Detection Of Carbapenemase Activity Research Articles

Multicenter Performance Evaluation of MALDI-TOF MS for Rapid Detection of Carbapenemase Activity in Enterobacterales: The Future of Networking Data Analysis With Online Software.

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: “software design,” “intercenter evaluation,” and “clinical validation.” First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy.

Prevalence of Multidrug-resistant and Carbapenemase-producing Klebsiella Pneumoniae and Pseudomonas Aeruginosa Isolates in Tertiary Care Hospital in Kathmandu, Nepal

Carbapenemases are the enzymes that catalyze β–lactam groups of antibiotics. The carbapenemase producers are resistant to β–lactam antibiotics and are usually multidrug-resistant bacteria challenging widely used therapeutics and treatment options. Therefore, the detection of carbapenemase activity among clinical isolates is of great therapeutic importance. We aimed to study the MDR and carbapenemase-producing Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from various clinical samples at a tertiary care hospital in Nepal. A total of 3,579 clinical samples were collected from the patients visiting the Department of Microbiology, B&B Hospital, Gwarko, Lalitpur. The samples were processed to isolate K. pneumoniae and P. aeruginosa and then subjected to antibiotic susceptibility testing (AST) by the Kirby-Bauer disk diffusion method. Phenotypic detection of carbapenemase activity was performed in the imipenem-resistant isolates by the modified Hodge test (MHT). Of the total samples, 1,067 (29.8%) samples showed significant growth positivity, out of which 190 (17.3%) isolates were K. pneumoniae and 121 (11.3%) were P. aeruginosa. Multidrug resistance was seen in 70.5% of the K. pneumoniae isolates and 65.3% of the P. aeruginosa isolates. Carbapenemase production was confirmed in 11.9%, and 12.2% of the imipenem-resistant K. pneumoniae and P. aeruginosa isolates, respectively, by the MHT. This study determined the higher prevalence of MDR among K. pneumoniae and P. aeruginosa; however, carbapenemase production was relatively low.

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.

Modified CIM test as a useful tool to detect carbapenemase activity among extensively drug-resistant Klebsiella pneumoniae, Escherichia coli and Acinetobacter baumannii

PurposeTimely detection of carbapenemases is essential for developing strategies to control the spread of infections by carbapenem-resistant isolates. The purpose of this study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant isolates of Acinetobacter baumannii, Klebsiella pneumoniae, and Escherichia coli. In addition, the efficacy of the modified Hodge test (MHT), Carba NP test, and modified carbapenem inactivation method (mCIM) were compared.MethodsA total of 122 carbapenem-resistant clinical isolates including 77 K. pneumoniae, 39 A. baumannii, and six E. coli were collected from hospitalized patients. Three phenotypic methods, including the MHT, Carba NP test, and mCIM were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was performed to detect carbapenemase-encoding genes.ResultThe sensitivity and specificity of the MHT were 75.0% and 100%, respectively. In addition, Carba NP displayed 80.8% sensitivity and 100% specificity, whereas the sensitivity and specificity were 90.4% and 100% for the mCIM test, respectively. Among carbapenem-resistant isolates, 70, 84, and 87 isolates exhibited positive results according to the MHT, Carba NP test, and mCIM, respectively. PCR indicated the presence of one or more carbapenemase genes in 119 of carbapenem-resistant isolates, with blaKPC and blaVIM being the most commonly encountered. Co-production of ‘KPC and OXA-48’, ‘KPC and VIM’, and ‘KPC and IMP’ was detected in three, nine, and seven isolates, respectively.ConclusionOur results confirm that the mCIM test is a useful tool for the reliable detection of carbapenemase activity in enterobacterial isolates, especially in clinical microbiological laboratories with limited resources.

