Modified Carbapenem Inactivation Method Research Articles

Evaluation of Three Carbapenemase-Phenotypic Detection Methods and Emergence of Diverse VIM and GES Variants among Pseudomonas aeruginosa Isolates in Tunisia.

Background: Since 2012, few reports on the molecular epidemiology of Pseudomonas aeruginosa were reported in Tunisia. Objectives: This study aimed to evaluate carbapenem-resistance determinants and molecular epidemiology and to compare the carbapenemase-phenotypic detection methods of multidrug-resistant P. aeruginosa isolates. Methods: During a period of four years (2014 to 2017), all imipenem-ceftazidime-resistant P. aeruginosa isolates were retrospectively selected at the microbial laboratory of Charles Nicolle hospital of Tunis. These isolates were examined by the modified Hodge test, modified carbapenem inactivation method (mCIM), and another mCIM, called CIMTris, and their performance was evaluated using PCR analysis as the gold standard. Results: A total of 35 isolates were recovered among patients hospitalized in different units. All strains were colistin-susceptible.All carbapenem-resistant isolates showed a high-level resistance to carbapenems. CIMTris and mCIM showed 96.15% and 46.15% sensitivity and 44.44% and 100% specificity, respectively, for detecting carbapenemase production.Conclusions: CIMTris is a promising approach for detecting carbapenemase activity in P. aeruginosa and merits further testing. Moreover, this study described the first detection of GES-5- and GES-9-producing P. aeruginosa in Tunisia as well as the co-occurrence of the blaGES-5 and blaVIM-11 carbapenemase genes in one isolate. These findings are of great concern because the rapid dissemination of MDR strains represents a major therapeutic and epidemiological threat.

Use of the Combined Modified Carbapenem Inactivation Method and EDTA-modified Carbapenem Inactivation Method for Detection of Carbapenemase-Producing Enterobacteriaceae Causing Ventilator-associated Respiratory Infections

There is an alarming rise in carbapenem-resistant Enterobacteriaceae (CRE) causing nosocomial infections such as ventilator-associated respiratory infections (VARIs). The use of rapid phenotypic methods for the detection and differentiation of carbapenemases elaborated by these CRE would be helpful in providing timely empirical therapeutic options for management of these infections and preventing spread of these CRE strains in hospital settings. Hence, this study aimed to detect CRE among pathogens isolated from the endotracheal secretions recieved from suspected cases of VARIs and differentiate carbapenemases elaborated by these CRE using combined phenotypic methods, such as the modified carbapenem inactivation method (mCIM) and EDTA modified CIM (eCIM). This observational study was conducted over a period of 1 year in the Department of Microbiology and the intensive care unit of a tertiary care center. Carbapenem resistance was found in 75% of Klebsiella pneumoniae isolates and 50% of Escherichia coli isolates, of which 58.4% were metallo-β-lactamases and 41.6% were serine carbapenemase producers. In conclusion, the combination of the mCIM and eCIM could be useful as an epidemiological tool and be considered essential in deciding the initial antibiotic therapy, help reduce morbidity and mortality associated with VARIs, and guide hospital infection control practices.

Characterization of carbapenem-resistant gram-negative bacteria collected in the Sentinel Surveillance Program, 2018–2019

Background: Carbapenem resistance in gram-negative organisms is an important public health problem. The CDC conducted Sentinel surveillance in 2018–2019 to characterize these organisms from 9 facilities in 9 different states. Methods: Carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter spp (CRA) obtained from clinical samples of patients in acute-care or long-term care facilities were submitted to the CDC. Identification was confirmed using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and antimicrobial susceptibility testing (AST) was performed via broth microdilution for 27 antibiotics. All confirmed CRE and CRPA were tested for carbapenemase production (CP) using the modified carbapenem inactivation method (mCIM). The isolates that were mCIM-positive were assessed by real-time PCR for presence of blaKPC, blaNDM, blaVIM, and blaIMP. CP-CRE were also assessed for blaOXA-48-like. All confirmed CRA were tested for the same genes as CRPA and blaOXA-23–like, blaOXA-24/40-like, blaOXA-58–like, and blaOXA-235–like genes. Difficult-to-treat resistance (DTR) was defined as resistance to all β-lactams (excluding newer β-lactam combination agents) and quinolones tested. Results: The CDC confirmed 208 CRE, 161 CRPA, and 94 CRA. Table 1 summarizes AST results for a selection of drugs. We identified 112 (53.8%) mCIM-positive CRE and 6 (3.7%) mCIM-positive CRPA. The PCR results are summarized in Table 2. One mCIM-positive and PCR-negative isolate was positive in a metallo-β-lactamase screen. Conclusions: Resistance among CRE and CRPA to newer β-lactam combination agents was detected. Options for treating CRA are limited. Of 112 CP-CRE, 85.7% harbored blaKPC; CP-CRPA were rare (3.7%); and most CRA harbored blaOXA-23-like (55.3%) or blaOXA-24/40-like (30.9%). Whole-genome sequencing is planned to better understand gene variants, sequence types, and additional resistance markers present among the isolates.Funding: NoneDisclosures: None

