Detection Of Carbapenemase-producing Enterobacteriaceae Research Articles

Carbapenemase-Producing Enterobacteriaceae detected in a Large Canadian Tertiary Care Hospital: Five-year retrospective study

Background: The prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing worldwide. In Canada, where rates of healthcare-associated (HA) transmission of CPE remains relatively low, there is a need to share early experience of universal screening programs and risk factors for HA acquisition. Method: In 2018, universal screening was introduced throughout our large Canadian tertiary care hospital across, all critical care and oncology units. Additionally, risk-factor based screening was applied in all other inpatient units, with further targeted screening of roommate exposures or all inpatients on unit following identification of a single HA case. A retrospective cohort study was carried out on CPE cases detected between January 2018 and December 2023. We assessed the proportion of HA CPE cases, defined as CPE identified in patients with prior admission to our facility or after >72 hours after admission. HA cases were examined for relevant risk factors, including known roommate with CPE, the presence of other CPE on the unit, exposure to outbreak units, prior travel history, travel by a family member, and antibiotic exposure within the past 90 days. Result: A total of 150 CPE cases were identified, with 66 (44%) classified as HA. Among these HA cases, 14 (21%) were associated with presence of known case on the unit. The remaining 52 (79%) represented sporadic nosocomial cases without a known exposure or further transmission on the unit. Upon further retrospective review, 6 (9.2%) HA cases had documented travel history or exposure to a family member with recent travel to China, India, Sri Lanka, or the United States within the past year. Nearly all HA cases (62, 95.4%) had antibiotic exposure within 90 days of CPE detection; specifically, 47 (72.3%) received beta-lactams, 42 (64.6%) cephalosporin, 25 (38.5%) glycopeptide, 20 (30.8%) carbapenem, and 8 (12.3%) macrolide. Conclusion: HA CPE acquisition identified during the first 5-years of universal screening were mostly sporadic and not associated with known exposures or other risk factors. Receipt of prior antibiotics was present in nearly all cases.

Verification of direct methods for detection of carbapenemase producing Enterobacteriaceae from blood cultures

Verification of direct methods for detection of carbapenemase producing Enterobacteriaceae from blood cultures

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

Detection of Carbapenemase Producing Enterobacteriaceae from Clinical Samples and their Susceptibility to Conventional Antibiotics and Medicinal Plant Extracts

Carbapenamases are enzymes that renders inactive, the carbapenems antibiotics which are often considered as the last treatment option for all serious infections. Carbapenemase-producing Enterobacteriaceae (CPE) has already been identified all over the world. In Kano and other part of Northern Nigeria, there are several reports that CPE is in circulation at a rate researchers have never imagined and is rapidly spreading to far rural communities. This study aims to detect Carbapenemase producing Enterobacteriaceae from clinical sample and determine their susceptibility to conventional antibiotics and Ficus ovata and Guiera senegalensis plant extracts. Isolates of Pseudomonas aeruginosa, E. coli and Klebsiella pneumoniae were collected from Murtala Muhammad Specialist Hospital, reconfirmed and screened for Carbapenemase production using the Modified Hodges Test. The highest occurrence of carbapenemase was observed in E. coli (14.3%), while the least was observed in K. pneumoniae 3(12%) and P. aeruginosa 2(6.7%). Ficus ovata and Guiera senegalensis Plants were extracted using ethanol, N-butanol and N-hexane as separate solvents. Of all the three plants extracts, highest yield was observed in N-Butanol extracts having 32.7% in Ficus ovata. Susceptibility test using antibiotics revealed a decreased sensitivity of the test isolates to some commonly used antibiotics; Cefotaxime (25μg), Cefixime (5μg), Levofloxacin (5μg), Amoxicillin (30μg) and Cefuroxime (30μg). Agar well diffusion test was employed at a concentration of 20, 10 and 5mg/ml. All the six extracts were observed to have the highest activity against P. aeruginosa with Guiera senegalensis having more inhibitory activity. This demonstrates the extracts potentials for extraction of chemotherapeutic agents

