Carbapenem-resistant Enterobacteriaceae Resistance Research Articles

Development of a multiplex real-time PCR assay for simultaneous detection of common colistin and carbapenemase genes

Carbapenem and colistin are often used as last-resort treatment for Gram-negative multi-drug resistant (MDR) bacteria. Nevertheless, co-resistance of these drugs is threatening the global healthcare system. Rapid and accurate detection of carbapenem and colistin resistant bacteria is critical for adequate antibiotic therapy and infection control, particularly in the context of an outbreak. The presence of blaNDM, blaKPC, blaIMP-1 and blaOXA-48 is responsible for greater than 95% phenotypic resistance in carbapenem-resistant Enterobacteriaceae, while mcr-1 is the most prevalent and well disseminated of all mcr genes in colistin-resistant strains. In this study, we aim to develop a multiplex real time-PCR assay for simultaneous detection of the five genes blaNDM, blaKPC, blaIMP-1, blaOXA-48 and mcr-1. The melting curve-based multiplex real time PCR assay was established with the dissociation temperature range extended from 76°C to 87°C. The whole process is completed within one hour and half, allowing rapid screening of the five genes in cultured bacteria samples with a limit of detection of 10 CFU/ml. The proposed multiplex real-time PCR assay is a robust, reliable and rapid method for the detection of bacterial strains carrying blaOXA-48, blaIMP, blaNDM, blaKPC and mcr-1 gene individually or in cocktail of genes. This assay will be a valuable tool for surveillance and monitoring of MDR bacteria additionally resistant to either carbapenem or colistin or both drugs.

Genomic characterisation of a blaKPC-2- and mcr-10-co-harbouring Enterobacter kobei isolate with high-level resistance to colistin and carbapenems

The emergence and spread of colistin resistance in carbapenem-resistant Enterobacteriaceae pose a serious threat to human and animal health. This work aimed to characterize the genetic features of antimicrobial resistance of a carbapenem and colistin resistant Enterobacter kobei SCLZS19 from hospital sewage by using whole genome sequencing. Antimicrobial susceptibility tests were performed by using the disk diffusion method. Whole genome sequencing of SCLZS19 was carried out on the HiSeq 2000 combined with PacBio RSII platforms. Sequence type, plasmid incompatibility types, resistance genes, and insertion elements were identified using the MLST, PlasmidFinder, ResFinder, and ISfinder, respectively. Conjugation assays were performed using both broth- and filter-based methods with the azide-resistant Escherichia coli J53 as the recipient. The function of the mcr-9-like variant was determined by gene cloning. E. kobei SCLZS19 has a 4,862,177-bp circular chromosome, and nine circular plasmids ranging in size from 4,120 bp to 282,472 bp. It carries 11 antibiotic resistance genes (ARGs), and 10 of them are located on plasmids. The colistin resistance gene mcr-10 is located on a 118,766-bp non-transferable IncF (Y3:A-:B-) plasmid. The carbapenemase gene blaKPC-2 is carried by a self-transmissible IncP6 plasmid which is epidemic in China. In addition, SCLZS19 also carries a mcr-9-like variant on a IncHI2 (ST1) plasmid. Cloning assay shows that the mcr-9-like variant does not mediate colistin resistance in E. coli DH5α. The findings highlight that carbapenem- and colistin-resistant Enterobacterales from water environment may serve as a reservoir for clinically significant ARGs, and continuous surveillance is required.

Multidrug-resistant VAP before and during the COVID-19 pandemic among hospitalized patients in a tertiary private hospital.