Verification and application of a modified carbapenem inactivation method (mCIM) on Pseudomonas aeruginosa: a potential screening methodology on carbapenemases phenotype in Bacillus cereus

ABSTRACT Antimicrobial resistance (AMR) has been a leading issue for human health globally threatening the achievement of several of the Sustainable Development Goals (SDGs). Originated from Bacillus cereus, carbapenemases phenotype has been considered to be a major concern in AMR. In this study, the AMR identification rate of P. aeruginosa isolates and infections in FAHJU showed an obvious upward trend from 2012 to 2016. All 88 carbapenem-resistant P. aeruginosa strains were screened for carbapenemase phenotype by modified Carbapenem Inactivation Method (mCIM), and these results of mCIM were compared with traditional PCR results. The isolates of P. aeruginosa and infected patients showed obvious upward trend from 2012 to 2016. The drug resistance to common clinical antibiotics was serious that the clinical rational use of antibiotics should be strengthened, which is in accordance with the Global Antimicrobial Resistance and Use Surveillance System (GLASS) report. In comparison, the results of mCIM showed that 18 out of 88 CRPA strains were carbapenemase positive, which were completely consistent with the results yielded by PCR method. Therefore, it is convinced that this mCIM methodology is a simple and quick method for detected carbapenemases producing P. aeruginosa and has a potential capability in carbapenemases phenotype of pathogen like B. cereus, which will undoubtedly aid in the AMR therapy.

Three Novel Sequence Types Carbapenem-Resistant Klebsiella pneumoniae Strains ST5365, ST5587, ST5647 Isolated from Two Tertiary Teaching General Hospitals in Shanxi Province, in North China: Molecular Characteristics, Resistance and Virulence Factors.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) represents a significant threat to public health and has already drawn worldwide attention. Hence, we aim to comprehensively analyze the case condition, as well as molecular epidemiology, resistance and virulence of three CRKP isolates with new sequence types (STs).MethodsThree CRKP were collected from November 2019 to April 2021. The three patients’ clinical characteristics were analyzed through His system. In order to screen phenotype of metallo-carbapenemase, the modified Carbapenem Inactivation Method (mCIM) and EDTA-modified Carbapenem Inactivation Method (eCIM) were conducted. Three isolates were subjected to antimicrobial susceptibility testing (AST) using the agar dilution method or minimal broth dilution method. The string test, the sedimentation assay, biofilm formation and the serum resistance assay were performed as phenotypic experiments to assist in evaluating virulence. The presence of resistance and virulence genes were detected by Whole-Genome Sequencing (WGS). Serotypes and new STs were compared and determined by multi-locus sequence typing (MLST).ResultsOverall, all Klebsiella pneumoniae isolates were multi-resistant, but sensitive to tigecycline and colistin. Among them, all formed biofilms, strain 1 and strain 2 were classified as moderate-producers, while strain 3 as weak-producer. The results of the serum resistance assay indicated that only strain 2 was resistant. From WGS analysis, it showed that all isolates co-harbored multiple resistance genes, such as carbapenemase genes, sulfonamides, fluoroquinolones, aminoglycosides, and tetracyclines. Meanwhile, several virulence genes were also contained, including siderophores, fimbriae, capsule and lipopolysaccharides-associated genes. The serotypes of strain 1 and strain 2 manifested K35 and KL47, respectively.ConclusionThree novel ST5365, ST5587, ST5647 were first discovered in North China. Our study suggested that we should pay more attention to their resistance. And the results will help treat CRKP infections caused by these novel STs.

Detection of blaKPC and blaNDM carbapenemase genes among Klebsiella pneumoniae isolates in Addis Ababa, Ethiopia: Dominance of blaNDM