817. Appropriate Sites for the Active Surveillance of Carbapenemase-producing Enterobacteriaceae

Abstract Background Active surveillance tests for carbapenemase-producing Enterobacteriaceae (CPE) are recommended in patients showing risk factors for colonization by these bacteria. There are limited data however on whether surveillance tests for anatomic sites other than the stool would be useful to detect CPE colonization, and we investigated this in our present study. Methods Retrospective analysis was performed on cases at our tertiary care hospital during a 5-year period. The study patients with CPE colonization had been admitted to our surgical intensive care unit (SICU) or sub-ICU for liver transplantation in this period and undergone surveillance tests for both the stool and other sites. Patients were grouped as stool CPE negative (but which included CPE positive cases from initial sputum and other site tests) or positive. Results Among the total study cohort of 158 patients, 138 (87.3%) were included in the stool CPE positive group and the remaining 20 (12.7%) in the stool CPE negative group. While the sensitivity of CPE surveillance testing of the stool was 87.3% (95% CI 81.1-92.1), the sensitivity when combining stool and sputum samples was 93.7% (88.7-96.9). The transmission rates were similar for patients showing CPE positivity in the stool, sputum and other sites, at 4.8% (27/557), 4.7% (3/64), and 6.7% (1/15), respectively (p = 0.95). Conclusion The sensitivity of CPE detection in a stool sample is suboptimal for ruling out CPE colonization and the transmission rates are similar between stool-positive or -negative cases. Combining surveillance of the stool with other sites may be needed for detecting CPE. Disclosures All Authors: No reported disclosures

Multidrug-Resistant and Carbapenemase-Producing Enterobacteriaceae in Addis Ababa, Ethiopia.

Background The emergence and spread of multi-drug resistant (MDR) bacteria have become a public health problem in recent years. For the last many years, carbapenem antibiotics have been used successfully to treat infections caused by MDR Enterobacteriaceae. However, recently, Enterobacteriaceae producing carbapenemases have emerged, which confer broad resistance to most β-lactam antibiotics including carbapenems. Therefore, this study is aimed at determining the magnitude of MDR and carbapenemase-producing Enterobacteriaceae (CPE) isolated from various clinical specimens in Addis Ababa, Ethiopia. Methods A cross-sectional study was conducted from January to April 2018. A total of 312 Enterobacteriaceae isolates were identified from various clinical specimens. The Phoenix automated system (BD Phoenix100) was used for bacterial identification and antimicrobial susceptibility testing. Potential carbapenemase producers were confirmed by the modified carbapenem inactivation test, and KPC, MBL, and OXA-48 were phenotypically characterized by the disk diffusion method. The data obtained were entered and analyzed using SPSS version 20 software. Descriptive statistics, chi square, bivariate and multivariable logistic regression analyses were performed. P value ≤ 0.05 with corresponding 95% confidence interval was considered for statistical significance. Results A total of 312 Enterobacteriaceae were recovered. Of these isolates, 68.6% were MDR and 2.6% were CPE with different classes including OXA-48 1.6% (5/312), MBL 0.6% (2/312), and KPC and OXA-48 0.3% (1/312). The predominant bacterial isolates were E. coli 72.4% (226/312) followed by K. pneumoniae 13.8% (43/312). The antibiotic resistance rates of CPE isolates were significantly higher than other MDRE including ampicillin (100% versus 77.6%), cefoxitin (75% versus 20.6%), and piperacillin/tazobactam (50% versus 13.1%). Conclusion In this study, a relatively higher prevalence of MDR was observed, and the highest resistance was recorded against ampicillin, amoxicillin with clavulanic acid, and sulfamethoxazole-trimethoprim. Detection of CPE is important for implementing appropriate antimicrobial therapy and in controlling the spread of the infection. Furthermore, continuous screening and investigations, including genotypic characterization of CPE, are required for the prevention and control of the spread of antimicrobial-resistant pathogens.

Isolation Procedure for CP E. coli from Caeca Samples under Review towards an Increased Sensitivity.