There is limited data on ventilator-associated pneumonia (VAP) and multidrug-resistant VAP (MDR VAP) among COVID-19 patients. A retrospective study in a single, tertiary, private hospital in the Philippines was conducted comparing the incidence, profile, and patient outcomes of MDR VAP during the pre-COVID-19 (2018-2019) and COVID-19 (2020-2021) periods. In total, 80/362 (22%) patients developed VAP, 27/204 (33.75%) from pre-COVID-19 and 53/158 (66.25%) from the COVID-19 period, respectively. The majority were male [20/27 (74%) vs 34/53 (64%)], with a median age of 66 (range 35-90) and 67 (range 32-92) years in each period, respectively. Comorbidities were similar, except cardiovascular disease (14/27 vs 11/53 patients, p-value 0.005) and chronic lung disease (14/27 vs 9/53 patients, p-value 0.0012). VAP incidence density was 19.3/1000 and 27.8/1000 ventilator days (p-value 0.9819)]; median length of stay before VAP for pre- and COVID-19 periods was 17 and 10 days, respectively (p-value <0.0001). Extended-spectrum β lactamase (ESBL)-producing resistance increased significantly [1/27 (3.7%) pre-COVID-19 vs 15/53 (28.3%)]during COVID-19, while Carbapenem-resistant Enterobacteriaceae resistance was higher in the pre-COVID-19 period(15/27 [56%] vs 10/53 [19%]). Mortality was high in both periods at 93% and 83%, respectively. On multivariate analysis, only female gender was associated with MDR VAP in the COVID-19 period (OR =3.47, [CI 1.019, 11.824], p-value < 0.047). The frequency of VAP and MDR VAP increased during the COVID-19 period, despite a shorter duration of hospital stay. The mortality of VAP was extremely high. Factors associated with increased risk of VAP and COVID-19 need to be studied further, and preventive measures should be prioritized.

Carbapenem Resistance Mechanisms, Carbapenemase Genes Dissemination , and Laboratory Detection Methods: A Review

In recent years, carbapenem-resistant Enterobacteriaceae (CRE) has expanded quickly throughout the world, posing a serious threat to public health. They are also very difficult to treat and are associated with a high fatality rate. The class of antimicrobials known as carbapenems is regarded as the most trustworthy and last line of defense. It is possible to distinguish between CRE resistance mechanisms that produce carbapenemase enzymes and those that do not. The three classes of &beta;-lactama

The Resistance Mechanism Governs Physiological Adaptation of Escherichia coli to Growth With Sublethal Concentrations of Carbapenem.

Factors governing resistance in carbapenem-resistant Enterobacteriaceae are manifold. Despite ample research efforts, underlying molecular mechanisms are still only partly understood. Furthermore, little is known on (eco)physiological consequences from resistance acquisition originating from distinct mechanisms in respective bacteria.In this study, we examined physiological adaptation of Escherichia coli clinical isolates exhibiting two distinct resistance mechanisms–either carrying a carbapenemase (n = 4, CARB) or alterations in porin-encoding genes (n = 6, POR)–during growth with sublethal concentrations of ertapenem in chemostat culture. Basic growth parameters based on optical density and flow-cytometric analyses as well as global gene expression patterns using RNA-Seq were recorded. We demonstrate that strategies to deal with the antibiotic were distinct between strains of the two groups, where (increased) expression of carbapenemases was the major response in CARB, whereas wide-spread alterations in gene-expression that promoted a survival-like phenotype was observed in POR. The response in POR was accompanied with “costs of resistance” resulting in reduced growth efficiencies compared with CARB that are intrinsic to that group and were also observed during growth without antibiotic challenge, however, at lower levels. All strains showed similar minimal inhibitory concentrations and did not form phylogenetic groups, indicating that results cannot be attributed to distinct resistance levels or phylogenetic relationships, but are indeed based on the resistance mechanism.

Transcriptome-based design of antisense inhibitors potentiates carbapenem efficacy in CRE Escherichia coli

In recent years, the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has risen substantially, and the study of CRE resistance mechanisms has become increasingly important for antibiotic development. Although much research has focused on genomic resistance factors, relatively few studies have examined CRE pathogens through changes in gene expression. In this study, we examined the gene expression profile of a CRE Escherichia coli clinical isolate that is sensitive to meropenem but resistant to ertapenem to explore transcriptomic contributions to resistance and to identify gene knockdown targets for carbapenem potentiation. We sequenced total and short RNA to analyze the gene expression response to ertapenem or meropenem treatment and found significant expression changes in genes related to motility, maltodextrin metabolism, the formate hydrogenlyase complex, and the general stress response. To validate these findings, we used our laboratory's Facile Accelerated Specific Therapeutic (FAST) platform to create antisense peptide nucleic acids (PNAs), gene-specific molecules designed to inhibit protein translation. PNAs were designed to inhibit the pathways identified in our transcriptomic analysis, and each PNA was then tested in combination with each carbapenem to assess its effect on the antibiotics' minimum inhibitory concentrations. We observed significant PNA-antibiotic interaction with five different PNAs across six combinations. Inhibition of the genes hycA, dsrB, and bolA potentiated carbapenem efficacy in CRE E. coli, whereas inhibition of the genes flhC and ygaC conferred added resistance. Our results identify resistance factors and demonstrate that transcriptomic analysis is a potent tool for designing antibiotic PNA.