BackgroundInfections caused by Klebsiella pneumoniae have been difficult to control because of the worldwide emergence of carbapenem-resistant isolates mainly due to carbapenemase production. Information regarding carbapenemase-producing K. pneumoniae is still scarce in Ethiopia. Therefore, the current study aimed to determine the prevalence of carbapenemase-producing K. pneumoniae and to assess the occurrence of blaNDM and blaKPC carbapenemase genes.MethodsA cross-sectional study was conducted from September 2018 to February 2019 at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia. A total of 132 non-duplicate K. pneumoniae isolates were studied. Phenotypic confirmation of carbapenemase production was done by modified Carbapenem Inactivation Method (mCIM). Multiplex PCR was performed for the detection of carbapenemase-encoding genes blaKPC, and blaNDM.ResultsOut of the total 132 K. pneumoniae isolates, 39 (29.6%) were non-susceptible to one or more carbapenems. The prevalence of carbapenemase-producing isolates from the total was 28 (21.2%) with mCIM of which the most dominant gene was blaNDM 26 (92.9%) and one isolate carried blaKPC concomitantly. Carbapenemase-producing K. pneumoniae isolates were 100% non-susceptible to half of the antimicrobials used in the study, including meropenem and ertapenem. Previous use of carbapenems was associated with carbapenemase production (P = 0.004).ConclusionsThe prevalence of carbapenemase-producing K. pneumoniae isolates was worrying in the study area. To our knowledge, the study described the emergence of blaNDM and blaKPC gene carrying K. pneumoniae in Ethiopia for the first time. Further large-scale molecular-based studies, including other carbapenemase genes and sequencing of K. pneumoniae, are warranted to have a clear awareness about the presence of antimicrobial resistance high-risk clones in Ethiopia.

Direct Identification, Antimicrobial Susceptibility Testing, and Extended-Spectrum β-Lactamase and Carbapenemase Detection in Gram-Negative Bacteria Isolated from Blood Cultures.

PurposeTo shorten the turnaround time for blood culture (BC) analyses, a rapid method was developed for the direct identification, antimicrobial susceptibility testing (AST), and multidrug resistance testing of bacteria-positive BCs.Materials and MethodsThe mixtures in BC bottles were treated with the multistep centrifugation method developed here and the conventional culture-based method. The bacterial sediment obtained after centrifugation was analyzed directly with MALDI-TOF MS and Vitek 2 Compact, and AST was performed directly with the Kirby–Bauer (K–B) disk diffusion, VITEK 2 Compact, and E-test methods. Extended spectrum lactamases (ESBLs) were detected with discs containing cefotaxime, cefotaxime/clavulanate, ceftazidime, and ceftazidime/clavulanate, and carbapenemase was detected with the modified carbapenem inactivation method (mCIM) and EDTA-mCIM (eCIM).ResultsAll the results of direct testing were compared to those of the conventional methods, to evaluate the accuracy of the direct methods. The accuracies of the direct Vitek 2 Compact and MALDI-TOF MS methods were 95.5% (214/224) and 90.2% (202/224), respectively. Direct AST with K–B, Vitek 2, and E-test showed category agreement of 96.0% (2611/2721), 96.1% (2614/2721), and 97.4% (2650/2721), respectively, and the major errors and very major errors were < 2% for all three methods. In the direct determination of ESBLs, the results for cefotaxime combined with cefotaxime/clavulanate were completely consistent with those after the standard isolation method. The carbapenemase detection rate with direct mCIM and eCIM was exactly the same as that with the standard method.ConclusionThese direct procedures based on multistep centrifugation are not only highly accurate but are appropriate for clinical laboratory use because the turnaround time is shorter.

Community Fecal Carriage and Molecular Epidemiology of Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Escherichia coli from Healthy Children in the Central South China.

BackgroundFecal carriage of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) and carbapenemase-producing E. coli (CP-EC) is well reported among hospitalized adults and children. However, there are few studies on the carriage prevalence and ESBL-EC and CP-EC genotypes among healthy children in China.Patients and MethodsStool samples were collected from 330 students in 2021 from three randomly selected primary schools in Changsha, China. ESBL-EC and CP-EC were screened using CHROMagarTM chromogenic plates. ESBL and carbapenemase production was confirmed using the double-disc synergy test and a modified carbapenem inactivation method, respectively. Antimicrobial susceptibility was tested using the broth microdilution method. Resistance determinants, virulence factors, and phylogenetic groups were determined by PCR and sequencing. Multi-locus sequence typing (MLST) was performed (seven housekeeping genes were amplified and sequenced) on the phylogenic group B2 E. coli to detect high-risk clonal strains such as ST131 E. coli. Then, ST131 E. coli were characterized based on ST131 clades, O-type, and fimH alleles.ResultsIn total, 118 (35.8%) ESBL-EC and 3 (0.9%) CP-EC were isolated. blaCTX-M was the most common genotype (27.1%), identified in all ESBL-EC, except one, which carried blaSHV-12. One isolate with mcr-1 was found amongst ESBL-EC, whereas all three CP-EC carried blaNDM-1. The predominant sequence type (ST) clones in group B2 were ST131 and ST1193. The prevalence of ST131 E. coli was 9.9%, displaying serotypes O16 and O25b, fimH alleles 30, 41, and 89, and ST131 clades A and C1-M27.ConclusionIn this study, high carriage rate of ESBL-EC was found among healthy children, and the dominant ESBL was CTX-M-14. In addition, high-risk clones (ST131 and ST1193) were also detected. This emphasizes the importance of monitoring ESBL-EC in community settings.

High prevalence of fecal carriage of Extended-spectrum beta-lactamase and carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar, Northwest Ethiopia.