Direct Detection and Discrimination of Carbapenemases of Acinetobacter baumannii from Uncultured Tracheal Aspirates

Carbapenemases play important roles in conferring resistance to beta-lactam antibiotics, including the carbapenems. Detection of carbapenemase activity helps to understand the possible mechanism(s) of carbapenem resistance. Identification of carbapenemases is currently being done by various phenotypic methods and molecular methods. However, innovative biochemical and spectrophotometric methods are desirable as they will be easy to perform, affordable, and rapid. A novel chromogenic method called Carba NP test was introduced recently to screen for carbapenemases in clinical isolates of gram-negative pathogens. We adopted this assay (1) to detect the total carbapenemase activity, (2) to discriminate Class A, B, and D carbapenemases with inhibitors, (3) to compare with carbapenemase genotype, and (4) for direct differential diagnosis of carbapenemases in uncultured clinical sample such as tracheal aspirate. The study included 132 purulent tracheal aspirates. All samples were processed and screened by a protocol optimized in our laboratory, which showed good sensitivity and correlation with genotyping and conventional phenotyping. Our protocol not only offers the fastest way to identify the pathogen but also its carbapenemase profile, directly from uncultured clinical samples in less than 4 hr. Our protocol is currently being validated on other types of clinical specimens in our laboratory.

Blood-Modified Carbapenem Inactivation Method: a Phenotypic Method for Detecting Carbapenemase-Producing Enterobacteriaceae Directly from Positive Blood Culture Broths.

A variant of the modified carbapenem inactivation method (mCIM) was developed to detect carbapenemase activity directly from positive blood culture broths. The method, termed "Blood-mCIM," was evaluated using Bactec blood culture bottles (Becton, Dickinson and Company, Franklin Lakes, NJ) inoculated with 27 different carbapenemase-producing Enterobacteriaceae (CPE) isolates and 34 different non-CPE isolates. The assay was positive for all blood culture broths inoculated with CPE isolates and negative for all blood culture broths inoculated with non-CPE isolates, corresponding to a diagnostic sensitivity and specificity of 100%, respectively. This assay is inexpensive using "off the shelf" reagents, does not require centrifugation or mechanical lysis, and can be readily implemented in any clinical microbiology laboratory. The Blood-mCIM should facilitate expedient administration of antimicrobial therapy targeted toward CPE bloodstream infections and assist infection control and public health surveillance.

Detection of carbapenemase-producing Enterobacterales and the BD Phoenix CPO Detect panel

ABSTRACTIntroduction: Treatment options for infections with carbapenem-resistant Enterobacterales are strongly limited. New antimicrobials are not effective against all types of carbapenemases. Therefore, rapid and reliable antimicrobial susceptibility testing and identification of the resistance mechanism are important.Areas covered: We assess several methods to determine carbapenemase production of Enterobacterales in culture and discuss the value of the novel automated BD Phoenix CPO Detect (BDPCPO) panel for the detection and classification of carbapenemases.Expert opinion: The meropenem minimum inhibitory concentration (MIC) range used in the BDPCPO panel includes the EUCAST screening breakpoint for the detection of carbapenemase producers. The phenotypic, inhibitor-based assay for detection of carbapenemase activity in the BDPCPO panel displays high sensitivity for carbapenemase detection while its specificity is modest. Therefore, confirmation testing of positive results is warranted. Nevertheless, implementing the BDPCPO panel has the potential to reduce time-to-result for detection and classification of carbapenemase producers.

High incidence of MDR and XDR Pseudomonas aeruginosa isolates obtained from patients with ventilator-associated pneumonia in Greece, Italy and Spain as part of the MagicBullet clinical trial.

To characterize the antimicrobial susceptibility, molecular epidemiology and carbapenem resistance mechanisms in Pseudomonas aeruginosa isolates recovered from respiratory tract samples from patients with ventilator-associated pneumonia enrolled in the MagicBullet clinical trial. Isolates were collected from 53 patients from 12 hospitals in Spain, Italy and Greece. Susceptibility was determined using broth microdilution and Etest. MALDI-TOF MS was used to detect carbapenemase activity and carbapenemases were identified by PCR and sequencing. Molecular epidemiology was investigated using PFGE and MLST. Of the 53 isolates, 2 (3.8%) were considered pandrug resistant (PDR), 19 (35.8%) were XDR and 16 (30.2%) were MDR. Most (88.9%) of the isolates from Greece were MDR, XDR or PDR, whereas fewer of the isolates from Spain (33.3%) and Italy (43.5%) showed antibiotic resistance. Three Greek isolates were resistant to colistin. Overall, the rates of resistance of P. aeruginosa isolates to imipenem, ciprofloxacin, ceftolozane/tazobactam and ceftazidime/avibactam were 64.1%, 54.7%, 22.6% and 24.5%, respectively. All isolates resistant to ceftolozane/tazobactam and ceftazidime/avibactam (Greece, n = 10; and Italy, n = 2) carried blaVIM-2. Spanish isolates were susceptible to the new drug combinations. Forty-eight restriction patterns and 27 STs were documented. Sixty percent of isolates belonged to six STs, including the high-risk clones ST-111, ST-175 and ST-235. MDR/XDR isolates were highly prevalent, particularly in Greece. The most effective antibiotic against P. aeruginosa was colistin, followed by ceftolozane/tazobactam and ceftazidime/avibactam. blaVIM-2 is associated with resistance to ceftolozane/tazobactam and ceftazidime/avibactam, and related to highly resistant phenotypes. ST-111 was the most frequent and disseminated clone and the clonal diversity was lower in XDR and PDR strains.

