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

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

Detection of Carbapenem-resistance in CRE by Comparative Assessment of RAPIDEC® CARBA NP and Xpert™Carba-R Assay.

Introduction: Carbapenem-resistant Enterobacteriaceae (CRE) infections resist nearly most available antimicrobials, resulting in poor clinical outcomes. Saudi Arabia has a relatively high CRE prevalence. This study aims to evaluate the sensitivity of Rapidec Carba NP test and GeneXpert Carba-R assay compared with conventional manners for detection of carbapenemase-producing Enterobacteriaceae. Methods: This is a cross-sectional study including a total of 90 CRE isolates examined at two tertiary hospitals in KSA from October 2020 to December 2021. Gram-negative Enterobacteriaceae were identified by using Vitek 2 system and were furtherly tested for imipenem and meropenem susceptibility by E- test strips, followed by Rapidec Carba NP test and the Xpert™Carba-R assay. Results: Carbapenem-resistant K. pneumoniae (78.9%) and carbapenem-resistant E. coli (14.4%) were the two most common isolates species. Colistin (98.9%) and tigecycline (88.9%) were the most effective antibiotics against CRE isolates, followed by amikacin (52.2%), gentamicin (33.3%), cotrimoxazole (15.6%), and ciprofloxacin (8.9%). blaOXA-48 was the predominant carbapenemase gene (44.4%), followed by blaNDM (32.2%). blaKPC gene was not detected. The Rapidec Carba NP and the Xpert™Carba-R demonstrated an overall sensitivity of 69.3% and 88%, respectively, in comparison to gold standard detection of meropenem and imipenem resistance by Vitek 2 system and E- test strips. Discussion: RAPIDEC® CARBA NP may be a beneficial screening test for detecting CRE, but for confirmation of the results, Xpert Carba-R assay is more sensitive, significantly lowering the turnaround time compared to reference traditional methods. The information on carbapenemase genes may be used for epidemiologic purposes and outbreak management.

Intestinal Colonization Due to Carbapenem-Resistant Enterobacteriaceae Among Hematological Malignancy Patients in India: Prevalence and Molecular Charecterisation.

Faecal carriage of Carbapenem-resistant Enterobacteriaceae (CRE) is being observed as an important risk factor for bacteremia among patients with hematological malignancies. A prospective surveillance study was conducted among these patients to determine the gut colonization of CRE. Rectal/perianal swabs were collected to isolate CRE. Carbapenem resistance was detected by disk diffusion, modified-Hodge, Carba-NP test, and PCR for bla NDM-1, bla KPC, bla OXA-48, bla VIM, bla IMP genes. A total of 209 CRE isolates were identified from 151 patients. E. coli was the most common (83.2%) CRE identified, followed by Klebsiella spp. (9.6%). The majority of CRE were observed resistant to ertapenem (86%). bla NDM-1 was the most common gene (57.3%), followed by bla OXA-48 (37.8%). 26.8% isolates found to carry both bla NDM-1 and bla OXA-48 genes. CRE is increasingly observed to cause bacteremia among hematological malignancy patients due to increased colonization. Screening for gut CRE colonization is necessary to guide empirical therapy and apply infection control measures among these patients.

The prediction values of carbapenemase detection methods and carbapenem susceptibility testing for clinical outcomes of patients with Acinetobacter bacteremia under carbapenem treatment.

BACKGROUND: Carbapenem-resistant Acinetobacter species have emerged as notorious pathogens causing nosocomial infections. Several phenotypic methods have been developed for detecting carbapenemase production in Enterobacteriaceae. The accuracy of these methods in the prediction of carbapenemase production in Acinetobacter species has not been studied well. METHODS: This retrospective study enrolled adult patients with Acinetobacter bacteremia from four medical centers in Taiwan between 2012 and 2016. Their demographics and clinical outcomes were recorded. The carbapenem susceptibility of the Acinetobacter species was determined using the agar diffusion method. The carbapenemase genes were detected by PCR. Four phenotypic methods, including the modified Hodge test (MHT), modified carbapenem inactivation method (mCIM), Carba NP test, and CarbAcineto NP test were carried out to determine the production of carbapenemase. RESULTS: We analyzed 257 adults who received initial carbapenem monotherapy for the treatment of Acinetobacter bacteremia. Shock within three days of bacteremia and acquisition of carbapenem non-susceptible isolates were independently associated with a higher 14-day and 30-day mortality in patients with Acinetobacter bacteremia. Among the four phenotypic tests for carbapenemase detection, MHT using the imipenem disc displayed the greatest sensitivity (94%; 95% confidence interval [CI], 89-97%) and specificity (81%; 95% CI, 73-88%) for predicting imipenem non-susceptibility. CONCLUSION: Carbapenem non-susceptibility and shock were independent risk factors for mortality in patients with Acinetobacter bacteremia. The MHT could predict the carbapenem susceptibility of Acinetobacter isolates. It is a cheap and quick assay, which could be applied in clinical practice.