Carbapenemase Gene Profiles in Carbapenem-Resistant Enterobacteriaceae—United States, January 2018–August 2019

Background: Carbapenem-resistant Enterobacteriaceae (CRE) cause significant morbidity and mortality each year in the United States. Treatment options for these infections are often limited, in part due to carbapenemases, which are mobile β-lactam-hydrolyzing enzymes that confer multidrug resistance in CRE. As part of the CDC’s Containment Strategy for Emerging Resistance, public health laboratories (PHLs) in the CDC Antibiotic Resistance Laboratory Network (AR Lab Network) have worked to characterize clinical isolates of CRE for rapid identification of carbapenemase genes. These data are then used by public health and healthcare partners to promote patient safety by decreasing the spread of resistance. We summarize carbapenemase gene profiles in CRE, by genus and geography, using data collected through the AR Lab Network from January 2018 through August 2019. Methods: CRE isolates were submitted to 55 PHLs, including those of all 50 states, 4 large cities, and Puerto Rico, in accordance with each jurisdiction’s reporting laws. PHLs performed phenotypic and molecular testing on isolates to detect targeted, emerging carbapenemase genes and reported results to submitters. Carbapenemase-positive (CP) isolates were defined as PCR positive for ≥1 carbapenemase gene tested: blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48–LIKE. PHLs submitted results to CDC monthly. Genera other than Enterobacter, Klebsiella, and Escherichia coli are categorized as other genera in this analysis. Data were compiled and analyzed using SAS v 9.4 software. Results: From January 2018 to August 2019, the AR Lab Network tested 25,705 CRE isolates; 8,864 of 25,705 CRE (34%) were CP. Klebsiella spp represented the largest proportion of CP-CRE at 68% (n = 6,063), followed by E. coli (12%, n = 1,052), Enterobacter spp (11%, n = 981), and other genera (9%, n = 768). Figure 1a shows the composition of CP-CRE carbapenemase genes by genus. The most common carbapenemase and genus profiles were blaKPC in Klebsiella (74%; 5,562 of 7,561 blaKPC-positive) blaNDM in E. coli (43%; 372 of 868 blaNDM-positive) blaVIM in Enterobacter spp (35%; 25 of 72 blaVIM-positive), and blaIMP among other genera (90%; 92 of 102 blaIMP-positive). Common CP-CRE genes and genera also varied by geography (Fig. 1b). Conclusions: The AR Lab Network has greatly enhanced our nation’s ability to detect and characterize CP-CRE. Our data provide a snapshot of the organisms and regions where mobile carbapenemase genes are most often detected in CRE. Geographic variation in CP gene profiles provides actionable data to inform local priorities for detection and infection control and provide clinicians with situational awareness of the genes and organisms that are circulating in their region.Funding: NoneDisclosures: In this presentation, the authors discuss the drug combination aztreonam-avibactam and acknowledge that this drug combination is not currently FDA-approved.

Identification of an extensively drug-resistant Escherichia coli clinical strain harboring mcr-1 and blaNDM-1 in Korea.

The development of colistin resistance in carbapenem-resistant strains poses a serious public health problem. In this study, we collected 249 carbapenem-resistant Escherichia coli isolates from patients in Seoul in 2018, and screened all isolates for colistin resistance and for the presence of mobile colistin resistance (mcr) genes. Colistin-resistant strains were further analyzed using multilocus sequence typing, antimicrobial susceptibility testing, detection of antibiotic resistance determinants, plasmid transconjugation, and whole-genome sequencing. Three of the 249 carbapenem-resistant isolates were resistant to colistin, and mcr-1 was detected in one isolate (SECR18-0888), which belonged to sequence type 156 and was resistant to all antibiotics tested except tigecycline. The mcr-1.1 gene was located on an ~62 kb self-transferable IncI2 plasmid along with the blaCTX-M-55 gene, and the blaNDM-1, blaTEM, qepA1, and rmtB genes were additionally detected in SECR18-0888. As an extensively drug-resistant E. coli strain producing MCR-1 and NDM-1 was identified in Korea for the first time, continued monitoring of colistin resistance in carbapenem-resistant Enterobacteriaceae should be reinforced.