Evaluation of the automated BD Phoenix CPO Detect panel in combination with the β-CARBA assay for detection and classification of carbapenemase-producing Enterobacterales

Recently, the CPO Detect panel for the detection of carbapenemase-producing, Gram-negative bacteria was introduced for the Phoenix semi-automated antimicrobial susceptibility testing system. The CPO Detect assay aims to detect carbapenemase activity (P/N test) and to type carbapenemase producers according to the Ambler classification (Ambler test). The P/N test-based detection of carbapenemase producers was 100% sensitive and 55.3% specific in the assessment of 57 carbapenemase-producing and 38 non-carbapenemase-producing Enterobacterales. False-positive test isolates in the P/N test arose from carbapenemase-non-producing, but carbapenem-non-susceptible isolates. In contrast, using the Ambler test-based approach for carbapenemase detection resulted in a specificity of 100% and a sensitivity of 79%. In order to improve the overall performance, we established an algorithm that additionally included the colorimetric β-CARBA assay as downstream test for P/N test-positive isolates, which remained un-typed in the Ambler test. This algorithm displayed an overall sensitivity and specificity of 98.3% and 100%, respectively. Our data demonstrate that the combination of the CPO Detect assay with the β-CARBA test allows for rapid detection and classification of carbapenemase-producing Enterobacterales.

Rapid detection of carbapenemase activity of Enterobacteriaceae isolated from positive blood cultures by MALDI-TOF MS

BackgroundMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proved to be a useful tool for identification of pathogens directly isolated from blood cultures in clinical microbiology laboratories. β-Lactam antibiotics are commonly used for treatment of bloodstream infections caused by Enterobacteriaceae strains, and carbapenem is the superlative class of β-lactam antibiotics. Since the carbapenem resistance rate of Enterobacteriaceae strains raised year by year, efficient detection of carbapenemase activity and timely delivery of carbapenem susceptibility reports of Enterobacteriaceae strains isolated from blood cultures is important for clinicians.MethodsWe used 64 simulated blood cultures to establish the method of MALDI-TOF MS based ertapenem hydrolysis assay. The cutoff value of logRQ calculated from the peaks intensity of ertapenem and its hydrolysate was first set to identify the strains with carbapenemase activity. Then, we detected and calculated the logRQ values of 385 Enterobacteriaceae strains from positive clinical blood cultures to distinguish the carbapenemase producers and noncarbapenemase producers.ResultsThe mean logRQ value of 32 noncarbapenemase producers was − 0.85 ± 0.14 in simulated blood cultures, while the logRQ value of 32 carbapenemase producers was 0.87 ± 0.55. Thus, the cutoff value of logRQ was set at − 0.45 with sensitivity of 100% and specificity of 100%. In 385 clinical positive blood cultures, the logRQ values of all carbapenem-susceptible Enterobacteriaceae strains (81.3%, 313/385) were < − 0.45. Comparing with the detection of carbapenemase genes, carbapenem-resistant Enterobacteriaceae strains (18.7%, 72/385) were well distinguished by MALDI-TOF MS based ertapenem hydrolysis assay with a sensitivity of 92.5% and specificity of 100%.ConclusionsOur data show that MALDI-TOF MS based ertapenem hydrolysis assay is a rapid and accurate method to detect carbapenemase activity of Enterobacteriaceae strains from positive blood cultures, and can be routinely performed in clinical microbiology laboratories.