Phenotypic Detection of Carbapenemase Production in Carbapenem-Resistant Enterobacteriaceae by Modified Hodge Test and Modified Strip Carba NP Test.

Objective Carbapenems are last resort antibiotics for multidrug-resistant Enterobacteriaceae . However, resistance to carbapenem is increasing at an alarming rate worldwide leading to major therapeutic failures and increased mortality rate. Early and effective detection of carbapenemase producing carbapenem-resistant Enterobacteriaceae (CRE) is therefore key to control dissemination of carbapenem resistance in nosocomial as well as community-acquired infection. The aim of present study was to evaluate efficacy of Modified strip Carba NP (CNP) test against Modified Hodge test (MHT) for early detection of carbapenemase producing Enterobacteriaceae (CPE). Material and Methods Enterobacteriaceae isolated from various clinical samples were screened for carbapenem resistance. A total of 107 CRE were subjected to MHT and Modified strip CNP test for the detection of CPE. Statistical Analysis It was done on Statistical Package for the Social Sciences (SPSS) software, IBM India; version V26. Nonparametric test chi-square and Z -test were used to analyze the results within a 95% level of confidence. Results Out of 107 CRE, 94 (88%) were phenotypically confirmed as carbapenemase producer by Modified strip CNP test and 46 (43%) were confirmed by Modified Hodge Test (MHT). Thirty-eight (36%) isolates showed carbapenemase production by both MHT and CNP test, 56 isolates (52%) were CNP test positive but MHT negative, eight (7%) isolates were MHT positive but CNP test negative and five (5%) isolates were both MHT and CNP test negative. There is statistically significant difference in efficiency of Modified CNP test and MHT ( p < 0.05). Conclusion Modified strip CNP test is simple and inexpensive test which is easy to perform and interpret and gives rapid results in less than 5 minutes. It has high degree of sensitivity and specificity. Modified strip CNP test shows significantly higher detection capacity for carbapenemase producers as compared with MHT.

Evaluation of NitroSpeed-Carba NP test for rapid identification among different classes of carbapenemases in Enterobacterales and Pseudomonas aeruginosa.

OBJECTIVES: This study aimed to evaluate the performance of the NitroSpeed-Carba NP test for detecting carbapenemases in the clinical strains of Enterobacterales and Pseudomonas aeruginosa (P. aeruginosa), and analyze its advantages and limitations. METHODS: The antimicrobial susceptibility tests were performed according to the agar dilution method. Using the modified carbapenemase inactivation method (mCIM), polymerase chain reaction (PCR), and sequencing, the production of carbapenemase and the prevalence of genes were studied. The NitroSpeed-Carba NP test was performed to detect different types of carbapenemases in Enterobacterales and P. aeruginosa. The results of PCR and sequencing were used as the gold standard. RESULTS: Among 144 carbapenemase-producing and 54 carbapenemase-negative strains of Enterobacterales and P. aeruginosa, the NitroSpeed-Carba NP test correctly detected 143 of 144 carbapenemase producers and 51 of 54 non-carbapenemase producers. Moreover, the sensitivity and specificity of all tested isolates were 99.31% and 94.44%, respectively (99.28% and 92.86% for Enterobacterales; 100% and 100% for P. aeruginosa). The sensitivity was 100% for class A (56 of 56), 100% for class B (60 of 60), and 100% for class D (27 of 27). CONCLUSIONS: The results suggest that NitroSpeed-Carba NP test is a simple and valuable assay that could be used as a rapid and stable detection method to identify the carbapenemases in Enterobacterales and P. aeruginosa strains.

Multicentre study of the main carbapenem resistance mechanisms in important members of the Enterobacteriaceae family in Iran.

