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Combined use of the modified Hodge test and carbapenemase inhibition test for detection of carbapenemase-producing Enterobacteriaceae and metallo-beta-lactamase-producing Pseudomonas spp.
Abstract and Figures
Background:
We evaluated the combined use of the modified Hodge test (MHT) and carbapenemase inhibition test (CIT) using phenylboronic acid (PBA) and EDTA to detect carbapenemase-producing Enterobacteriaceae (CPE) and metallo-β-lactamase (MBL)-producing Pseudomonas spp.
Methods:
A total of 49 isolates of CPE (15 Klebsiella pneumoniae carbapenemase [KPC], 5 Guiana extended-spectrum β-lactamase [GES]-5, 9 New Delhi metallo-β-lactamase [NDM]-1, 5 Verona integron-encoded metallo-β-lactamase [VIM]-2, 3 imipenem-hydrolyzing β-lactamase [IMP], and 12 oxacillinase [OXA]-48-like), 25 isolates of MBL-producing Pseudomonas spp. (14 VIM-2 and 11 IMP), and 35 carbapenemase-negative controls were included. The MHT was performed for all isolates as recommended by the Clinical and Laboratory Standards Institute. Enhanced growth of the indicator strain was measured in mm with a ruler. The CIT was performed by directly dripping PBA and EDTA solutions onto carbapenem disks that were placed on Mueller-Hinton agar plates seeded with the test strain.
Results:
Considering the results of the MHT with the ertapenem disk in Enterobacteriaceae and Pseudomonas spp., the CIT with the meropenem disk in Enterobacteriaceae, and the imipenem disk in Pseudomonas spp., three combined disk tests, namely MHT-positive plus PBA-positive, EDTA-positive, and MHT-positive plus PBA-negative plus EDTA-negative, had excellent sensitivity and specificity for the detection of KPC- (100% sensitivity and 100% specificity), MBL- (94% sensitivity and 100% specificity), and OXA-48-like-producing isolates (100% sensitivity and 100% specificity), respectively.
Conclusions:
Combined use of the MHT and CIT with PBA and EDTA, for the detection of CPE and MBL-producing Pseudomonas spp., is effective in detecting and characterizing carbapenemases in routine laboratories.
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... The synthesis of carbapenemase was assessed using the carbapenemase inhibition test 12 . AmpC enzymes in CREco isolates were detected using the modi ed three-dimensional extract test 13 . ...
Objective: The objective of this study was to conduct a molecular epidemiological study of carbapenem-resistant Escherichia coli (CREco) within a tertiary hospital situated in the Dabie Mountains region of China, while also elucidating the underlying mechanisms of antimicrobial resistance.
Patients and methods: Between 2018 and 2022, a total of 33 CREco isolates were isolated from 33 patients in a tertiary hospital situated in the Dabie Mountains region of China. Subsequently, the next-generation sequencing of CREco isolates was performed, and the clinical retrospective analysis and the comprehensive bioinformatic analysis were presented. Phenotypic identification of carbapenemase and AmpC-type β-lactamase were also conducted.
Results: Two kinds of carbapenemase genes blaNDM (n = 30) and blaKPC-2 (n = 2) were identified in 33 CREco isolates. Of blaNDM-positive isolates, 24 belonged to blaNDM-5, and the remaining were blaNDM-13 (n = 4), blaNDM-1 (n = 1), and blaNDM-6 (n = 1). The predominant STs of these isolates were ST410 (13.3%, 4/30), ST692 (10.0%, 3/30), and ST156 (10.0%, 3/30). Except for carbapenemase genes, the most prevalent resistance genes were sulfonamide (97%, 32/33), and aminoglycoside (94%, 31/33) genes. By the disserted annotation of the core genetic envirionment of blaNDM and blaKPC, we identified that blaNDM and blaKPC were harbored by Tn125 and Tn6296, respectively. Although the core genetic environment of them were conserved, but the different truncations were underwent in the upstream/downstream of the prototype of Tn125 and Tn6296.