Genetic characterization of novel class 1 Integrons In0, In1069 and In1287 to In1290, and the inference of In1069-associated integron evolution in Enterobacteriaceae

BackgroundThis study aims to characterize genetically related class 1 integrons In1069, In893 and In1287 to In1290, and to further propose a scheme of stepwise integration or excision of individual gene cassettes (GCs) to generation of these integron variations.MethodsSix of 139 non-redundant Enterobacteriaceae strains were studied by bacterial antimicrobial susceptibility testing, detection of carbapenemase activity, and integron sequencing and sequence comparison.ResultsSix novel class 1 integrons, In0, In1069, and In1287 to In1290, together with the previously characterized In893, were determined from the above strains. An unusual blaKPC-2-carrying In0 and the blaIMP-30-carrying In1069 coexists in a single isolate of Escherichia coli. In0 contains a PcH1 promoter and a truncated aacA4’-3 gene cassette (GCaacA4’-3), as well as a blaKPC-2-containing region of Tn6296 integrated between PcH1 and GCaacA4’-3. In1069 carries GCblaIMP-30 and GCaacA4’-3 in this order. The other five integrons, In893 and In1287 to In1290, are genetically related to In1069, and all possess a core GCaacA4’-3. The integration or excision of one or more individual gene cassettes, such as GCblaIMP-30, GCaadA16, GCcatB3, GCarr3 and GCdfrA27, upstream or downstream of GCaacA4’-3 generates various gene cassettes arrays among these five integrons.ConclusionsThese findings provide the insight into stepwise and parallel evolution of In1069-associated integron variations likely under antibiotic selection pressure in clinical settings.

FRI-2 carbapenemase-producing Enterobacter cloacae complex in the UK.

Detection of rarer carbapenemases is challenging, as it requires molecular assays with comprehensive coverage or the use of phenotypic methods for the detection of carbapenemase activity. We describe a new class A carbapenemase, FRI-2, in an Enterobacter cloacae complex isolate following implementation of an in-house multiplex PCR for the detection of 'rare' class A carbapenemases. MICs were determined by agar dilution. A carbapenem-resistant E. cloacae complex isolate was tested by PCR for the class A carbapenemases blaKPC, blaFRI, blaIMI, blaGES and blaSME. Carbapenemase activity was assessed using Carba NP and the carbapenem inactivation method. Whole genome and plasmid analyses of the clinical isolate and the FRI-2 transformant were performed by WGS, respectively. Typing was carried out by PFGE. The E. cloacae complex isolate showed resistance to imipenem (MIC = 16 mg/L), meropenem (MIC = 8 mg/L) and ertapenem (MIC = 8 mg/L), but remained susceptible to piperacillin/tazobactam (MIC = 8 mg/L). Carbapenemase activity was confirmed in the isolate by both phenotypic methods. A blaFRI-1-like gene was detected by PCR and analysis of WGS data of the clinical isolate identified an ORF of 885 bp, which showed 97% nucleotide identity with blaFRI-1 and was named blaFRI-2. WGS of the transformant indicated blaFRI-2 was located on a 108 kb IncF/IncR plasmid. The FRI-2-positive E. cloacae complex isolate belonged to a novel ST (ST829). The possible circulation of rarer carbapenemases in clinical settings highlights the role of phenotypic tests to detect carbapenemase activity when molecular assays are negative for the 'big 5' carbapenemase families.

Evaluation of a modified meropenem hydrolysis assay on a large cohort of KPC and VIM carbapenemase-producing Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) have spread globally and represent a serious and growing threat to public health. The introduction of rapid and sensitive methods for the detection of carbapenemase-producing bacteria is of increasing importance. The carbapenemase production can be detected using non-molecular methods (such as the modified Hodge test, the synergy test, the Carba NP test and the antibiotic hydrolysis assays) and DNA-based methods. In this study, we propose a modified version of a previously described meropenem hydrolysis assay (MHA) by MALDI-TOF MS for the phenotypic detection in 2h of carbapenemase-producing Enterobacteriaceae. The MHA was successfully applied to detect carbapenemase activity in 981 well-characterized Enterobacteriaceae strains producing KPC or VIM carbapenemases, and in 146 carbapenem fully susceptible strains. This assay, applied also to NDM and OXA-48-producing strains and to CRE with resistance mechanisms other than carbapenemase production, has proved to be able to distinguish between carbapenemase-producing and -nonproducing Enterobacteriaceae.As already stated and as observed in our hands, MHA by MALDI-TOF MS analysis is independent from the type of carbapenemases involved, it is faster and easier to perform/interpret than culture-based methods. On the other hand, it cannot detect other carbapenem resistance mechanisms, such as porin alterations and efflux mechanisms.