Resistance to carbapenems has been increasingly reported from the Enterobacteriaceae family, with different mechanisms in different geographic parts of the world. This study investigated the mechanisms of carbapenem resistance in Escherichia coli, Klebsiella pneumoniae and Enterobacter spp. carried out as a multicentre study (n = 10). All third-generation cephalosporin-resistant E. coli, K. pneumoniae and Enterobacter spp. that had been recovered from the selected provinces were included. Modified Hodge test and Carba NP test were done as a phenotypical method for detection of carbapenemase; the most common carbapenemase was detected by PCR. We evaluated the presence of an active efflux pump by using cyanide 3-chlorophenylhydrazone. Overexpression of AcrA/B and presence of OqxAB was detected by real-time PCR and conventional PCR respectively. Microorganisms in this study included 58 E. coli, 95 K. pneumoniae and 60 Enterobacter spp. Modified Hodge test showed a sensitivity of 41% and a specificity of 83%, and the Carba NP test showed a sensitivity of 26% and a specificity of 92% for detection of carbapenemase. OXA-48 was the most frequently detected carbapenemase, followed by NDM-1. Thirty-nine percent and 27% of positive cyanide 3-chlorophenylhydrazone test organisms included active AcrA/B and OqxAB efflux pumps respectively. The result showed the Carba NP test was more specific than MHT. Data confirmed the involvement of AcrA/B and OqxAB efflux pump as a carbapenem resistance mechanism in selected bacteria. Similar to other reports from the Middle East, we found OXA-48 and NDM-1 to be the most frequent carbapenemase.

On-chip Carba NP test for accurate and high throughput detection of carbapenemase-producing Enterobacteriaceae.

Given the great threat posed by clinical Carbapenemase-producing Enterobacteriaceae (CPE), its rapid and accurate detection is highly required to guide infection treatment and to prevent serious outbreaks. While the Carba NP test (CNPt) is a popular and methodologically simple colorimetric assay for detecting carbapenemase activity, its clinical application is limited by low throughput, poor sensitivity and obscure color interpretation due to the lack of quantitative information on time- and dose-dependent color changes. To address these limitations, we developed a multiplexed microfluidic device (1280 chambers) that is able to automatically generate 16 bacterial concentration gradients and uses green channel intensity to extract quantitative color changes as a function of time. Here we examined the dynamic color changing profiles of 3 reference and 8 clinical bacteria isolates. The results demonstrated linear correlations between color developments and time (within 0-120 min) or bacterial doses (within OD600 of 0.8-8.0), enabling accurate and reproducible detection of CPE with over 105 fold improved sensitivity for an NDM-producer (LOD of 2.4 × 103 cells) and over 104 fold improved sensitivity for an OXA-48-producer (LOD of 7.2 × 104 cells) as compared to the conventional CNPt method (LOD above 1.3 × 109 cells). As the first digital microfluidic platform for CPE, this on-chip CNPt compared favourably with the conventional bench-based method by significantly improved sensitivity, high throughput, low consumption and automatization, favouring its potential clinical applications for rapid screening of CPE with a low cost. Furthermore, the results also revealed dynamic signature associated with bacteria, which may hold promise for a new approach to study divergent bacterial responses to antibiotic treatments.

Molecular epidemiology & therapeutic options of carbapenem-resistant Gram-negative bacteria.

Background & objectives: The growing incidence and the wide diversity of carbapenemase-producing bacterial strains is a major concern as only a few antimicrobial agents are active on carbapenem-resistant bacteria. This study was designed to study molecular epidemiology of carbapenem-resistant Gram-negative bacterial (GNB) isolates from the community and hospital settings. Methods: In this study, non-duplicate GNB were isolated from clinical specimens, and phenotypic test such as modified Hodge test, metallo ß-lactamase E-strip test, etc. were performed on carbapenem-resistant bacteria. Multiplex PCR was performed to identify the presence of blaIMP, blaVIM, blaKPC, blaOXA48, blaOXA23, blaSPM, blaGIM, blaSIM and blaNDM. Minimum inhibitory concentration (MIC) of colistin, fosfomycin, minocycline, chloramphenicol and tigecycline was also determined. Results: Of the 3414 GNB studied, carbapenem resistance was 9.20 per cent and maximum resistance (11.2%) was present at tertiary care centre, followed by secondary care (4%) and primary centre (2.1%). Among the carbapenem-resistant bacteria, overall, the most common isolate was Pseudomonas aeruginosa (24%). On multiplex PCR 90.3 per cent carbapenem-resistant isolates were positive for carbapenemase gene. The blaNDM(63%) was the most prevalent gene followed by blaVIM(18.4%). MIC results showed that 88 per cent carbapenem-resistant Enterobacteriaceae were sensitive to fosfomycin, whereas 78 per cent of P. aeruginosa and 85 per cent Acinetobacter spp. were sensitive to colistin. Interpretation & conclusions: Carbapenem resistance in GNB isolates from the community and hospital settings was found to be on the rise and should be closely monitored. In the absence of new antibiotics in pipeline and limited therapeutic options, prudent use of antibiotics and strict infection control practices should be followed in hospital to limit the emergence and spread of multidrug-resistant bacteria.