Conclusion: The blaNDM-5 was the principal carbapenem resistance mechanism of CREco isolates in the Dabie Mountains region in China. Besides, two rare NDM variants blaNDM-6 and blaNDM-13 were detected herein, and the dissemination risk of these two genes was needed to be attention and the further surveillance was needed in China.
... These discrepancies could appear due to the type of tested bacteria, as the most addressed pathogens were part of the Enterobacterales, not P. aeruginosa, or due to the subjective interpretation of the results [51]. Even though many previous studies presented the MHT as a good method to identify carbapenemase production at P. aeruginosa [52][53][54], formal organizations do not necessarily agree with this. For example, the standard laboratories guides such as CLSI (Clinical Laboratory Standards Institute) or EUCAST (The European Committee on Antimicrobial Susceptibility Testing) do not consider this phenotypic method for carbapenemase detection reliable, due to the difficulties in interpretation of the results or the poor sensibility and specificity [55,56]. ...
1) Background: The purpose of the study was to describe the activity of mex efflux pumps in Multidrug-Resistant (MDR) clinical isolates of Pseudomonas aeruginosa and to compare the carbapenem-resistance identification tests with PCR; (2) Methods: Sixty MDR P. aeruginosa were analyzed for detection of carbapenemase by disk diffusion inhibitory method, carbapenem inactivation method and Modified Hodge Test. Endpoint PCR was used to detect 7 carbapenemase genes (bla KPC , bla OXA48-like , bla NDM , bla GES-2 , bla SPM , bla IMP , bla VIM) and mcr-1 for colistin resistance. The expression of mexA, mexB, mexC, mexE and mexX genes corresponding to the four main efflux pumps was also evaluated; (3) Results: From the tested strains, 71.66% presented at least one carbapenemase gene, with bla GES-2 as the most occurring gene (63.3%). Compared with the PCR, the accuracy of phenotypic tests did not exceed 25% for P. aeruginosa. The efflux pump genes were present in all strains except one. In 85% of the isolates, an overactivity of mexA, mexB and mostly mexC was detected. Previous treatment with ceftriaxone increased the activity of mexC by more than 160 times; (4) Conclusions: In our MDR P. aeruginosa clinical isolates, the carbapenem resistance is not accurately detected by phenotypic tests, due to the overexpression of mex efflux pumps and in a lesser amount, due to carbapenemase production.
... Sensitivity, 62.5 % Specificity, 62.5% PPV, 31.3% NPV and an overall accuracy of 41.7 22 . Similarly, a study demonstrated that, the modified Hodge test has an excellent sensitivity for detecting enterobacterial isolates producing Ambler class A (KPC) and class D (OXA-48) carbapenemases. ...
... In Colombia and other Latin American countries, different phenotypic tests are used for detecting carbapenemases such as the modified Hodge test (MHT) (10), the carbapenem inactivation method (CIM) (11,12), and the modified CIM method (mCIM) (13), as well as synergy tests with boronic acid for detecting serine-carbapenemases like KPC and ethylenediaminetetraacetic acid (EDTA) for detecting metallo-beta-lactamases such as NDM or VIM (14). Although these methodologies are inexpensive, they are limited by long turnaround times (18 h to 24 h), false positives caused by membrane permeabilization (e.g., EDTA) or hyperproduction of AmpC (e.g., boronic acid), and false negatives because of adjustment of the distance between disks, low levels of enzyme expression, or co-production of carbapenemases. ...