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

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 modified carbapenem inactivation method with CARBA NP, MHT and real-time PCR for detection of carbapenemase-producing Enterobacteriaceae

Background: Carbapenemase-producing Enterobacteriaceae (CPE) are a global emerging threat with high transmissibility, multidrug-resistance and limited treatment options. Reliable detection is important for prevention, containment of these pathogens as well as for antimicrobial stewardship. The modified carbapenem inactivation method (mCIM) is an inexpensive and straightforward phenotypic tool for detecting carbapenemase activity. The aim of this study was to evaluate the performance of mCIM for detecting carbapenemase activity by comparing with CARBA NP, Modified Hodge Test (MHT) and Real-time PCR. Methods and materials: A total of 94 carbapenem-nonsusceptible Enterobacteriaceae comprising of E. coli (n = 43), Klebsiella species (n = 33) and Enterobacter species (n = 18) were analyzed by mCIM, MHT and CARBA NP according to the Clinical and Standard Institute (CLSI) guidelines (M100-S27). In-house Real-time PCR was performed to confirm carbapenemase genes (KPC, NDM, VIM, IMP, OXA-48). The sensitivity and specificity of mCIM, MHT and CARBA NP was determined by comparing the results with Real-time PCR. Klebsiella pneumoniae BAA-1706 strain and Klebsiella pneumoniae ATCC 700603 both carbapenemase non-producers were used as negative controls. Results: Of the ninety-four isolates analyzed by real-time PCR, 47 isolates expressed NDM and 5 isolates expressed OXA. 38 isolates showed co-expression of NDM and OXA. Two showed co-expression of KPC and NDM. One Enterobacter aerogenes showed co-expression of NDM and VIM, while another showed co-expression of NDM and IMP. The sensitivity of mCIM, CARBA NP and MHT were 100%, 84% and 74% respectively. mCIM showed higher sensitivity than CARBA NP & MHT. The mCIM assay perfectly correlated with the genotypic findings of the 94 isolates with 100% sensitivity and specificity for CPE detection. Conclusion: The results of our study show that mCIM is suitable and reliable for detecting carbapenemase-producing Enterobacteriaceae than CARBA NP and MHT. Compared to molecular method, mCIM is less expensive and can be readily adopted in clinical microbiology laboratory.

Development of a new spectrophotometric assay for rapid detection and differentiation of KPC, MBL and OXA-48 carbapenemase-producing Klebsiella pneumoniae clinical isolates

The increased prevalence of carbapenemase-producing Enterobacteriaceae (CPE) has made essential the design of quicker tests for CPE detection. In the present study, a simple and rapid assay was developed based on measurement of the hydrolytic activity of imipenem at a final concentration of 65 µg/mL (100 µM) through ultraviolet-visible (UV-Vis) spectrophotometry. All measurements were conducted at 297 nm. A total of 83 carbapenem-non-susceptible CPE, consisting of Klebsiella pneumoniae clinical isolates and genotypically characterised as KPC-, VIM-, NDM- or OXA-48-producers, were tested. For comparison, 30 carbapenem-non-susceptible clinical isolates, consisting of Escherichia coli and K. pneumoniae and genotypically confirmed as non-CPE, were also examined. The spectrophotometric assay enabled efficient discrimination of CPE from non-CPE isolates even in 45 min (P < 0.0001). Moreover, the presence of phenylboronic acid (PBA) or ethylene diamine tetra-acetic acid (EDTA) in the reaction mixture was able to inhibit the hydrolytic capacity of KPC- or metallo-β-lactamase (MBL)-producers, respectively, while the hydrolytic activity of OXA-48-producing strains was not affected by the presence of these inhibitors (P < 0.001). The newly developed assay presented 100% sensitivity and specificity to detect and differentiate KPC-, MBL- and OXA-48-producers compared with genotypic characterisation. Thus, the proposed spectrophotometric method can be considered as an easy, fast, accurate and cost-effective diagnostic tool for screening carbapenem-non-susceptible K. pneumoniae isolates in the clinical laboratory.