Evaluation of the carbapenem inactivation method (CIM) for detecting carbapenemase activity in enterobacteria

ObjectivesThe objective of this study was to evaluate the accuracy of the CIM test in the detection of carbapenemase activity in 124 strains of Enterobacteriaceae. MethodsA panel of 124 previously characterized Enterobacteriaceae was tested: 77 strains producing the following carbapenemase families: KPC (n = 14), GES (n = 22), NDM (n = 19), VIM (n = 4), IMP (n = 4) and OXA-48 (n = 14) and 47 non-carbapenemase producers. For the CIM method, an active susceptibility meropenem disc was exposed to a bacterial suspension of a test strain; when a carbapenemase is produced, the antibiotic is inactivated allowing uninhibited growth of an indicator strain after overnight incubation. A clear inhibition zone (≥20 mm) was considered indicative of no-carbapenemase activity. ResultsAll KPC, NDM, VIM, IMP or OXA-48 producing strains were unequivocally detected with the CIM test. CIM false negative results were obtained with eleven Enterobacter cloacae producing GES-6. Two other E. cloacae not producing carbapenemase (one with SHV-12, one hyperproducing AmpC) were positive by the test. The sensitivity and specificity of the assay compared to those of molecular methods were 85.7% and 95.7%, respectively. ConclusionsThe CIM method proved to be inexpensive and easy to interpret. It provided less than optimal results in the detection of GES-6 activity.

Rapid direct detection of carbapenemase-producing Enterobacteriaceae in clinical urine samples by MALDI-TOF MS analysis.

Development of an automated MALDI-TOF MS-based method for the rapid, direct detection of carbapenemase-producing Enterobacteriaceae in clinical urine samples within 90 min of sample reception. A total of 3041 urine samples were processed by flow cytometry, and a cut-off value of ≥1.5 × 10 5 bacteria/mL was used to select samples for the study. Following these criteria, 608 samples were selected for direct bacterial identification. Detection of carbapenemase activity by MALDI-TOF MS analysis was only performed after reliable direct identification of Gram-negative bacilli. A novel protocol was developed for extracting bacteria from urine samples by using the Sepsityper Kit (Bruker Daltonik, Germany). Carbapenem resistance was detected with imipenem as an antibiotic marker and the results were automatically interpreted using the STAR-BL module of MALDI-TOF Biotyper Compass software (Bruker Daltonik, Germany). The MALDI-TOF MS-based assay yielded direct reliable identification of 91% (503/553) of the samples. The assay showed 100% sensitivity (30/30) and specificity (454/454) for detecting carbapenemase activity within 90 min of sample reception. Isolates included in the study were further characterized by PCR and sequencing, and bla OXA-48 was detected from all isolates that tested positive in the MALDI-TOF MS-based resistance assay. The proposed protocol for the direct analysis of urine samples by MALDI-TOF MS is suitable for use in clinical laboratories to identify bacteria and detect carbapenemase activity, thus saving at least 24-48 h relative to current routine methods.

Detection of carbapenemase activity in Enterobacteriaceae using LC-MS/MS in comparison with the neo-rapid CARB kit using direct visual assessment and colorimetry

It has been described that the sensitivity of the Carba NP test may be low in the case of OXA-48-like carbapenamases and mass spectrometry based methods as well as a colorimetry based method have been described as alternatives. We evaluated 84 Enterobacteriaceae isolates including 31 OXA-48-like producing isolates and 13 isolates that produced either an imipenemase (IMP; n=8), New Delhi metallo-β-lactamase (NDM; n=3), or Klebsiella pneumoniae carbapenemase (KPC; n=2), as well as 40 carbapenemase negative Enterobacteriaceae isolates. We used the Neo-Rapid CARB kit, assessing the results with the unaided eye and compared it with a colorimetric approach. Furthermore, we incubated the isolates in growth media with meropenem and measured the remaining meropenem after one and 2h of incubation, respectively, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Whilst all carbapenemase producing isolates with the exception of the OXA-244 producer tested positive for both the Neo-rapid CARB test using the unaided eye or colorimetry, and the 13 isolates producing either IMP, NDM or KPC hydrolysed the meropenem in the media almost completely after 2h of incubation, the 31 OXA-48-like producing isolates exhibited very variable hydrolytic activity when incubated in growth media with meropenem. In our study, the Neo-Rapid CARB test yielded a sensitivity of 98% for both the traditional and the colorimetric approach with a specificity of 95% and 100% respectively. Our results indicate that the Neo-Rapid CARB test may have use for the detection of OXA-48 type carbapenemases and that it may be particularly important to ensure bacterial lysis for the detection of these weaker hydrolysers.