The spread of carbapenem-resistant Pseudomonas aeruginosa and carbapenemase-producing Enterobacterales (CPE) has dramatically impacted morbidity and mortality. COVID-19 pandemic has favored 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 personal protective equipment. Therefore, early CPE detection is considered essential for their control. We aimed to compare conventional phenotypic synergy tests and two lateral flow immunoassays for detecting carbapenemases in Enterobacterales and P. aeruginosa. We analyzed 100 carbapenem-resistant Gram-negative bacilli isolates, 80 Enterobacterales, and 20 P. 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). In total, 76 KPC, seven VIM, one NDM, one OXA-48, and one isolate coproducing KPC + NDM enzymes were included. The concordance of different methods estimated by the 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. Conventional phenotypic synergy tests with boronic acid and EDTA for detecting carbapenemases are suboptimal, and their routine use should be reconsidered. These tests 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. IMPORTANCE Infections due to multidrug-resistant pathogens are a growing problem worldwide. The production of carbapenemases in Pseudomonas aeruginosa and Enterobacterales cause a high impact on the mortality of infected patients. Therefore, it is of great importance to have methods that allow the early detection of these multi-resistant microorganisms, achieving the confirmation of the type of carbapenemase present, with high sensitivity and specificity, with the aim of improving epidemiological control, dissemination, the clinical course to through targeted antibiotic therapy and promoting infection control in hospitals.
... Most of the strains with decreased susceptibility to carbapenems and carbapenemaseproducers were Klebsiella pneumoniae in our study, in accordance with data from the literature (13,14). ...
Carbapenemase-producing Enterobacteriaceae have the potential to rapid dissemination in healthcare settings, becoming a major infection control and public health concern. PCR remains the reference method for identifying these strains, but is still expensive. We evaluated the performance of four phenotypic tests for identifying OXA-48-producing (Oxacillinase-48) Enterobacteriaceae. These were modified Hodge test (MHT), a combination disk test with meropenem alone or in combination with various inhibitors and temocillin, an enzymatic test and an immunochromatographic test. The tests were performed and interpreted according to guidelines or manufacturer’s recommendations. The most frequent microorganism included in the study was Klebsiella pneumoniae. All the tests had 100% specificity, except MHT (87,5%). Sensitivity was 91,25% for MHT, 90% for the combination disk test, 95% for the enzymatic test and 82,50% for the immunochromatographic test.
Background
Rapid and accurate identification of carbapenemase-producing organism (CPO) intestinal carriers is essential for infection prevention and control. Molecular diagnostic methods can produce results in as little as 1 h, but require special instrumentation and are expensive. Therefore, it is urgent to find an alternative method. The broth enrichment-multiplex lateral flow immunochromatographic assay was recently reported, but using it to directly detect CPO intestinal carriers in rectal swabs still requires the evaluation of many samples. The aim of this study was to compare the performance of these two methods, and to explore the control measures of CPO infection.
Methods
Through CPO selective culture, PCR and DNA sequencing, 100 rectal swabs confirmed to be CPO-positive and 100 rectal swabs with negative results were collected continuously. After eluting the rectal swabs with saline, three aliquots were used: one for counting, one for detection by Xpert Carba-R, and one for culture in broth for 0 h, 1 h, 2 h, 3 h and 4 h, followed by NG-Test CARBA 5 assessment. The sensitivity and specificity of the NG-Test CARBA 5 method after different incubation times were calculated. The limit of detection (LoD) of this assay after 4 h broth incubation was estimated by examining the bacterial suspensions and simulated faecal suspensions prepared with CPOs producing different types of carbapenemases.
Results
Xpert Carba-R demonstrated a combined sensitivity of 99.0% and specificity of 98.0%. The sensitivity and specificity were higher than 90.0% for the different enzyme types. The specificities of five common carbapenemases detected by the broth enrichment NG-Test CARBA 5 combined method after different incubation times were 100%. The sensitivities increased with increasing incubation time. At 4 h, the Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), imipenemase (IMP), Verona integron-encoded metallo-beta-lactamase (VIM), and oxacillinase (OXA) -48 detection sensitivities were 93.0%, 96.3%, 100%, 100% and 85.7%, respectively. The LoDs were between 10² and 10⁴ CFU/mL for all five enzymes after 4 h of incubation.
Conclusions
This investigation highlighted that the broth enrichment-multiplex lateral flow immunochromatographic assay can be used as a new method for screening CPOs in rectal swabs.