Comparative evaluation of in-house Carba NP test with other phenotypic tests for rapid detection of carbapenem-resistant Enterobacteriaceae.

BACKGROUND: The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is alarming worldwide causing serious infections. Rapid and accurate identification of CRE is crucial to reduce the mortality and morbidity. In this study, we tried to develop an in-house Carba NP test for detection of CRE and evaluate its performance with others. METHODS: A prospective study was conducted with 40 nonrepeating Enterobacteriaceae isolates over a period of 3 months. All the isolates were screened for carbapenem resistance as per CLSI 2016 guidelines followed by PCR for blaNDM-1, blaOXA-48, blaKPC, blaVIM, and blaIMP genes. All the isolates were subjected to five phenotypic tests, that is, in-house Carba NP (iCarba NP), commercial Carba NP (cCarba NP), Blue-Carba, modified Hodge test (MHT), and CHROMagar. RESULTS: Among the 40 isolates, 87.5% were identified as Escherichia coli, 7.5% were Klebsiella pneumoniae, 2.5% were Enterobacter cloacae, and 2.5% were Citrobacter freundii. Thirty-three of 40 (82.5%) isolates were found to harbor one or more resistant genes. Considering PCR to be the gold standard test, sensitivity of the phenotypic methods for CRE detection ranged from 63.6% (MHT) to 96.9% (CHROMagar). Both cCarba NP and iCarba NP observed to have highest specificity. The performance of iCarba NP was found comparable with cCarba NP by kappa score 1 and found approximately 10 times less expensive than cCarba NP. CONCLUSION: CHROMagar was observed most sensitive assay for detection of CRE followed by both Carba NP tests. iCarba NP was proved cheaper and equally good as cCarba NP for detection of CRE.

Rectal Carriage of Carbapenem-Resistant Enterobacteriaceae: A Menace to Highly Vulnerable Patients.

BACKGROUND: Bloodstream infection (BSI) due to carbapenem-resistant enterobacteriaceae (CRE) is the leading cause of morbidity and mortality in patients with hematological malignancy. These patients receive chemotherapy during treatment, which lead to severe mucositis of gastrointestinal tract and myelosuppression. It was hypothesized that the gut colonizer translocate into the blood circulation causing BSI. Colonization rate with CRE among these patients in India is unknown. AIM: This study aims to determine the carriage rate of CRE in cancer patients. SETTING AND DESIGN: A prospective study was conducted in a tertiary care hospital of India. MATERIALS AND METHODS: Rectal swab of 93 patients were collected and processed as per the Center for Disease Control and Prevention protocol for detection of CRE. The isolate CREs were identified by standard phenotypic tests and confirmed for carbapenem resistance by disk diffusion test using carbapenem disk (imipenem, meropenem, doripenem, and ertapenem), Carba-NP test and modified Hodge test. Resistant to any of the carbapenem disc is considered as CRE. RESULTS: A total of 86 isolates were detected from 93 patients. Seventy-six isolates were identified as CRE, and 10 isolates were Gram-positive cocci and other Gram-negative bacilli. Acute myeloid leukemia was the most common clinical presentation followed by acute lymphoid leukemia. Thirty-nine out of 93 patients were on chemotherapy. Sixty-seven out of 76 isolates of CRE were observed positive for carbapenemase production by Carba-NP test. CONCLUSION: This study highlights very high rate of CRE carriage among the hematological malignancy patients; who are highly vulnerable to infection. This confirms the need of infection control prevention activities among the hematological malignancy patients.