Fecal carriage of extended-spectrum beta-lactamase and Carbapenemase-producing Enterobacteriaceae is a potential risk for the transmission of infection with resistant strains. Understanding the burden of these resistant strains in asymptomatic people is essential to reduce the chain of infection transmission. However, data on the fecal carriage of Extended-spectrum Beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers were limited in developing countries especially in Ethiopia. The aim of the present study is, therefore, to assess fecal carriage rate, associated factors, and antimicrobial resistance patterns of Extended-spectrum Beta-lactamase and Carbapenemase-producing Enterobacteriaceae among food handlers at the University of Gondar Cafeterias, Northwest Ethiopia. An institution-based cross-sectional study was conducted from February to June 2021 at the University of Gondar cafeterias. A total of 290 stool samples were collected, transported using Cary Blair transport medium, and processed. All isolates were cultured and identified by using MacConkey agar, and routine biochemical tests. Antimicrobial susceptibility testing was done to each isolate following the Kirby Bauer disk diffusion method. If the zone of inhibition was ≤ 22 mm for ceftazidime, ≤25 mm for ceftriaxone, and ≤27 for cefotaxime they were considered as potential ESBL strain and selected for a further phenotypic confirmatory. Moreover, the double-disc diffusion test and the modified carbapenem inactivation method were used for confirmations of Extended-spectrum β-lactamase and Carbapenemase-producing Enterobacteriaceae respectively. If a ≥5mm difference in zone diameter for either antimicrobial agent in combination with clavulanic acid versus the zone diameter of the agent when tested alone (without B-lactamase inhibitor), was confirmed as ESBL-PE and if the zone of inhibition diameter between 6-15mm and 16- 18mm with a pinpoint colony, it was considered as carbapenem resistance Enterobacteriaceae. Data were entered using Epi-data version 4.6 and then exported to SPSS version 26 for analysis. Potential risk factors were assessed using multivariable logistic regression and a p-value less than 0.05 was considered statistically significant. Out of 290 stool samples, 63 (21.7%) and 7 (2.4%) were confirmed as Extended-spectrum β-lactamase and Carbapenemase-producing Enterobacteriaceae. The most predominant ESBL-PE was E. coli 43 (14.8%) followed by K. pneumoniae 17 (5.9%). Most of the Extended-spectrum β-lactamase and Carbapenemase-producing isolates were resistant to tetracycline, cefotaxime, ceftazidime, and ceftriaxone (100% each). In contrast, a low resistance level was recorded for Meropenem and cefoxitin. The overall Multi-drug resistant Enterobacteriaceae (MDR) was 147 (42.3%). Antibiotics usage in the last 3 months and drinking unpasteurized milk were associated with the carriage of the Extended-spectrum beta-lactamase-Producing Enterobacteriaceae. The high fecal carriage rate of Multi-drug resistance isolate, Extended-spectrum β-lactamase, and Carbapenemase-producing Enterobacteriaceae were recorded among food handlers. Therefore, this study gives signals in the spread of drug-resistant bacteria easily to the community. Hence, the need for adjusting and promotion of infection prevention measures to prevent the spread of drug-resistant bacteria should not be underestimated.

A Longitudinal Nine-Year Study of the Molecular Epidemiology of Carbapenemase-Producing Enterobacterales Isolated From a Regional Hospital in Taiwan: Predominance of Carbapenemase KPC-2 and OXA-48.

Enterobacterales clinical isolates are now being resistant to clinically achievable concentrations of most commonly used antibiotics that makes treatment of hospitalized patients very challenging. We hereby determine the molecular characteristics of carbapenemase genes in carbapenem-resistant Enterobacterales (CRE) isolates in Taiwan. A total of 455 CRE isolates were identified between August 2011 to July 2020. Minimum inhibitory concentrations for selected carbapenems were tested using Vitek 2, and carbapenemase genes were determined using polymerase chain reaction in combination with sequencing. Phenotypic detection of carbapenemase was determined by modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) to validate our PCR screening results. Pulsed-field gel electrophoresis (PFGE) was used to determine the clonality of carbapenemase-producing Enterobacterales (CPE) isolates, and the transferability of carbapenemase-carrying plasmids was determined by conjugation assays. A slight increase in carbapenem-resistant E. coli (CREC) was observed, however, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) was steady, during 2011–2020. The dominant species among our CRE was K. pneumoniae (270/455, 59.3%), followed by E. coli (81/455, 17.8%), Morganella morganii (32/455, 7.0%), and Enterobacter cloacae (25/455, 5.5%). From 2011 to 2020, the total percentage of CPE increased steadily, accounting for 61.0% of CRE in 2020. Moreover, 122 of 455 CRE isolates (26.8%) were CPE. Among the CPE isolates, the dominant carbapenemase gene was blaOXA–48–like (54/122, 44.3%), and the second most common carbapenemase gene was blaKPC–2 (47/122, 38.5%). The sensitivity and specificity for mCIM to detect carbapenemase in the 455 isolates were both 100% in this study. The PFGE results showed that 39 carbapenemase-producing E. coli and 69 carbapenemase-producing K. pneumoniae isolates carrying blaKPC–2 and/or blaNDM–5 could be classified into 5 and 12 clusters, respectively. In conclusion, our results showed an increase in CPE isolates in Taiwan. Moreover, the distribution of carbapenemase and antimicrobial susceptibility in CPE were associated with PFGE typing.

