susceptibility to carbapenems.

Emergence of pandrug-resistant carbapenemase-producing Enterobacterales in dogs and cats: a cross-sectional study in Egypt.

One of the most important emerging health problems is the increasing role of animals in the rapid global rise in resistance to last-resort antibiotics, such as carbapenems. However, there is limited information on the role of pet animals in harboring and spreading pandrug-resistant (PDR) carbapenemase-producing Enterobacterales (CPE), especially in Egypt. This cross-sectional study was conducted to screen for CPE in healthy and diseased pets using phenotypic and molecular methods and the NG-Test CARBA 5 immunochromatographic assay. Rectal swabs were collected from 62 dogs and 48 cats, incubated overnight in tryptic soy broth containing 10 µg of meropenem disc and subsequently cultured on MacConkey agar supplemented with meropenem (1 mg/L). Sixty-six isolates (60.6%), including 56 Klebsiella pneumoniae, seven Escherichia coli, and three K. oxytoca isolates, were confirmed to be carbapenem-resistant Enterobacterales (CRE) by the disc diffusion method, broth microdilution test, CNPt-direct, and PCR assay targeting carbapenemase genes. Forty-three (65.2%) dogs and 23 (34.8%) cats carried CPE. Of these, 35 (70.0%) were healthy (including 27 dogs and 8 cats) and 31 (52.5%) were diseased (including 16 dogs and 15 cats). bla OXA-181 was the most common gene detected (42/66, 63.6%), followed by bla IMP (40/66, 60.6%), bla OXA-48-like (29/66, 43.9%), bla KPC and bla VIM (20/66, 30.3% each), and bla NDM (17/66, 25.8%). The identified genotypes were bla KPC-2, bla IMP-1, bla VIM-1, bla NDM-1, and bla NDM-5. The CARBA 5 assay showed higher sensitivity and specificity for the detection of NDM, OXA and KPC than that for VIM and IMP genes. Antimicrobial resistance profiles of CRE isolates revealed 20 PDR, 30 extensively drug-resistant (XDR), and 16 multidrug-resistant (MDR) phenotypes. This study provides evidence of colonization with PDR CPE in dogs and cats. To manage the infection or colonization of pets in veterinary clinical settings, extended surveillance systems should be considered, and the use of critical antibiotics should be strictly controlled.

Evaluation of the BD Phoenix CPO Detect Panel for Detection and Classification of Carbapenemase Producing Enterobacterales.

Carbapenem-resistant Enterobacterales (CRE) pose a serious public health threat due to their resistance to most antibiotics. Rapid and correct detection of carbapenemase producing organisms (CPOs) can help inform clinician decision making on antibiotic therapy. The BD Phoenix™ CPO detect panel, as part of antimicrobial susceptibility testing (AST), detects carbapenemase activity (P/N) and categorizes CPOs according to Ambler classes. We evaluated a CPO detect panel against 109 carbapenemase producing Enterobacterales (CPE) clinical isolates from Korea. The panel correctly detected carbapenemases production in 98.2% (n = 107/109) isolates and identified 78.8% (n = 26/33) class A, 65.9% (n = 29/44) class B, and 56.3% (n = 18/32) class D carbapenemase producers as harboring their corresponding Ambler classes. Specifically, the panel correctly classified 81.3% (n = 13/16) of K. pneumoniae KPC isolates to class A. However, the panel failed to classify 40.0% (n = 4/10) IMP and 63.6% (n = 7/11) VIM isolates to class B. Despite 27.5% (n = 30/109) CPE not being assigned Ambler classes, all of them tested carbapenemase positive. Our results demonstrate that the CPO detect panel is a sensitive test for detecting CPE and classifying KPC as class A, helping with antibiotics selection, but one-third of CPE remained unclassified for Ambler classes.

Companion Animals-An Overlooked and Misdiagnosed Reservoir of Carbapenem Resistance.

The dissemination of antimicrobial-resistance is a major global threat affecting both human and animal health. Carbapenems are human use ß-lactams of last resort; thus. the dissemination of carbapenemase-producing (CP) bacteria creates severe limitations for the treatment of multidrug-resistant bacteria in hospitalized patients. Even though carbapenems are not routinely used in veterinary medicine, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals are being reported. NDM-5 and OXA-48-like carbapenemases are among the most frequently reported in companion animals. Like in humans, Escherichia coli and Klebsiella pneumoniae are the most represented CP Enterobacterales found in companion animals, alongside with Acinetobacter baumannii. Considering that the detection of carbapenemase-producing Enterobacterales presents several difficulties, misdiagnosis of CP bacteria in companion animals may lead to important animal and public-health consequences. It is of the upmost importance to ensure an adequate monitoring and detection of CP bacteria in veterinary microbiology in order to safeguard animal health and minimise its dissemination to humans and the environment. This review encompasses an overview of the carbapenemase detection methods currently available, aiming to guide veterinary microbiologists on the best practices to improve its detection for clinical or research purposes.

