An increase of carbapenemase-producing Bacteroides fragilis infections is observed. To detect such a resistance in B. fragilis, several tests exist that are expensive or show poor sensitivity and specificity. Therefore, we upgraded the Anaerobic Carbapenem Inactivation Method (Ana-CIM) to easily screen for carbapenemase-producing B. fragilis. The presence of carbapenemase cfiA gene was identified in 50 B. fragilis isolates by PCR. We modified the Ana-CIM by (1) increasing the bacterial inoculum, and (2) measuring the differences in diameter between the negative control and the testing disc. We correctly classified the cfiA-negative and positive isolates and could define a cut-off of positivity at 2 mm. Our modified Ana-CIM allowed to correctly discriminate the 31 cfiA-positive with meropenem MICs ranging from 1 to > 32 µg/mL. We anticipate that our modified Ana-CIM could be used in most clinical laboratories to easily screen for carbapenemase-producing B. fragilis, even at low levels.

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.

Carbapenem-resistant Enterobacterales are a growing problem in healthcare systems worldwide. While whole-genome sequencing (WGS) has become a powerful tool for analyzing transmission and possible outbreaks, it remains laborious, and the limitations in diagnostic workflows are not well studied. The aim of this study was to compare the performance of WGS and real-time multiplex PCR (RT-qPCR) for diagnosing carbapenem-resistant Enterobacterales. In this study, we analyzed 92 phenotypically carbapenem-resistant Enterobacterales, sent to the University Hospital Heidelberg in 2019, by the carbapenem inactivation method (CIM) and compared WGS and RT-qPCR as genotypic carbapenemase detection methods. In total, 80.4% of the collected isolates were identified as carbapenemase producers. For six isolates, discordant results were recorded for WGS, PCR and CIM, as the carbapenemase genes were initially not detected by WGS. A reanalysis using raw reads, rather than assembly, highlighted a coverage issue with failure to detect carbapenemases located in contigs with a coverage lower than 10×, which were then discarded. Our study shows that multiplex RT-qPCR and CIM can be a simple alternative to WGS for basic surveillance of carbapenemase-producing Enterobacterales. Using WGS in clinical workflow has some limitations, especially regarding coverage and sensitivity. We demonstrate that antimicrobial resistance gene detection should be performed on the raw reads or non-curated draft genome to increase sensitivity.

UNASSIGNED: To compare the sensitivity and specificity before and after the addition of Triton X-100 in the modified Hodge test (MHT) and carbapenem inactivation method (CIM) for the detection of carbapenemase in Acinetobacter baumannii.
UNASSIGNED: A total of 135 isolates of A. baumannii (83 carbapenem-resistant and 52 carbapenem-sensitive) were selected and the carbapenemase genotypes were detected using PCR. Carbapenemase phenotypes were tested using the MHT, Triton-MHT (THT), CIM, modified CIM (mCIM), and Triton-CIM (TCIM). Different concentrations (0.05, 0.1, 0.25, and 0.5% v/v) of Triton X-100 were used in the TCIM.
UNASSIGNED: The sensitivity was determined to be 59.03% (MHT), 100% (THT), 6.02% (CIM), 8.43% (mCIM), 71.08% (TCIM 0.05%), 100% (TCIM 0.1%), 97.59% (TCIM 0.25%), and 96.38% (TCIM 0.5%) in 83 carbapenemase-producing isolates, and the specificity for each of these methods was 100%.
UNASSIGNED: The addition of Triton X-100 while using the MHT and CIM could significantly improve the sensitivity in the detection of A. baumannii carbapenemase with a specificity of 100%. A concentration of 0.1% v/v Triton X-100 showed the best results in TCIM.

Infections caused by carbapenem-resistant Enterobacterales (CRE) present an important therapeutic problem, as there are limited number of effective therapeutic alternatives available. In this study, phenotypic and genotypic methods were used to characterize carbapenemase-production and other resistance-determinants (AmpC and ESBL-production, efflux pump-overexpression) in 50 isolates (Klebsiella spp. n = 35, Escherichia coli n = 12 and Enterobacter cloacae complex n = 3) collected at the Albert Szent-Györgyi Clinical Center (University of Szeged) between 2014 and 2017. Minimum inhibitory concentrations of meropenem, sulfamethoxazole/trimethoprim, tigecycline, amikacin, moxifloxacin, colistin and fosfomycin were also determined. 24% of isolates were AmpC-producers, while 30% carried blaCTX-M ESBL-genes. Carbapenemase-genes were detected in 18 (36%) of the tested isolates: in 2 isolates blaNDM, in 6 isolates blaOXA-48-like and in 12 isolates, blaVIM was detected by PCR. The species-distribution for isolates positive for carbapenemase-genes was the following: Klebsiella pneumoniae n = 11, Klebsiella oxytoca n = 1, E. coli n = 5, E. cloacae complex n = 1. Efflux pump-overexpression based on the PAβN-screening agar was shown in n = 3 of the tested strains. In nine isolates (18%), carbapenemase and ESBL-genes were detected simultaneously. Highest levels of resistance were noted for fosfomycin (74%) and moxifloxacin (70%), while all isolates were susceptible to colistin. Among applied phenotypic tests in this study the modified carbapenem inactivation method (mCIM) proved to be the most accurate one compared to that of PCR results.