Extended-Spectrum Beta-Lactamase- and Carbapenemase-Producing Enterobacteriaceae Family of Bacteria from Diarrheal Stool Samples in Northwest Ethiopia.

Background Resistance among the commensal flora is a serious threat because they are highly populated ecosystems like the gut, maybe a source of extraintestinal infections. Infections due to extended-spectrum beta-lactamase (ESBL)- and carbapenemase (CPM)-producing Enterobacteriaceae family of bacteria impose a major global issue because they are usually resistant to multiple antimicrobial agents. Data on the fecal ESBL- and CPM-producing group of bacteria in developing countries including Ethiopia are limited mainly due to resource constraints. Thus, this study aimed to determine the prevalence of multidrug-resistant (MDR)-, ESBL-, and CPM-producing Enterobacteriaceae family of bacteria from diarrheal stool samples at the University Hospital, Northwest Ethiopia. Materials and Methods A hospital-based cross-sectional study was conducted involving a total of 384 study participants having gastrointestinal complaints from January to April 2019. A diarrheal stool sample was aseptically collected and inoculated on a MacConkey agar plate. After getting pure colonies, biochemical and antimicrobial susceptibility testing was done following standard microbiological techniques. ESBL production was screened using ceftazidime and cefotaxime and confirmed using a combined disk diffusion test. Carbapenemases were screened by meropenem disk and confirmed by the modified carbapenem inactivation method. Data were checked, cleaned, and entered using Epi Info version 7.1 and transferred to SPSS version 20 for analysis. Result A total of 404 Enterobacteriaceae groups of bacteria were isolated from 384 diarrheal stool samples. The overall prevalence of fecal MDR-, ESBL-, and CPM-producing group of Enterobacteriaceae was 196 (48.5%), 66 (16.3%), and 4 (1%), respectively. Of the total ESBL-producing Enterobacteriaceae, E. coli (41/66 (62.1%)) and K. pneumoniae (18/66 (27.3%)) were the most predominant isolates. One half of CPE has been observed in Citrobacter species and the rest in E. coli (25%) and P. vulgaris (25%). Conclusion and Recommendation. Finding the high rate of ESBL-producing Enterobacteriaceae and CPE requires strict infection control measures and careful selection of empirical therapy in the study area. Therefore, active surveillance with large sample size and better infection prevention control is needed.

Prevalence and Molecular Characterization of Extended Spectrum β-Lactamase and Carbapenemase-Producing Enterobacteriaceae Isolates from Bloodstream Infection Suspected Patients in Addis Ababa, Ethiopia.

BackgroundProduction of Extended spectrum beta-lactamase (ESBL) and Carbapenemase is the most common strategy for drug resistance in clinical isolates of Enterobacteriaceae. This study was conducted to determine the magnitude of ESBL and Carbapenemase production (CPE) among clinical isolates of Enterobacteriaceae causing bloodstream infections (BSI) in Ethiopia.MethodsA cross-sectional study was performed from September 2018 to January 2019 in Ethiopia. A total of 2397 BSI suspected patients were enrolled and blood culture was performed using a BacT/Alert instrument in combination with conventional methods for identification. After antimicrobial susceptibility test, phenotypic confirmation of ESBLs was done by combined disc-diffusion. Meanwhile carbapenemase production was done by modified carbapenem inactivation method. Multiplex PCR was conducted to detect the presence of blaCTX-M,blaSHV, blaTEM, blaKPC and blaNDM genes.ResultsA total of 104 (4.3%) Enterobacteriaceae were isolated from 2397 BSI suspected patients. Klebsiella pneumoniae (55/104, 52%) was the predominant isolate followed by E. coli, (19.2%, 20/104) and K.oxytoca (17.3%, 18/104). ESBL and carbapenemase production were observed from 70 (67.3%, 57.4 −76.2% at 95% CI) and 8 (7.7%, 3.4–14.6% at 95% CI) isolates respectively. The highest frequency of ESBL and carbapenemase production was observed in K. pneumoniae 78.2% (43/55) and 9.1% (5/55), respectively. All the 70 isolates confirmed as ESBL producers harbored at least one of the ESBL genes and the majority of them carried multiple beta-lactamase genes (84.3%), where blaCTX-M, type was the most predominant (67.3%). Similarly, the entire eight isolates positive for carbapenemase carried blaNDM but none of them carried blaKPC.ConclusionIn our study, the rate of ESBL production among BSI-causing Enterobacteriaceae was alarming and most of the isolates carried multiple types of ESBL genes. A significant magnitude of CPE isolates causing BSI was recorded.