[Triton X-100 Enhanced the Detection of Hodge Test and Carba NP Test for Carbapenemase-producing Acinetobacter baumannii Complex].

OBJECTIVE: To find out highly effective phenotypic methods to detect carbapenemase-producing Acinetobacter baumannii (A. baumannii complex) so as to support the epidemiological investigation and clinical application. METHODS: We included 113 A. baumannii complex and compared the detection performance of modified Hodge test, Carba NP test, Triton Hodge test, and the simplified Carba NP-direct test with Tritont X-100. RESULTS: We tested 83 carbapenemase-producing A. baumannii complex and 30 non-carbapenemase producers. The sensitivity and specificity of Hodge test were significantly higher than those of Carba NP test (71.1% versus 35.0%, 100% versus 86.7%, P<0.05, respectively). The sensitivity of Triton Hodge test and Carba NP-direct test was respectively significantly higher than Hodge test and Carba NP test (98.8% versus 71.1%, 85.5% versus 35.0%, P<0.001, respectively ). However, the specificities were comparable (P>0.05). The positive additive effects of the two methods with Triton X-100 were more obvious than those of the methods without Triton X-100 (P<0.001). CONCLUSION: Triton X-100 could increase the sensitivity and positive additive effect on phenotypic detection of A. baumannii complex. Triton Hodge test and Carba NP-direct test were more applicable for clinical routine procedure.

Phenotypic Detection of Carbapenemase-Producing Organisms from Clinical Isolates.

The rapid spread of multidrug-resistant Gram-negative organisms constitutes one of the greatest challenges to global health. While Gram-negative organisms have developed several mechanisms to avert the bactericidal effects of commonly prescribed antibiotic agents, the increasing prevalence of carbapenemase-producing organisms (CPO) is particularly concerning given the rapid spread of mobile genetic elements containing carbapenemase genes, the limited treatment options for infections caused by these organisms, and the high mortality rates associated with CPO infections. Understanding if an organism is carbapenemase producing and, if so, the class of carbapenemase(s) produced has treatment implications, as some agents preferentially have activity against specific carbapenemases. Furthermore, CPO disseminate between patients with greater ease than non-CP-carbapenem-resistant organisms and warrant more intensive infection control measures than would be employed in the absence of carbapenemase production. Phenotypic assays currently used in clinical practice to detect CPO consist of the following: (i) growth-based assays which measure carbapenem resistance based on organism growth in the presence of a carbapenem antibiotic (e.g., modified Hodge test and modified carbapenem inactivation method), (ii) hydrolysis methods which detect carbapenem degradation products (e.g., Carba NP test and matrix-assisted laser desorption-ionization time of flight mass spectrometry), and (iii) lateral flow immunoassays which detect carbapenemase enzymes through the use of specific antibodies. Although there is no single phenotypic test that meets all specifications of the ideal test, as we describe in this review, there are a number of tests that are user-friendly, affordable, accurate, and feasible for implementation in clinical microbiology laboratories of all sizes.

Comparative Evaluation of Four Phenotypic Methods for Detection of Class A and B Carbapenemase-Producing Enterobacteriaceae in China.

The objective of this study was to evaluate the performance of four phenotypic methods in the detection of carbapenemase-producing Enterobacteriaceae (CPE) in China. We evaluated the performance of four carbapenemase detection methods, the modified Hodge test (MHT), the Carba NP test, the meropenem hydrolysis assay (MHA) with 1- and 2-h incubation, and the modified carbapenem inactivation method (mCIM) with meropenem, imipenem, and ertapenem, on 342 carbapenem-resistant Enterobacteriaceae isolates (CRE) in China. PCR was used as the gold standard. The 2-h-incubation MHA performed the best in carbapenemase detection (overall sensitivity, specificity, positive predictive value, and negative predictive value all 100%). Second was the Carba NP test, with a sensitivity of 99.6%. The 1-h-incubation MHA performed poorly in Klebsiella pneumoniae carbapenemase (KPC) detection (sensitivity, 71.3%). For mCIM, the best performance was observed with the meropenem disk. The MHT exhibited the worst performance, with a specificity of 88.8%. All assays except 1-h-incubation MHA, which failed to identify 68 KPC-2s, had a sensitivity of >98% in the detection of 172 KPCs. Likewise, all assays had a sensitivity of >95% in the detection of 70 class B carbapenemases, except for MHT (82.9%). The 2-h-incubation MHA significantly improved the accuracy in CPE detection compared with that for 1-h incubation and performed the best in the detection of class A and B carbapenemases. Our findings suggest that the MHA is the most practical assay for carbapenemase detection. For those who cannot afford the associated equipment, both the Carba NP test and mCIM are good alternatives with regard to the practical requirements of time and cost.

Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae from Bangkok, Thailand, and Their Detection by the Carba NP and Modified Carbapenem Inactivation Method Tests.

AIM: The purpose of the study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant Enterobacteriaceae and evaluate the Carba NP and modified carbapenem inactivation method (mCIM) tests in their detection. MATERIALS AND METHODS: A total of 287 nonduplicated Enterobacteriaceae isolates, which were at least resistant to one of the carbapenems, were identified and detected for carbapenemase genes by multiplex PCR covering blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like. All positive genes were then sequenced. These isolates were phenotypically tested for the production of carbapenemases by mCIM and Carba NP tests to evaluate the efficacy of these methods. RESULTS: Seven species of carbapenem-resistant isolates mainly Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae were detected. Of these isolates, three families of carbapenemase genes, including blaNDM (blaNDM-1, -4, -5, -9), blaOXA (blaOXA-48, -181, -232), and blaIMP-14, were found. Of these, 223 (77.70%) carried at least one of the carbapenemase genes. The blaNDM was detected in 160/223 (71.75%) isolates, of which 153/160 (95.63%) were the blaNDM-1. Three types of the blaOXA-48-like group, blaOXA-48, blaOXA-181, and blaOXA-232, were found, 91/104 (87.5%) harbored the blaOXA-232. In addition, 25.11% (56/223) of the carbapenemase-producing isolates harbored a combination of blaNDM and blaOXA-48-like. Phenotypic detection methods, mCIM and Carba NP, showed 100% sensitivity and specificity to blaNDM, blaIMP-14, and blaOXA-48, while the mCIM was positive in all blaOXA-181 and blaOXA-232 isolates, only 12.5% (1/8) and 28.95% (11/38), respectively, were detected by the Carba NP test. CONCLUSIONS: This study revealed a unique prevalence of carbapenemase genes in Bangkok, Thailand, as well as demonstrated the efficacy and limitation of phenotypic detection methods of carbapenemase in the area where blaNDM-1 and blaOXA-232 were predominant.

Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae.

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory.

Study on Carbapenemase Genotype of Imipenem-resistant Gram-negative Bacilli in Intensive Care Unit of 3 Third Grade Class A Hospitals from Qingdao Area / 中国药房

OBJECTIVE: To analyze carbapenemases genotype of imipenem-resistant Gram-negative bacilli in intensive care unit (ICU) of 3 third grade class A hospitals from Qingdao area, so as to provide reference for drug-resistant bacteria infection prevention and treatment in clinic. METHODS: From Jan. 2013 to Jun. 2016, each 60 strains of imipenem-resistant Klebsiella pneumoniae (IRKP), imipenem-resistant Pseudomonas aeruginosa (IRPA) and imipenem-resistant Acinetobacter baumanii (IRAB) were collected from 3 third grade class A hospitals from Qingdao area. Drug sensitivity test was performed by using Kirby-Bauer method. Phenotypes of carbapenemases were determined by Carba NP trial. PCR was applied to amplify carbapenemase gene; Sanger seqnencing method was adopted for bi-directional sequencing; Blast comparison with GenBank database was conducted. RESULTS: Three kinds of imipenem-resistant Gram-negative bacilli showed high drug resistance to majority commonly used antibiotics as piperacillin, cefazolin, imipenem and cilastatin sodium, gentamicin, etc., but were sensitive to polymyxin B (resistance rate of 0). Among 180 drug-resistant strains, there were 52 strains of class A carbapenems, 13 strains of class B carbapenems and 39 strains of class D carbapenems; the detection rates of them were 28. 89％, 7. 22％ and 21. 67％, respectively. There were 52 strains of KPC-2 gene (IRKP), 4 strains of IMP-1 gene (IRPA), 8 strains of VIM-2 gene (7 strains of IRPA, 1 strain of IRAB), 39 strains of OXA-23 gene (IRAB); the detection rates of them were 28. 89％, 2. 22％, 4. 44％, 21. 67％; all strains were not detected 1MP-2, VIM-1, NDM-1, OXA-24, OXA-58 genes. Results of Blast comparison showed that above detected genes were absolutely homology with the corresponding genes in GenBank database. CONCLUSIONS: Drug resistance of imipenem-resistant Gram-negative bacilli in ICU of 3 third grade class A hospitals is serious in this region, which are nearly no-sensitive to most of commonly used antibiotics in clinic. Main genotypes included KPC-2 (K. pneumoniae), OXA-23 (A. baumanii) and IMP-1 and VIM-2 (P. aeruginosa).

Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria.

PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-ß-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.

Evaluation of the RAPIDEC® CARBA NP and ß-CARBA® tests for rapid detection of Carbapenemase-producing Enterobacteriaceae.

The analytical performances of the RAPIDEC CARBA NP® (bioMérieux) and the ß-CARBA® (Bio-Rad) tests were evaluated for the rapid detection of any known type of carbapenemases in Enterobacteriaceae. An international collection of 149 enterobacterial isolates comprising 111 Carbapenemase-Producing Enterobacteriaceae (CPE) and 38 non-carbapenemase producers was used. CPE included 32 carbapenemase producers of class A (18 KPC-2, 1 FRI-1, 5 SME and 8 IMI), 33 of class B (13 NDM, 11 VIM and 9 IMP) and 46 of class D (15 OXA-48, 14 OXA-181, 10 OXA-204, 3 OXA-232 and 4 OXA-162). The RAPIDEC CARBA NP® and the ß-CARBA® tests were performed in strict accordance to the manufacturer's instructions and results were read within 2 h and 30 min, respectively. RAPIDEC CARBA NP® detected 104/111 CPE isolates compared to 72/111 for the ß-CARBA® test. The overall sensitivity and specificity were 93.7% and 100%, respectively, for the RAPIDEC CARBA NP® test, and 64.9% and 90%, respectively, for the ß-CARBA® test. The ß-CARBA® test failed to detect all the non-KPC class A carbapenemases (14/14) and most (24/31) of the OXA-48-like producers (OXA-162, OXA-181, OXA-204 and OXA-232), and detected 1/1 OXA-163 and 1/1 OXA-405 as carbapenemase producers whereas these enzymes are rather defined as non carbapenemases. RAPIDEC CARBA NP® test exhibited better performances than those of the ß-CARBA® test and confirmed to be a reliable tool for the detection of CPE, especially for OXA-48-like producers.

Transcriptional Response of Multiple ESBL Genes Within Escherichia coli Under Oxyimino-Cephalosporin Stress.

The expression of extended-spectrum beta-lactamases directly interferes with the treatment options in a clinical setting. It is not clearly defined why bacteria acquire multiple beta-lactamases and how they are being expressed in antibiotic stress. With this key question, the study was designed to understand the transcriptional response in Escherichia coli harboring multiple blaESBLs against different oxyimino-cephalosporin stress. A total of 169 consecutive, nonduplicate oxyimino-cephalosporin-resistant isolates of E. coli were screened and were ESBL positive. Among them six isolates were found to harbor multiple beta-lactamase genes and we, as per our objective, selected them for this study. Molecular characterization was done by multiplex polymerase chain reaction (PCR) assay. Minimum inhibitory concentration, transcriptional expression, transferability, and plasmid incompatibility typing of multiple blaESBLs were carried out. Plasmid stability and antibiotic susceptibility of donor and transconjugants were performed. A total of six isolates were found to be harboring multiple ESBL genes and MIC above the breakpoint level against all the tested antibiotics. Quantitative real-time PCR showed that in basal level without antibiotic stress, SHV-148 expressed more, but with ceftriaxone stressed, expression of CTX-M-15 and SHV-148 was high. In case of PER-1, expression was high with ceftazidime stress. blaESBLs were horizontally transferable and originated through multiple inc types. Plasmids were stable till 115 serial passages. Pulsed-field gel electrophoresis results showed that multiple ESBL genes were spread through six pulsotypes. Our study concludes that acquisition of multiple ESBL genes in E. coli was a specific adaptation for survival against multiple oxyimino-cephalosporin stress in this clinical setting.