Phenotypic detection of carbapenemase production in carbapenem-resistant isolates with the rapid carbapenemase detection method (rCDM).

INTRODUCTION: Carbapenem antibiotics are widely used for the treatment of infections caused by multidrug-resistant bacteria. As a result of this, resistance to carbapenems is gradually increasing. Identification of carbapenemase production, one of the reasons for resistance, through molecular methods is expensive and time-consuming. In the present study, it was aimed to investigate the sensitivity of the newly developed rapid carbapenemase detection method (rCDM) as compared to the gold standard molecular method and to evaluate its consistency with another phenotypical method, the modified carbapenem inactivation method (mCIM). MATERIAL AND METHODS: In our study, a total of 152 Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli) isolated from various clinical samples of which 50 were controls were included. Strain identification was done by using VITEK®MS (bioMérieux, Marcy-I'Étoile, France), carbapenem sensitivity was tested by using VITEK®2 (bioMérieux). For carbapenem-resistant isolates, carbapenem resistance genes were detected with multiplex PCR [Carbapenem and Colistin Resistance qPCR kit (Bioeksen, Istanbul, Turkey)] kit by a molecular method. All included isolates were evaluated by the rCDM and mCIM tests in order to detect carbapenemase phenotypically. The molecular method was accepted to be the gold standard and the sensitivity of rCDM was calculated. The McNemar test was applied to analyze the difference between two phenotypic tests (rCDM and mCIM) and Cohen's Kappa analysis was applied to determine consistency. RESULTS: Out of 102 carbapenem-resistant isolates, at least one of the resistance genes in the multiplex PCR panel (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-51, blaOXA23/58, blaOXA-48) was detected in 92 and blaOXA-48 was the most common (90.2%). The sensitivity of the rapid carbapenemase detection method was found to be 100%. When the results of the two phenotypic methods were compared, no statistically significant difference could be found (PMcNemar:1, Kappa coefficient:1.00). CONCLUSION: The rapid carbapenemase detection method was found to be suitable to use in routine laboratory analysis as its sensitivity was found to be high, exhibited a good performance for detection of frequent carbapenemase types in our country (Turkey), a high consistency with mCIM, and also it is an easily applied and rapid method.

A Variant Carbapenem Inactivation Method (CIM) for Acinetobacter baumannii Group with Shortened Time-to-Result: rCIM-A.

Carbapenem-resistant Acinetobacter baumannii group organisms (CRAB) are challenging because the choice between targeted, new antibiotic drug options and hygiene measures should be guided by a timely identification of resistance mechanisms. In CRAB, acquired class-D carbapenemases (CHDLs) are active against meropenem and imipenem. If PCR methods are not the first choice, phenotypic methods have to be implemented. While promising, the carbapenemase inactivation method (CIM) using meropenem-hydrolysis is, however, hampered by poor performance or overly long time-to-result. We developed a rapid CIM (rCIM-A) with good performance using ertapenem, imipenem, and meropenem disks, 2-h permeabilization and incubation with the test strain in trypticase soy broth, and a read-out of residual carbapenem activity after 6 h, and optionally after 16-18 h. Using clinical isolates and type-strains of Acinetobacter (n = 67) not harboring carbapenemases (n = 28) or harboring acquired carbapenemases (n = 39), the sensitivity of detection was 97.4% with the imipenem disk after 6 h at a specificity of 92.9%. If the inhibition zone around the ertapenem disk at 6 h was 6 or ≤26 mm at 16-18 h, or ≤25.5 mm for meropenem, the specificity was 100%. Because of the high negative predictive value, the rCIM-A seems particularly appropriate in areas of lower CRAB-frequency.

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

OBJECTIVES: The 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. METHODS: Bacterial 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. RESULTS: The 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%. CONCLUSION: This 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.

Detection of KPC, NDM and VIM-Producing Organisms Directly from Rectal Swabs by a Multiplex Lateral Flow Immunoassay.

We report a preliminary evaluation of the NG-Test CARBA 5 immunochromatographic assay for detecting carbapenemases directly from rectal swabs on the same day of sampling. Thirty fecal swabs were examined for carbapenemase-producing organisms (CPOs) by conventional culture, PCR, and NG-Test CARBA 5. Each sample was tested by the immunochromatographic assay five times, including direct testing and incubation in trypticase soy broth for 1, 2, 3, and 4 h. Twenty patients yielded CPOs by culture. Immunochromatographic and PCR results were concordant and detected the same 25 carbapenemases (11 KPC, 8 VIM, and 6 NDM). In five cases, we detected co-carriage of KPC and VIM. Compared with PCR, the sensitivity of NG-Test CARBA 5 for the detection of KPC, VIM, and NDM was 80% without incubation, 88% with one hour, 92% with two, and 100% with three hours incubation, while specificity was 100% for all time points. All samples containing adequate fecal content were detected by NG-Test CARBA 5 concordantly with PCR, without incubation. NG-Test CARBA 5 is a reliable test that rapidly detects the presence of carbapenemases at the same day of sampling, directly from rectal swabs. It thus provides early information to guide antimicrobial treatment and infection control interventions.