Comparison of the Performance of Phenotypic Methods for the Detection of Carbapenem-Resistant Enterobacteriaceae (CRE) in Clinical Practice

In order to investigate the diagnostic performance characteristics of four phenotypic assays in detecting carbapenem-resistant Enterobacteriaceae (CRE), we collected the CRE strains from infected patients. The results of carbapenemase gene detection, bla KPC-2, bla OXA-23, bla NDM-1, bla NDM-4, bla NDM-5, bla IMP-4, and bla IMP-8, were used as a standard to evaluate the performances of combined disk test (CDT), modified carbapenem inactivation method(mCIM)/EDTA-modified carbapenem inactivation method(eCIM), NG-Test CARBA 5 (CARBA), and color developing immunoassay (CDI). The compliance of phenotype results based on CDT, mCIM/eCIM, CARBA, and CDI with genetic detection results was 94% (231/247), 95% (235/247), 98% (242/247), and 99% (246/247), respectively. CDT demonstrated a low specificity for carbapenemase detection, low negative predictive value (NPV), and low sensitivity for metallo-β-lactamase (79%, 55%, and 88%, respectively); it also failed to accurately detect IMP. The mCIM/eCIM assay had serious problems in detecting OXA-23-like carbapenemases. The sensitivity and specificity of CARBA and CDI were higher than those of the first two methods. However, CARBA did not cover the detection of OXA-23, while CDI cannot detect IMP-8, resulting in low NPVs (70% and 88%, respectively). In conclusion, CARBA and CDI assays are highly accurate except individual rare genes and allow direct genotype detections. CDT and mCIM/eCIM assays are moderately accurate and can only distinguish serine-β-lactamases from metallo-β-lactamases. Laboratories should choose the appropriate method that meets their needs based on its characteristic.

The Antibiotic Resistance Profile of Pseudomonas Aeruginosa in a Tertiary Medical Center from Malaysia

Background: Pseudomonas aeruginosa is a notorious gram-negative bacterium that has become a globalpublic health concern owing to the emergence of multi- and pandrug-resistant strains. This study sought todetermine the antibiotic susceptibility profile of P. aeruginosa in a tertiary medical center from Malaysia. Materials and Methods: Each isolate’s identity was confirmed using the VITEK 2 GN kit, and subjectedto antibiotic susceptibility testing using the VITEK 2 AST-N374 card (for testing against piperacillintazobactam,ceftazidime, cefepime, imipenem, meropenem, amikacin, gentamicin and ciprofloxacin) andEtest strips (for testing against doripenem and polymyxin B). Isolates which were not susceptible to &gt;1carbapenem were tested for carbapenemase production using the modified carbapenem inactivationMethod (mCIM). Results: Out of 102 isolates studied, 64 (62.7%) were fully susceptible to all the antibiotics tested.Twenty-six (25.5%) were resistant to &gt;1 antibiotic from &gt;2 antibiotic classes, and 21 (20.6%) wereresistant to &gt;1 antibiotic from &gt;3 classes. Susceptibility was highest with polymyxin B (100%) and lowestwith piperacillin-tazobactam (64.7%). Carbapenem susceptibility was between 78.4% to 81.4%. Out of 22isolates which were not susceptible to &gt;1 carbapenem, 18 (81.8%) were not susceptible to all threecarbapenems. Conclusion: More than half of our P. aeruginosa isolates were fully susceptible to all the anti-pseudomonalantibiotics tested. Multidrug-resistant strains accounted for between 20% to 25% of all our P. aeruginosaisolates. Through mCIM testing, carbapenemase production did not appear to be the dominant resistancemechanism. J MEDICINE 2022; 23: 54-60

Evaluating NG-Test CARBA 5 Multiplex Immunochromatographic and Cepheid Xpert CARBA-R Assays among Carbapenem-Resistant Enterobacterales Isolates Associated with Bloodstream Infection.