Functional categorization of carbapenemase-mediated resistance by a combined genotyping and two-tiered Modified Hodge Test approach.

The functional relationship between the detection of carbapenemase activity and phenotypic resistance in Gram-negative bacterial pathogens is often ill-defined. To address this issue, we developed a two-tiered Modified Hodge Test approach for carbapenemase detection and typing, in which the use of Pseudomonas aeruginosa strain PAO1 and Escherichia coli as indicator strains conferred two levels of sensitivities to carbapenemases. When applied alongside PCR genotyping tests for existence of known carbapenemase genes in 92 carbapenem resistant clinical isolates, this method is extremely useful in elucidating the relative role by which different enzymes contributed to the prevalent carbapenem-resistance phenotypes. With this study approach, we showed that the proportion of P. aeruginosa and Acinetobacter baumannii strains whose carbapenem resistance phenotypes could at least be partially attributed to carbapenemase were 34 and 89%, respectively. Our data also facilitates detailed functional categorization of carbapenem resistance phenotypes on the basis of the types and activities of detectable carbapenemase produced by the test organism. For example, six A. baumannii isolates harboring the blaOXA-51/23-like gene without detectable enzymatic activities were identified, suggesting that other resistance mechanisms may be involved. On the other hand, there were seven P. aeruginosa strains which produced carbapenemase phenotype without harboring known carbapenemase genes, inferring the existence of some hitherto unknown resistance determinants. Findings in this work therefore provide a comprehensive view on the cellular basis of carbapenem resistance phenotypes in major Gram-negative bacterial species, paving the way for development of novel strategies to reverse the effects of the major resistance mechanisms concerned.

The carbapenem inactivation method (CIM), a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

Rapid, sensitive and specific detection of OXA-48-like-producing Enterobacteriaceae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The emergence of Enterobacteriaceae producing the class D carbapenemase OXA-48 has become a public health concern, particularly in southern Europe and in the Mediterranean area. The existing MALDI-TOF MS assays that detect carbapenemase activity have disregarded the Enterobacteriaceae producing OXA-48-like enzymes. Here, we assessed the ability of an optimized MALDI-TOF MS imipenem hydrolysis assay to detect OXA-48-like-producing Enterobacteriaceae.We tested 372 non duplicate isolates of Enterobacteriaceae from 9 species, among which 92 harbored a blaOXA-48-like gene and 180 were non-susceptible to ertapenem. The vast majority (98.9%) of the OXA-48-like producers were detected and 97.8% of the ertapenem non-susceptible isolates were correctly classified. From the agar plate to the final result, the turnaround time of the test was 90min.This quick, easy and accurate MALDI-TOF MS test can expedite the implementation of hygiene measures to prevent dissemination of OXA-48-like-producing Enterobacteriaceae and limit outbreaks.

Determining Carbapenemase Activity with 18O Labeling and Targeted Mass Spectrometry

Carbapenems are broad spectrum antibiotics considered as a "last resort" medicine to treat bacterial infections. Carbapenem-hydrolyzing β-lactamases (also called carbapenemases), however, can confer bacterial resistance and represent a serious health threat. Here, we report a novel approach using (18)O labeling and selected reaction monitoring to detect carbapenemase activity from pathogenic microorganisms in a rapid and quantitative manner. Four model bacterial strains bearing various classes of β-lactamases were tested for their capability to hydrolyze Meropenem, an FDA-approved carbapenem drug. We were able to predict the Meropenem resistance of these bacteria on the basis of their carbapenemase activity, suggesting the great potential of our method in clinical diagnostics.