Carbapenemase detection testing in the era of ceftazidime/avibactam-resistant KPC-producing Enterobacterales: A 2-year experience.

OBJECTIVES: The aim of this study was to investigate the prevalence of ceftazidime/avibactam (CZA) resistance among carbapenemase-producing Enterobacterales (CPE) blood culture isolates as well as the performance of the main carbapenemase phenotypic detection methods to identify KPC variants associated with CZA resistance. METHODS: Non-duplicate CPE strains isolated from blood cultures during 2018-2020 were tested for antimicrobial susceptibility. Molecular testing was used to identify carbapenemase-producers. Strains harbouring blaKPC and with a CZA minimum inhibitory concentration (MIC) ≥8 mg/L were investigated by sequencing. Subsequentially, five phenotypic carbapenemase detection methods were evaluated on these strains, namely the modified carbapenem inactivation method (mCIM), Rapidec® Carba NP, the disk diffusion synergy test, NG-Test CARBA® 5 and RESIST-5 O.O.K.N.V. RESULTS: Overall, the CZA resistance rate was high (13.7%) and remained relevant (5.9%) excluding metallo-ß-lactamases-producers. All isolates harbouringblaKPC mutants (n = 8) were associated with reduced carbapenem MICs and negative results by all detection methods based on revelation of enzyme activity. Lateral flow immunoassays failed to detect KPC-31 (n = 4) and KPC-33 (n = 2) but correctly identified KPC-14 (n = 2). Conversely, isolates harbouring wild-type KPC genes (n = 3) were associated with high-level CZA resistance and carbapenem resistance and tested positive by all of the evaluated methods. CONCLUSION: In the era of CZA-based therapies, molecular blaKPC identification followed by a carbapenem hydrolysis-based phenotypic assay could be the most reasonable diagnostic algorithm to detect all KPC-producers and to identify mutants associated with impaired carbapenemase activity and CZA resistance.

An Application of Imipenem Discs or P. aeruginosa ATCC 27853 Reference Strain Increases Sensitivity of Carbapenem Inactivation Method for Non-Fermenting Gram-Negative Bacteria.

Non-fermenting Gram-negative rods are one of the most commonly isolated bacteria from human infections. These microorganisms are typically opportunistic pathogens that pose a serious threat to public health due to possibility of transmission in the human population. Resistance to beta-lactams, due to carbapenemases synthesis, is one of the most important antimicrobial resistance mechanisms amongst them. The aim of this study was to evaluate the usefulness of the Carbapenem Inactivation Method (CIM), and its modifications, for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. This research involved 81 strains of Gram-negative rods. Of the tested strains, 55 (67.9%) synthesized carbapenemases. For non-fermenting rods, 100% sensitivity and specificity was obtained in the version of the CIM test using imipenem discs and E. coli ATCC 25922 strain. The CIM test allows for differentiation of carbapenems resistance mechanisms resulting from carbapenemase synthesis from other resistance types. It is a reliable diagnostic method for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. Application of imipenem discs and P. aeruginosa ATCC 27853 reference strain increases CIM results sensitivity, while imipenem discs and E. coli ATCC 25922 strain use maintains full precision of the test for non-fermenting rods.

Evaluation of the usefulness of selected methods for the detection of carbapenemases in Klebsiella strains.

Introduction. Klebsiella rods, belonging to the family Enterobacteriaceae, are generally opportunistic pathogens commonly associated with nosocomial infections, especially in intensive care units. Interestingly, strains of this genus also show multi-drug resistance. In recent years, multiple studies have indicated that the prevalence of carbapenem resistance has increased rapidly among Klebsiella representatives.Aim. The aim of this study was to assess the usefulness of selected phenotypic and genotypic methods for the detection of the most important carbapenemases in Klebsiella strains.Methodology. The study involved 51 Klebsiella strains. The ability to produce carbapenemases was determined by phenotypic methods (double disc synergy test, test with four discs and three inhibitors, CarbaNP test, culture on chromogenic medium, panels of automatic method - Phoenix, CIM test and modified Hodge test). The potential for carbapenemase synthesis was also evaluated using real-time PCR, detecting bla VIM/IMP, bla KPC, bla NDM and bla OXA-48 genes.Results. Using the phenotypic methods, positive results were obtained for all of the analysed strains. Using PCR, carbapenemase synthesis potential was confirmed on the molecular level; the bla VIM gene was detected in 23 strains, the bla NDM gene in 26 strains and the bla OXA-48 gene in two strains.Conclusion. There was complete agreement between the carbapenemases detected by the genetic method and the results obtained with phenotypic methods.