ABSTRACTDecreased susceptibility to carbapenems in Enterobacterales is an emerging concern. Conventional methods with short turnaround times are crucial for therapeutic decisions and infection control. In the current study, we used the Xpert CARBA-R (Cepheid, Sunnyvale, CA, USA) and the NG-Test CARBA 5 (NG Biotech, Guipry, France) assays for carbapenemase detection in 214 carbapenem-resistant Enterobacterales (CRE) blood isolates. We used the modified carbapenem inactivation method, conventional PCR, and sequencing to determine the production of five common carbapenemase families and their subtypes. We performed wzc-genotyping for all CR-Klebsiella pneumoniae (CRKP) and multilocus sequence typing for all carbapenemase-producing CRE isolates to reveal their genetic relatedness. The results showed a sensitivity of 99.8% and a specificity of 100% by the Xpert assay, and a sensitivity of 100% and a specificity of 99% by the NG-Test in detecting carbapenemases of 84 CRKP isolates with only one (VIM-1+IMP-8) failure in both tests. For CR-Escherichia coli, four carbapenemase-producing isolates were detected accurately for their subtypes. The two major clones of carbapenemase-producing CRKP isolates in Taiwan were ST11-K47 producing KPC-2 (n = 47) and ST11-K64 producing OXA-48-like (n = 9). Our results support the use of either test in routine laboratories for the rapid detection of common carbapenemases. Caution should be taken using the Xpert assay in areas with a high prevalence of CRE carrying blaIMP-8.IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) are emerging worldwide, causing nosocomial outbreaks and even community-acquired infections since their appearance 2 decades ago. Our previous national surveillance of CPE isolates in Taiwan identified five carbapenemase families (KPC, OXA, NDM, VIM, and IMP) with the KPC-2 and OXA-48-like types predominant. Timely detection and classification of carbapenemases in CPE may be a useful test to guide optimal therapy and infection control. Genetic detection methods using the Xpert CARBA-R assay and the immunochromatographic assay using the NG-Test CARBA 5 have been validated with the advantage of short turnaround time. Our study demonstrated that the NG and Xpert assays are convenient methods to accurately identify carbapenemases in carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Escherichia coli blood isolates. Detecting IMP variants remains challenging, and the results of Xpert CARBA-R assay should be carefully interpreted.

Comparison of the In vitro Activities of Ceftazidime/Avibactam with Other Drugs Used to Treat Carbapenem-resistant Enterobacteriaceae

Background &amp; Objective:: Our goal was to compare the in vitro activities of ceftazidime/avibactam (CZA) to those observed in response to other drugs used to treat carbapenem-resistant Enterobacteriaceae (CRE). Methods:: Bacterial isolates collected from intensive care unit patients with hospital-acquired infections were identified using the VITEK 2TMCompact 15. Susceptibility testing for Escherichia coli and Klebsiella spp. was performed using Kirby- Bauer disk diffusion and the VITEK2 system. Minimum inhibitory concentration (MIC) E-test strips were used to examine susceptibility to fosfomycin, colistin, and CZA. Detection of carbapenemase in isolates of Enterobacteriaceae was performed using the modified carbapenem inactivation method (mCIM) and multiplex polymerase chain reaction. Results:: A full 62.5% of the Enterobacteriaceae isolates were CRE. The sensitivity and specificity of mCIM were 98.3% and 100%, respectively, with positive predictive and negative predictive values and accuracy at 100%, 97.3%, and 98.96%, respectively. The most prevalent of the carbapenemase genes was blaKPC which was detected in 46.7% of all isolates and in 83.3% of those identified as Klebsiella pneumoniae. CRE were most sensitive to colistin (96.7%), CZA (80%) and tigecycline (71.7%) (p&lt;0.001). The MIC50 for CZA (≤0.016 μg/mL) was the lowest of all the drugs evaluated, followed by colistin (1 μg/mL) and tigecycline (2 μg/mL). All metallo-ß-lactamase-negative isolates were sensitive to CZA. Conclusions:: mCIM is sensitive, specific, inexpensive and easy to perform; as such, it an ideal method for identification of carbapenem-resistant strains. Our results suggest that CZA may be a good choice for treating infections with CRE if therapeutic options are limited.

Evaluación de métodos fenotípicos para la detección de carbapenemasas aplicables a laboratorios de poca complejidad

The spread of carbapenemase-producing gram-negative bacilli is a global public health problem. Several authors have proposed phenotypic assays to presumptively detect these enzymes applicable to low and medium complexity laboratories. In the present study, we have developed and compared different phenotypic techniques using strains genetically identified as carbapenemase-producing. All the tested methods detected the presence of carbapenemases. The carbapenem inactivation method (MIC) and the modified carbapenem inactivation method with and without EDTA (mMIC-eMIC) were the simplest and easiest to interpret but their disadvantage was on the time required to obtain results. The direct Carba NP and Carba-Blue colourimetric methods were the fastest but they depend on reagent preparation and accurate pH adjustment of the solutions. Synergy methods with EDTA discs, boronic acid and the Triton Hodge Test (THT) require technical expertise to evaluate true synergism. Whereas, the Disk Carbapenemase Test (DCT) was the method that presented the greatest technical difficulties.

Comparison of sCIM and Other Phenotypic Detection Methods for Carbapenemase-Producing Enterobacterales.