Polymyxin-carbapenem-resistant Klebsiella pneumoniae (PCR-Kp) with pan (PDR)- or extensively drug-resistant phenotypes has been increasingly described worldwide. Here, we report a PCR-Kp outbreak causing untreatable infections descriptively correlated with bacterial genomes. Hospital-wide surveillance of PCR-Kp was initiated in December-2014, after the first detection of a K. pneumoniae phenotype initially classified as PDR, recovered from close spatiotemporal cases of a sentinel hospital in Rio de Janeiro. Whole-genome sequencing of clinical PCR-Kp was performed to investigate similarities and dissimilarities in phylogeny, resistance and virulence genes, plasmid structures and genetic polymorphisms. A target phenotypic profile was detected in 10% (12/117) of the tested K. pneumoniae complex bacteria recovered from patients (8.5%, 8/94) who had epidemiological links and were involved in intractable infections and death, with combined therapeutic drugs failing to meet synergy. Two resistant bacterial clades belong to the same transmission cluster (ST437) or might have different sources (ST11). The severity of infection was likely related to patients’ comorbidities, lack of antimicrobial therapy and predicted bacterial genes related to high resistance, survival, and proliferation. This report contributes to the actual knowledge about the natural history of PCR-Kp infection, while reporting from a time when there were no licensed drugs in the world to treat some of these infections. More studies comparing clinical findings with bacterial genetic markers during clonal spread are needed.
Background:
The emergence of carbapenemase-producing Enterobacteriaceae (CPE) represents a major clinical problem. Recently, the occurrence of CPE has increased globally, but epidemiological patterns vary across region. We report the trends in the genotypic distribution and antimicrobial susceptibility of CPE isolated from rectal and clinical samples during a four-year period.
Methods:
Between January 2016 and December 2019, 1,254 nonduplicated CPE isolates were obtained from four university hospitals in Korea. Carbapenemase genotypes were determined by multiplex real-time PCR. Antimicrobial susceptibility was profiled using the Vitek 2 system (bioMérieux, Hazelwood, MO, USA) or MicroScan Walkaway-96 system (Siemens West Sacramento, CA, USA). The proportions of carbapenemase genotypes and nonsusceptibility were analyzed using Pearson's chi-square test.
Results:
Among the 1,254 CPE isolates, 486 (38.8%), 371 (29.6%), 357 (28.5%), 8 (0.6%), 8 (0.6%), and 24 (1.9%) were Klebsiella pneumoniae carbapenemase (KPC), oxacillinase (OXA)-48-like, New Delhi metallo-β-lactamase (NDM), imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), and multiple producers, respectively. The predominant species was K. pneumoniae (72.6%), followed by Escherichia coli (6.5%). More than 90% of the isolates harboring KPC, NDM, and OXA-48-like were nonsusceptible to cephalosporins, aztreonam, and carbapenems.
Conclusions:
The impact of CPE is primarily due to KPC-, NDM-, and OXA-48-like-producing K. pneumoniae isolates. Isolates carrying these carbapenemase are mostly multidrug-resistant. Control strategies based on these genotypic distributions and antimicrobial susceptibilities of CPE isolates are required.
Background
The emergence and spread of antimicrobial resistance and infectious agents have challenged hospitals in recent decades. Our aim was to investigate the circulation of target infectious agents using Geographic Information System (GIS) and spatial–temporal statistics to improve surveillance and control of healthcare-associated infection and of antimicrobial resistance (AMR), using Klebsiella pneumoniae complex as a model.
Methods
A retrospective study carried out in a 450-bed federal, tertiary hospital, located in Rio de Janeiro. All isolates of K. pneumoniae complex from clinical and surveillance cultures of hospitalized patients between 2014 and 2016, identified by the use of Vitek-2 system (BioMérieux), were extracted from the hospital's microbiology laboratory database. A basic scaled map of the hospital’s physical structure was created in AutoCAD and converted to QGis software (version 2.18). Thereafter, bacteria according to resistance profiles and patients with carbapenem-resistant K. pneumoniae (CRKp) complex were georeferenced by intensive and nonintensive care wards. Space–time permutation probability scan tests were used for cluster signals detection.