Results of screening for emergent highly resistant bacteria in Tunisian intensive care units

Abstract Background The infections caused by emergent highly resistant bacteria (eHBR) that develop in intensive care units (ICUs) may result in significant patient illnesses and deaths, extend the duration of hospital stays and generate added costs. Facing this problem, the screening that emphasizes early identification of colonized patients, reduces the prevalence and incidence of infection, improves patient outcomes and reduces healthcare costs. In this context, we have implemented a screening for eHBR in ICUs of Sahloul university hospital of Sousse (Tunisia), which we report in this study the first six-months outcomes. Methods Rectal swab cultures were collected to detect Vancomycin resistant enterococcus (VRE) and Carbapenemase producing Enterobacteriaceae (CPE) among patients admitted in six ICUs of Sahloul university hospital of Sousse (Tunisia) and more than three times, at least one week apart, between 1 June and 31 December 2018. Results During the study period 174 patients were screened. Of them, 69.5% were male and 73.6% were admitted in surgical ICU. In total, 161 and 152 samples were realized respectively for the detection of CPE and VRE. These samples were positive in 15% and 8.5% respectively for CPE and VRE. Klebsiella pneumoniae OXA 48 was the most isolated CPE (80%). Conclusions Our screening program helped us in infection control by early identification of patients, thereby facilitating an informed decision about infection prevention interventions. Moreover, these results encouraged us to improve and generalize this program throughout the hospital. Key messages eHRB screening becomes an important axis in the prevention of eHRB infections in our facilities. eHRB screening allows the reinforcement of the basic infection prevention and control measures.

ChromID® CARBA Agar Fails to Detect Carbapenem-Resistant Enterobacteriaceae With Slightly Reduced Susceptibility to Carbapenems.

After first detections of carbapenemase-producing Enterobacteriaceae (CPE) in animals, the European Union Reference Laboratory for Antimicrobial Resistance has provided a protocol for the isolation of carbapenemase-producing Escherichia (E.) coli from cecum content and meat. Up to now, only few isolates were recovered using this procedure. In our experience, the choice of the selective agar is important for the efficacy of the method. Currently, the use of the prevailing method fails to detect CPE that exhibit a low resistance against carbapenems. Thus, this study aims to evaluate the suitability of selective media with antibiotic supplements and commercial ChromID® CARBA agar for a reliable CPE detection. For comparative investigations, detection of freeze-dried carbapenemase-resistant bacteria was studied on different batches of the ChromID® CARBA agar as well as on MacConkey agar supplemented with 1 mg/L cefotaxime and 0.125 mg/L meropenem (McC+CTX+MEM). The suitability of the different media was assessed within a time of 25 weeks, starting at least six weeks before expiration of the media. Carbapenem-resistant isolates exhibiting a serine-based hydrolytic resistance mechanism (e.g., blaKPC genes) were consistently detected over 25 weeks on the different media. In contrast, carbapenemase producers with only slightly reduced susceptibility and exhibiting a zinc-catalyzed activity (e.g., blaVIM, blaNDM, and blaIMP) could only be cultivated on long-time expired ChromID® CARBA, but within the whole test period on McC+CTX+MEM. Thus, ChromID® CARBA agar appears to be not suitable for the detection of CPE with slightly increased minimum inhibitory concentrations (MIC) against carbapenems, which have been detected in German livestock and thus, are of main interest in the national monitoring programs. Our data are in concordance with the results of eleven state laboratories that had participated in this study with their ChromID® CARBA batches routinely used for the German CPE monitoring. Based on the determined CPE detection rate, we recommend the use of McC+CTX+MEM for monitoring purposes. This study indicates that the use of ChromID® CARBA agar might lead to an underestimation of the current CPE occurrence in food and livestock samples.

Trends and molecular characteristics of carbapenemase-producing Enterobacteriaceae in Japanese hospital from 2006 to 2015