BACKGROUND: The emergence and spread of carbapenemase-producing Enterobacterales (CPE) is a worldwide public health threat. Rapid and accurate detection of CPE is essential to prevent their dissemination within health care settings. The aim of this study was to evaluate the performance of CIM, mCIM and mCIM with ammonium bicarbonate (mCIM-A) methods by using different interpretation criteria for detection of carbapenemases.
METHODS: One hundred and fifty-three Klebsiella pneumoniae isolates previously characterized by molecular tests, including 133 carbapenemase producers and 20 non-carbapenemase producers, were collected in this study. CIM and mCIM tests were performed as described previously. mCIM-A by adding 50 mM ammonium bicarbonate to the bacterial suspension prepared in tryptic soy broth. The inhibition zone diameter of around meropenem disc was measured and interpreted as positive according to i) Pierce and colleagues (<19 mm), ii) EUCAST meropenem susceptibility breakpoint (<22).
RESULTS: CIM, although seems to be good for carbapenemases other than OXA-48-like and NDM, is not satisfactory (42.3% and 83.4%, respectively) for those enzymes with any of the interpretation criteria. OXA-48-like and NDM were detected with a better performance (88.7% and 92.8, respectively) with mCIM when results were interpreted according to <22 mm zone diameter for OXA-48-like and NDM. The best results were obtained with mCIM-A using <22 mm criteria without any difference in the results of other enzymes and negative strains.
CONCLUSIONS: mCIM-A method interpreted with <22 mm meropenem zone diameter seems to be preferable compared to CIM and mCIM. mCIM-A is simple and useful tool for identification of CPEs in clinical microbiology laboratories.

Objectives: There is an urgent need for accurate and fast diagnostic tests to identify carbapenemase-producing bacteria. Here we used Bacillus stearothermophilus as an indicator strain in the format of the carbapenem inactivation method (CIM) procedure to develop a rapid carbapenemase phenotype detection method: CIMB.S. Methods: The CIMB.S test was derived from the mCIM, where B. stearothermophilus replaced Escherichia coli as the indicator strain. The test bacteria were incubated in the presence of imipenem for 30 min, and then, aliquots were placed on colorimetric plates, and incubation was continued for 3.5 h at 60°C. We examined 134 clinical strains to evaluate the CIMB.S performance. Results: The CIMB.S can be completed in 4 h, and we successfully identified 38/39 (97.4%) carbapenemase-producing Enterobacteriaceae, including 17/18 (94.4%) carbapenemase-producing Pseudomonas aeruginosa and 18/19 (94.7%) carbapenemase-producing Acinetobacter baumannii. All non-carbapenemase producers we tested were negative and included Enterobacteriaceae (n = 36), P. aeruginosa (n = 17), and A. baumannii (n = 5). Conclusions: The CIMB.S test is a rapid carbapenemase phenotype detection method requiring only 4 h of total work time and displays high sensitivity and specificity.

BACKGROUND: The carbapenem inactivation method (CIM) is a cost-effective assay for detecting carbapenemases. However, its interpretation is unclear for Pseudomonas spp. We evaluate its accuracy when meropenem is changed to imipenem.
METHODS: We analyzed 266 P. aeruginosa isolates. The CIM method consists of: resuspend bacterial colonies (a full 10μL loop) in 400μL water, in which a 10μg disk of meropenem/imipenem is immersed. After 2h of incubation (35°C), remove the disk, place it onto a Mueller-Hinton agar plate previously inoculated with Escherichia coli (ATCC 25922), and incubate at 35 ̊C between 18-24 h. Interpretation criteria (mm of inhibition zone): ≤19mm, positive; ≥25mm negative; 20-24mm, undetermined.
RESULTS: Imipenem improves the sensitivity and specificity of CIM when compared to meropenem (99.4% and 98.9%, vs. 91.9% and 94.7%, respectively).
CONCLUSIONS: The accuracy of CIM for carbapenemase detection in P. aeruginosa is increased with the use of imipenem.

The modified carbapenem inactivation method (mCIM) is a simple phenotypic screening method for detecting carbapenemase production by Enterobacteriaceae and Pseudomonas aeruginosa. We recently developed another modified carbapenem inactivation method (CIMTris), in which carbapenemase is extracted from bacteria with Tris-HCl buffer, to detect carbapenemase production by Acinetobacter and Pseudomonas species. This study describes an improved carbapenem inactivation method, CIMTrisII, for detecting carbapenemase production by Gram-negative pathogens, including Enterobacteriaceae, Acinetobacter and Pseudomonas species. CIMTrisII was different from CIMTris in the concentration of Meropenem disks (5-µg MEM disks vs. 10-µg MEM disks), the inoculum volume of the bacteria (a 5-µl loopful vs. a 10 µl loopful) and the incubation time (1 vs. 2 h). CIMTrisII showed an overall sensitivity of 99.3 % and an overall specificity of 95.0 % for tested isolates. In comparison, CIMTris showed a sensitivity of 96.1 % and a specificity of 96.3 %, and mCIM showed a sensitivity of 67.1 % and a specificity of 100 %. CIMTrisII is thus deemed useful for detecting carbapenemase production by Gram-negative pathogens.

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