Results
Of the total 759 studied isolates, a significant increase in the resistance profile of K. pneumoniae complex was detected during the studied years. We also identified two space–time clusters affecting adult and paediatric patients harbouring CRKp complex on different floors, unnoticed by regular antimicrobial resistance surveillance.
Conclusions
In-hospital GIS with space–time statistical analysis can be applied in hospitals. This spatial methodology has the potential to expand and facilitate early detection of hospital outbreaks and may become a new tool in combating AMR or hospital-acquired infection.
The biochemical-based Carba NP test has been evaluated to detect carbapenemase-producing Enterobacteriaceae (n = 193) directly from spiked blood cultures. It was able to rapidly detect KPC (n = 50), IMP (n = 27), VIM (n = 37), NDM (n = 33) and OXA-48-like producers (n = 46) with sensitivity and specificity of 97.9% and 100%, respectively. This cost-effective technique may be implemented in any microbiology laboratory and offers a reliable test for an early identification of carbapenemase-producing Enterobacteriaceae directly from blood culture that could be useful for the management of infected patients.
This study was designed to evaluate the performance of the broth microdilution (BMD) test in detecting the production of Klebsiella pneumoniae carbapenemases (KPCs) and metallo-β-lactamases (MBLs) in clinical isolates of gram-negative bacilli by using various β-lactamase inhibitors. A carbapenemase detection test, comprised of Mueller-Hinton broth containing serial twofold dilutions of imipenem or meropenem with and without aminophenylboronic acid (APB), phenylboronic acid (PB), cloxacillin (CLX), dipicolinic acid (DPA), or EDTA, was evaluated against 31 Klebsiella pneumoniae strains with KPC, 21 MBL-producers (3 Enterobacteriaceae and 18 non-fermenters), and 26 carbapenemase-non-producing Enterobacteriaceae strains. A threefold or greater decrease in the MIC of imipenem or meropenem with β-lactamase inhibitors, when compared with the imipenem or meropenem alone, was considered a positive result. Imipenem with and without PB had the most comparable sensitivity (93.5%) and specificity (93.6%) for the detection of K. pneumoniae with KPC enzymes if the additional criterion of a negative CLX result was included. Both DPA and EDTA had excellent sensitivity (imipenem with and without DPA vs. EDTA, 90.5% vs. 95.2%) and specificity (imipenem with and without DPA vs. EDTA, 98.2% vs. 100%) for the detection of MBL-producing gram-negative bacilli. The comparative study showed that, by using imipenem with and without PB, CLX, and EDTA, the BMD test was effective in detecting KPC and MBL enzymes. The BMD test could be applied for routine use in commercially available semiautomated systems for the detection of KPCs and MBLs in gram-negative bacilli.
Gram-negative bacilli with acquired metallo--lactamase (MBL) production have been increasingly re- ported in some countries, necessitating their detection. The aim of this study was to evaluate the performance of the Hodge test and those of the imipenem (IPM)-EDTA, ceftazidime (CAZ)-mercaptopropionic acid (MPA), and CAZ-sodium mercaptoacetic acid (SMA) double-disk synergy tests (DDSTs). The efficiencies of testing CAZ-resistant and IPM-nonsusceptible isolates were also compared. Strains used for the evaluation were known IMP-1 and VIM-2 MBL-producing isolates and consecutive and CAZ-nonsusceptible isolates of pseudo- monads and acinetobacters. The performance of the Hodge test was improved by addition of zinc sulfate (140 g/disk) to an IPM disk. In DDSTs, EDTA (ca. 1,900 g) disks were better at detecting MBL-producing strains among pseudomonads, while MPA (3 l) and SMA (3 mg) disks performed better for acinetobacters. EDTA (ca. 750 g)-plus-SMA (ca. 2 mg) disks performed better than EDTA, MPA, or SMA disks with both organisms. CAZ-SMA DDSTs failed to detect 22 of 80 (28%) MBL-producing acinetobacters. In conclusion, use of an IPM disk and an EDTA (750 g)-plus-SMA (2 mg) disk improves performance, and testing IPM- nonsusceptible isolates rather than CAZ-resistant isolates could reduce screening work. Further evaluation of the test is required for the detection of other types of MBL-producing gram-negative bacilli.