BackgroundThe increasing number of carbapenemase-producing Enterobacteriaceae (CPE) has become a global problem. Most carbapenemases detected in Japan are imipenemase, which is an imipenem-degrading enzyme with low ability; thus, CPE could have been overlooked. Therefore, this study aimed to detect and analyze CPE, without overlooking CPE showing the low minimum inhibitory concentration phenotype. MethodsCPE screening was conducted on 531 ceftazidime-resistant Enterobacteriaceae isolated from Kitasato University Hospital during 2006–2015. We confirmed the presence of the carbapenemase genes (blaIMP, blaVIM, blaKPC, blaNDM, and blaOXA-48) by multiplex polymerase chain reaction. The detected CPE strains were analyzed by antimicrobial susceptibility testing, multilocus sequence typing, conjugal experiments, replicon typing, and plasmid profiling by restriction enzyme treatment. ResultsThe CPE detection rate in Kitasato University Hospital within the past 10 years was 0.0003% (nine CPE strains). These nine CPE strains were identified to harbor 8 blaIMP-1 or 1 blaNDM-5. The CPE strains consisted of five species including Klebsiella pneumoniae and Citrobacter freundii. Six of eight blaIMP-1 were coded by IncHI2 plasmid, and the other two were coded by IncA/C plasmid. Plasmid profiling revealed that K. pneumoniae and C. freundii isolated from the same patient harbored the same plasmid. ConclusionThe CPE detection rate in this study was significantly lower than those previously reported in Japan. In one case, IncA/C plasmid transmission through different bacterial species within the body was speculated. Although the number of CPE detected was low, these results indicated that the resistance plasmid could spread to other bacterial species.

Rapid Detection of Carbapenemase-Producing Enterobacteriacae Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

The emergence and rapid spread of antibiotic resistance poses a serious threat to healthcare systems across the globe. The existence of carbapenemase-producing Enterobacteriaceae (CPE) such as Klebsiella pneumoniae renders the use of carbapenems, the last-resort class of β-lactam antibiotics, ineffective against bacterial infections, often leading to CPE-associated mortalities. Current methods of detection such as the Carba NP test and modified Hodge's test require hours to days to detect, which delays the response to isolate patients for rapid intervention. Here, we developed a surface-enhanced Raman scattering (SERS)-based detection scheme which utilizes gold nanostars conjugated to a β-lactam antibiotic ceftriaxone (CRO) as a beacon for rapid detection of bacterial β-lactamase secreted by Delhi metalloproteinase (NDM)-producing Escherichia coli as our CPE model with carbapenemase activity. The cleavage of β-lactam ring in CRO by NDM (Class B β-lactamase) caused a detectable reduction in SERS intensities at 722, 1358, and 1495 cm-1 within 25 min. Ratiometric analysis of the SERS peaks at 722, 1358, and 1495 cm-1 normalized against the Raman peak of polystyrene cuvette at 620 cm-1 showed the peak at 1358 cm-1 having the most significant change in intensity upon CPE detection. This reduced detection time has not been reported to date for CPE detection, and our novel approach using SERS could be extended to detect the activity of other classes of β-lactamases to broaden its clinical utility.

Towards a harmonised approach to Carbapenemase-producing Enterobacteriaceae (CPE) screening and patient management in North East and Cumbria hospitals

In mid-2018, a Carbapenemase-producing Enterobacteriaceae (CPE) cluster was investigated in the North East of England, in which transmission had occurred across several hospital trusts. We undertook a survey of current CPE detection and management practices in North East and Cumbria (NEC) trusts to inform a harmonised approach that could be adopted across the region. A semi-structured survey was carried out with one consultant microbiologist per trust by telephone interview (n=6) or electronic questionnaire (n=3) to determine CPE screening procedures, laboratory techniques and patient management practices. Screening policies were in place in all trusts, including screening protocols for patients previously admitted outside of the UK or those known to be CPE positive, in accordance with PHE guidelines. Policies for screening patients who had been admitted to other UK hospitals varied across trusts, as did laboratory methods used. Over 80% of trusts communicated CPE status to patients and contacts by information leaflet. However, there was no uniform way of communicating CPE status to social care settings, GPs and other hospital trusts in the event of discharge or inter-hospital transfer. The results of the survey suggest improvements could be made to the way information on patient CPE status is shared to ensure that the risk of transmission is reduced. This includes improved communication with residential settings, primary care and other hospital trusts in the event of a discharge or inter-hospital transfer. In addition, this work will inform further discussions across NEC towards developing a single regional Infection Prevention and control strategy for CPE.