ABSTRACTRapid detection and reporting of carbapenemase-producing Enterobacterales (CPE) is one of the top priorities of clinical microbiology laboratories. The Clinical and Laboratory Standards Institute recommends the modified carbapenem inactivation method (mCIM) as the preferred method for this purpose, but it requires a broth incubation process which can be cumbersome. Here, we compared the performance of mCIM with three alternative rapid CPE detection methods against a collection of genetically defined CPE, with most carrying blaIMP, and non-CPE clinical isolates. The sensitivities of mCIM, simplified carbapenem inactivation method (sCIM), Rapidec Carba NP, and NG-Test Carba 5 were 98.0%, 54.9%, 90.2%, and 72.5%, whereas the specificities were 89.5%, 84.2%, 89.5%, and 100%, respectively. Modification of the interpretive criteria of sCIM increased its sensitivity to 88.2% and specificity to 89.5%. The results suggest that mCIM is currently the optimal method for CPE detection in an epidemiological setting where CPE-producing IMP group carbapenemase is predominant. While sCIM is easier to perform, it requires further validation before it can be widely adopted as an alternative to mCIM in the clinical laboratory.IMPORTANCE Simple identification methods for carbapenemase-producing Enterobacterales are required for the clinical laboratory. The simplified carbapenem inactivation method (sCIM) is a carbapenemase detection method that can be performed with less hands-on time than mCIM, but its sensitivity and specificity were suboptimal compared with other phenotypic detection methods when tested against a collection of IMP-producing CPE. Insufficient inactivation of imipenem from inadequate inoculation was suspected as the cause. While sCIM is easier to perform, it requires optimization before it can be widely adopted as an alternative to mCIM in the clinical laboratory.

Comparative study of phenotypic-based detection assays for carbapenemase-producing Acinetobacter baumannii with a proposed algorithm in resource-limited settings

The increasing incidence of carbapenem resistance in Acinetobacter baumannii is a critical concern worldwide owing to the limitations of therapeutic alternatives. The most important carbapenem resistance mechanism for A. baumannii is the enzymatic hydrolysis mediated by carbapenemases, mostly OXA-type carbapenemases (class D) and, to a lesser extent, metallo-β-lactamases (class B). Therefore, early and accurate detection of carbapenemase-producing A. baumannii is required to achieve the therapeutic efficacy of such infections. Many methods for carbapenemase detection have been proposed as effective tests for A. baumannii; however, none of them are officially recommended. In this study, three carbapenemase detection methods, namely, CarbaAcineto NP test, modified carbapenem inactivation method (mCIM), and simplified carbapenem inactivation method (sCIM) were evaluated for phenotypic detection of clinically isolated A. baumannii. The MICs of imipenem, meropenem, and doripenem were determined for 123 clinically isolated A. baumannii strains before performing three phenotypic detections. The overall sensitivity and specificity values were 89.09%/100% for the carbAcineto NP test, 71.82%/100% for sCIM, and 32.73%/33.13% for mCIM. CarbAcineto NP test and sCIM performed excellently (100% sensitivity) when both Class B and Class D carbapenemases were present in the same isolate. Based on the results, the combined detection method of sCIM and CarbAcineto NP test was proposed to detect carbapenemase-producing A. baumannii rather than a single assay, significantly increasing the sensitivity of detection to 98.18%. The proposed algorithm was more reliable and cost-effective than the CarbAcineto NP test alone. It can be easily applied in routine microbiology laboratories for developing countries with limited resources.

Characteristics of Carbapenem-Resistant Gram-Negative Bacilli in Patients with Ventilator-Associated Pneumonia.

Carbapenem-resistant Gram-negative bacilli (CR-GNB) has become a global threat. In hospital settings, the association of CR-GNB with ventilator-associated pneumonia (VAP) is a critical public health concern owing to their high resistance rate to most antibiotics. The present study aims to identify the frequency of carbapenem-resistance and to determine the rate of multidrug resistance (MDR), extensive drug resistance (XDR) and pan-drug resistance (PDR) among CR-GNB infections in VAP. Antimicrobial susceptibility testing was carried out using the disk diffusion method and the detection of carbapenemases was screened using the imipenem-E test and the modified carbapenem-inactivation method (mCIM). The isolates were verified by polymerase chain reaction (PCR) for the presence of blaNDM, blaSPM, blaVIM, blaIMP and blaGIM genes. 89.5%, 14%, 17.5%, 10.5%, 3.5% of isolates exhibited the presence of blaNDM, blaVIM, blaSPM, blaIMP and blaGIM, respectively. 76%, 17% and 7% of isolates were PDR, XDR, and MDR, respectively. Carbapenem-resistance genes were identified in a significant percentage and blaNDM was the most predominant gene. All isolates were highly resistant to most antibiotics. This health concern has proven to be a big challenge in developing countries such as Egypt, as it is associated with high morbidity, high mortality, and raised healthcare costs.