A prospective survey was conducted on 862 Enterobacteriaceae isolates with reduced susceptibility to carbapenems. The Carba NP test, UV spectrophotometry, and a DNA microarray were used
to detect carbapenemase producers, and the results were compared to those from PCR and sequencing. The 172 carbapenemase producers
were detected using the Carba NP test and UV spectrophotometry, whereas the DNA microarray failed to detect IMI producers.
The use of the Carba NP test as a first screening, followed by the use of molecular techniques, has been determined to be
an efficient strategy for identifying carbapenemase-producing Enterobacteriaceae.
Accurate detection of metallo-β-lactamase (MBL)-producing Pseudomonas spp. and Acinetobacter spp. became very important with the increasing prevalence of carbapenem-nonsusceptible clinical isolates. The performance
of phenotypic MBL detection methods may depend on the types of MBL and the characteristics of the isolates. A high false-positive
rate is a problem with EDTA-based MBL detection methods. We evaluated the performance of double-disk potentiation tests (DDPTs)
and disk potentiation tests (DPTs) with dipicolinic acid (DPA) using 44 isolates of Pseudomonas spp. and Acinetobacter spp. producing IMP-1-like, VIM-2-like, and SIM-1 type MBLs. Also, we characterized P. aeruginosa isolates with positive imipenem (IPM)-DPA DDPT, but negative meropenem (MEM)-DPA DDPT, and determined possibility of improving
a DDPT by using MacConkey agar. Among five different DDPT methods, the IPM-DPA 250-μg method showed the highest sensitivity
(97.7%) and specificity (100%). Among four DPT tests, the highest sensitivity (100%) was shown by the IPM-EDTA 1,900-μg disk
method, but the specificity was very low (11.4%). Five of six P. aeruginosa isolates with false-negative DDPTs with MEM-DPA 250-μg disks carried blaIMP-6, and the high level resistance to MEM (MIC ≥ 512 μg/ml) was reduced by the presence of phenylalanine arginine β-naphtylamide.
Improvement of DDPTs was observed when MacConkey agar was used instead of Mueller-Hinton agar. In conclusion, DPA is a better
MBL inhibitor than EDTA for detection of Pseudomonas spp. and Acinetobacter spp. with IMP-1-like, VIM-2-like, and SIM-1-type MBLs. In DPA DDPTs, IPM disks perform better than MEM disks when the isolates
are highly resistant to MEM due to the overexpression of efflux pumps.
Dissemination of carbapenem resistance among Enterobacteriaceae poses a considerable threat to public health. Carbapenemase gene detection by molecular methods is the gold standard but is available in only a few laboratories. The aim of this study was to test phenotypic methods for the detection of metallo-β-lactamase (MBL)- or Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and associated mechanisms of β-lactam resistance against a panel of 30 genotypically characterized carbapenem-resistant Enterobacteriaceae : 9 MBL, 7 KPC, 6 OXA-48, and 8 extended-spectrum β-lactamase (ESBL) or AmpC β-lactamases associated with decreased permeability. We used carbapenemase inhibitor-impregnated agar to test for carbapenem-resistant strains. Differences in the inhibition zone sizes of the meropenem, imipenem, ertapenem, and doripenem disks were measured between control and inhibitor (EDTA or phenylboronic acid [PBA] with or without cloxacillin)-impregnated Mueller-Hinton agar with a cutoff of 10 mm. All 9 MBL- and 7 KPC-producing Enterobacteriaceae were identified from the differences in zone size in the presence and absence of specific inhibitors, regardless of the carbapenem MICs and including isolates with low-level resistance to carbapenems. We also detected their associated β-lactam resistance mechanisms (11 ESBL-type and 5 class A β-lactamase 2b). No differences in zone size were observed for OXA-48-producing strains or other carbapenem resistance mechanisms such as ESBL and decreased permeability. We propose a new strategy to detect carbapenemases (MBL- and KPC-type) and associated mechanisms of β-lactam resistance (ESBL or class A β-lactamase 2b) by the use of inhibitor-impregnated agar. A rapid phenotypic detection of resistance mechanisms is important for epidemiological purposes and for limiting the spread of resistant strains by implementing specific infection control measures.