Rapid detection of carbapenemase production in Enterobacteriaceae by different phenotypic methods

Introduction: Carbapenemase producing enterobacteriaceae (CPE) isolates have now emerged worldwide. Resistance to carbapenems is mainly due to production of beta-lactamases inactivating carbapenems (carbapenemases) included in classes A, B, C, or D of the Ambler classification. In Enterobacteriaceae carbapenemase encoding genes are often located on plasmids that contribute to a rapid spread among clinically relevant gram negative bacteria. Rapid and reliable detection of carbapenemase production is needed for therapeutic and control reasons. Aims: To compare the different phenotypic methods of carbapenemase detection namely, the modified paper strip carba NP method, the CLSI Carba NP method, the carbapenem inactivation method, and for rapid detection of CPE. Materials and methods: A total of 200 CPE from urine, pus, and blood cultures sent to the Microbiology Laboratory, R.G. Kar Medical College and Hospital (RGKMCH), were tested for the modified paper strip carba NP method, the CLSI Carba NP method, and the carbapenem inactivation method. Results and analysis: Out of 200 isolates, 108 isolates of Klebsiella spp and 92 isolates of Escherichia coli were compared for carbapenemase production by various phenotypic methods. In total, 100(93%) isolates of Klebsiella spp and 88 (95%) isolates of Escherichia coli showed positive results by paper strip and carba NP methods. A total of 96 (89%) isolates of Klebsiella spp and 84 (91%) isolates of Escherichia coli showed positive results by CLSI carba NP method. A total of 104 (96%) isolates of Klebsiella spp and 92 (100%) isolates of Escherichia coli showed positive results by CIM method. Conclusion: Rapid and accurate detection of carbapenemase producers are important for preventing their spread in health care settings. Although genotypic tests remain the gold standard but cannot practically be conducted because they are highly expensive and results are limited by the targets. The different phenotypic methods used in this study were inexpensive, rapid, highly sensitive, and specific. The modified paper strip Carba NP method in this study is a simple and rapid method compared to those performed by the CLSI method.

An agar plate-based modified carbapenem inactivation method (p-mCIM) for detection of carbapenemase-producing Enterobacteriaceae

Detecting carbapenemase-producing Enterobacteriaceae (CPE) has become increasingly difficult due to the emergence of diverse enzymes. The aim of the study was to evaluate an agar plate-based modified carbapenem inactivation method (p-mCIM) for detection of CPE. Stock strains and clinical isolates of CPE were used to evaluate the p-mCIM. The p-mCIM was performed as described for the mCIM, except that meropenem disks were placed on the lawn of test organisms on Mueller-Hinton agar (MHA) plates. Among 17 stock strains of CPE, six of eight KPC-2-like- and all six NDM-1-like carbapenemase-producing strains were positive by the p-mCIM without incubation in the carbapenem inactivation (CI) step. Among 380 CPE clinical isolates detected, 308 and 38 were KPC-2-like and NDM-1-like enzyme producers, respectively. The required incubation time in the CI step to show all isolates were positive by p-mCIM was 3 h for isolates with KPC-2-like enzyme and 1 h for isolates with metallo-β-lactamases. Twenty-eight of 30 isolates with OXA-48-like enzymes were p-mCIM positive. Sensitivities of both the p-mCIM and the mCIM (based on inhibition zone of ≤15 mm) for detection of CPE were 100%. All 70 ertapenem-nonsusceptible, but carbapenemase gene-negative isolates tested were both p-mCIM (based on inhibition zone of ≥21 mm) and mCIM negative. In conclusion, performance of the p-mCIM, which uses a lawn of bacterial colonies on MHA plate instead of a bacteria-suspended Tryptic soy broth tube in the CI step, is essentially identical to that of the CLSI-recommended mCIM in the detection of clinical isolates of Enterobacteriaceae producing carbapenemases including difficult to detect blaOXA-48-like enzymes.