We evaluated the ability of the modified Hodge test to discriminate between KPC- and metallo-beta-lactamase (MBL)-producing
Pseudomonas aeruginosa isolates and carbapenemase nonproducers. With Escherichia coli ATCC 25922 as the indicator strain, the MHT resulted in low sensitivity, specificity, and repeatability. Replacing the indicator
strain with Klebsiella pneumoniae ATCC 700603 led to an improved performance (100%, 97%, 0%, and 100% sensitivity, specificity, indeterminate results and repeatability,
respectively).
A carbapenem-resistant Escherichia coli strain (DVR22) was recovered from a stool specimen from a patient with traveler's diarrhea who had traveled to India. Molecular
screening led to the first identification of NDM-1 in Spain. The blaNDM-1 gene was located in a conjugative plasmid of ca. 300 kb that also contained the blaCTX-M-15, blaTEM-1, ΔblaDHA-1, and armA genes. In addition, blaNDM-1 was preceded by an ISAba125 insertion element only found in Acinetobacter spp.
A simple disk diffusion test was constructed for detection of IMP-1-type metallo-beta-lactamase-producing gram-negative bacteria. Two Kirby-Bauer disks containing ceftazidime (CAZ) and a filter disk containing a metallo-beta-lactamase inhibitor were used in this test. Several IMP-1 inhibitors such as thiol compounds including 2-mercaptopropionic acid, heavy metal salts, and EDTA were evaluated for this test. Two CAZ disks were placed on a Mueller-Hinton agar plate on which a bacterial suspension was spread according to the method recommended by the National Committee for Clinical Laboratory Standards. The distance between the disks was kept to about 4 to 5 cm, and a filter disk containing a metallo-beta-lactamase inhibitor was placed near one of the CAZ disks within a center-to-center distance of 1.0 to 2.5 cm. For IMP-1-producing strains, the growth-inhibitory zone between the two disks expanded, while no evident change in the shape of the growth-inhibitory zone was observed for CAZ-resistant strains producing serine beta-lactamases such as AmpC or SHV-12. As a result, 2 to 3 microliter of undiluted 2-mercaptopropionic acid or mercaptoacetic acid able to block IMP-1 activity gave the most reproducible and clearest results, and CAZ-resistant strains producing AmpC or extended-spectrum beta-lactamases were distinguishable from IMP-1 producers by this test. A similar observation was made with IMP-1-producing clinical isolates such as Serratia marcescens, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris, Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter spp., and Alcaligenes xylosoxidans. The specificity and sensitivity of this test were comparable to those of PCR analysis using bla(IMP)-specific primers. Therefore, this convenient test would be valuable for daily use in clinical laboratories.
Carbapenemases are β-lactamases that confer resistance to the carbapenems (e.g., imipenem, meropenem, ertapenem, and doripenem). Most often these enzymes confer resistance to the other β-lactam agents as well, including extended-spectrum cephalosporins. The enzymes are usually found in bacterial isolates that are already resistant to nearly all other antimicrobial agents, and treatment options for infections caused by them are significantly limited. This important mechanism of resistance is emerging in the United States, and the most common enzyme is the Klebsiella pneumoniae carbapenemase (KPC). The KPC enzyme is found in many different genera of Enterobacteriaceae, but most commonly in K. pneumoniae. Currently, KPC-producing isolates are commonly isolated in the northeastern part of the U.S., but reports of isolates from other locations are increasing. Prevention and control of this emerging resistance is complicated by the occurrence of KPC-positive isolates producing low-level carbapenem resistance that is hard to detect in the clinical microbiology laboratory. The most sensitive methods for detecting carbapenemase-producing isolates are to look for elevated but susceptible carbapenem susceptibility results. In addition to discussing KPC, this article will review the epidemiology of other carbapenemases and their potentials for dissemination in the U.S.