Rectal screening for carbapenemase-producing Enterobacteriaceae: a proposed workflow

ObjectivesActive screening is a crucial element for the prevention of carbapenemase-producing Enterobacteriaceae (CPE) transmission in healthcare settings. Here we propose a culture-based protocol for rectal swab CPE screening that combines CPE detection with identification of the carbapenemase type. MethodsThe workflow integrates an automatic digital analysis of selective chromogenic media (WASPLab®; Copan), with subsequent rapid tests for the confirmation of carbapenemase production [i.e. detection of Klebsiella pneumoniae carbapenemase (KPC)-specific peak by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) or a multiplex immunochromatographic assay identifying the five commonest carbapenemase types]. To evaluate the performance of this protocol in depth, data for 21 162 rectal swabs submitted for CPE screening to the Microbiology Unit of S. Orsola-Malpighi Hospital (Bologna, Italy) were analysed. ResultsConsidering its ability to correctly segregate plates with/without Enterobacteriaceae, WASPLab Image Analysis Software showed globally a sensitivity and specificity of 100% and 79.4%, respectively. Of the plates with bacterial growth (n = 901), 693 (76.9%) were found to be positive for CPE by MALDI-TOF/MS (KPC-specific peak for K. pneumoniae) or by immunochromatographic assay. Only 2.8% (16/570) of KPC-positive K. pneumoniae strains were missed by the specific MALDI-TOF/MS algorithm, being detected by the immunochromatographic assay. The mean turnaround time needed from sample arrival to the final report ranged between 18 and 24 h, representing a significant time saving compared with manual reading. ConclusionThis workflow proved to be fast and reliable, being particularly suitable for areas endemic for KPC-producing K. pneumoniae and for high-throughput laboratories.

Modification and evaluation of the Triton Hodge test for screening carbapenemase-producing Enterobacteriaceae

Detection of carbapenemase-producing Enterobacteriaceae (CPE) has become critical for appropriate antimicrobial therapy and for controlling the spread of infection. We evaluated Triton Hodge test (THT) for screening CPE. A spreader can be used to apply more constant volume of Triton on whole surface of Mueller–Hinton agar (MHA), or alternatively, a 10-μL inoculating loop can be used to apply a 20% Triton solution lineally. The THT procedure can be simplified by eliminating the 1/10 dilution step of indicator bacteria from the McFarland 0.5 turbidity suspension. The presence of Triton in the MHA plates significantly increased the enhanced growth size of not only Enterobacteriaceae producing NDM-1–like enzymes but also those producing the most prevalent KPC-2–like enzyme, resulting in 100% sensitivity of the test.

PCR-Dipstick-Oriented Surveillance and Characterization of mcr-1- and Carbapenemase-Carrying Enterobacteriaceae in a Thai Hospital.

Colistin is used as an alternative therapeutic for carbapenemase-producing Enterobacteriaceae (CPE) infections which are spreading at a very high rate due to the transfer of carbapenemase genes through mobile genetic elements. Due to the emergence of mcr-1, the plasmid-mediated colistin resistance gene, mcr-1-positive Enterobacteriaceae (MCRPEn) pose a high risk for the transfer of mcr-1-carrying plasmid to CPE, leading to a situation with no treatment alternatives for infections caused by Enterobacteriaceae possessing both mcr-1 and carbapenemase genes. Here, we report the application of PCR-dipstick-oriented surveillance strategy to control MCRPEn and CPE by conducting the PCR-dipstick technique for the detection of MCRPEn and CPE in a tertiary care hospital in Thailand and comparing its efficacy with conventional surveillance method. Our surveillance results showed a high MCRPEn (5.9%) and CPE (8.7%) carriage rate among the 219 rectal swab specimens examined. Three different CPE clones were determined by pulsed-field gel electrophoresis (PFGE) whereas only two MCRPEn isolates were found to be closely related as shown by single nucleotide polymorphism-based phylogenetic analysis. Whole genome sequencing (WGS) and plasmid analysis showed that MCRPEn carried mcr-1 in two plasmids types—IncX4 and IncI2 with ~99% identity to the previously reported mcr-1-carrying plasmids. The identification of both MCRPEn and CPE in the same specimen indicates the plausibility of plasmid-mediated transfer of mcr-1 genes leading to the emergence of colistin- and carbapenem-resistant Enterobacteriaceae. The rapidity (<2 h) and robust sensitivity (100%)/specificity (~99%) of PCR-dipstick show that this specimen-direct screening method could aid in implementing infection control measures at the earliest to control the dissemination of MCRPEn and CPE.