621. In vitro Ceftazidime: Avibactam Resistance in Carbapenem-Resistant Enterobacteriaceae Isolates

BackgroundCeftazidime–avibactam (CAZ-AVI) is a new antibiotic with activity against many Carbapenem-resistant Enterobacteriaceae (CRE). Although CAZ-AVI resistance in CRE has been reported, it is not consistently assessed. Our study aimed to assess the prevalence of CAZ-AVI resistance in CRE isolated from patients with and without prior exposure to CAZ-AVI.MethodsWe tested 116 CRE isolates for CAZ-AVI resistance by Kirby–Bauer (KB) disk diffusion susceptibility. Resistant isolates were verified by repeat KB and E-test performed by the Stony Brook Hospital laboratory. The blaKPC gene of resistant strains was amplified by PCR and sequenced. Patient data were used to determine whether patients were colonized or infected, and whether they were exposed to CAZ-AVI.ResultsOf the 116 CRE isolates from 86 patients (96 encounters), 50% were Klebsiella species, 23.2% were Enterobacter species, 10.3% Escherichia coli and 16.5% other CRE. They were recovered from colonized (37%) and infected (63%) patients of which 18% were treated with CAZ-AVI during their hospitalizations (median duration of therapy, 6 days). Two CRE isolates (1.7%) were found to be resistant on repeated testing. One isolate was K. pneumoniae derived from the sputum of a patient diagnosed with ventilator-associated pneumonia who received 40 days of CAZ-AVI therapy prior to isolation of the resistant isolate (KB diameter 20 mm, MIC > 512 μg/mL by E-Test). Sequencing of the strain’s blaKPC3 gene revealed a previously described Ambler-position D179Y mutation that has been shown to convey resistance. The second CAZ-AVI-resistant K. pneumoniae (KB diameter 19 mm, MIC 64 μg/mL by E-test) was isolated from the urine of a colonized patient naïve to CAZ-AVI therapy. The strain’s blaKPC10 gene had no mutations.ConclusionIn our strain collection, the rate of resistance to CAZ-AVI remains low <2%. Although we found one mutation (D179Y) previously linked to CAZ-AVI resistance we also discovered one K. pneumoniae isolate with in vitro resistance to CAZ-AVI that did not exhibit any blaKPC mutations conveying CAZ-AVI resistance. Interestingly, this strain was derived from a patient with no prior CAZ-AVI exposure. Whole-genome sequencing will be performed to identify other genes or mutations that may confer resistance.Disclosures All authors: No reported disclosures.

Diversity of resistance mechanisms in carbapenem-resistant Enterobacteriaceae at a health care system in Northern California, from 2013 to 2016

The mechanism of resistance in carbapenem-resistant Enterobacteriaceae (CRE) has therapeutic implications. We comprehensively characterized emerging mechanisms of resistance in CRE between 2013 and 2016 at a health system in Northern California. A total of 38.7% (24/62) of CRE isolates were carbapenemase gene-positive, comprising 25.0% (6/24) blaOXA-48 like, 20.8% (5/24) blaKPC, 20.8% (5/24) blaNDM, 20.8% (5/24) blaSME, 8.3% (2/24) blaIMP, and 4.2% (1/24) blaVIM. Between carbapenemases and porin loss, the resistance mechanism was identified in 95.2% (59/62) of CRE isolates. Isolates expressing blaKPC were 100% susceptible to ceftazidime–avibactam, meropenem–vaborbactam, and imipenem–relebactam; blaOXA-48 like–positive isolates were 100% susceptible to ceftazidime–avibactam; and metallo β-lactamase–positive isolates were nearly all nonsusceptible to above antibiotics. Carbapenemase gene-negative CRE were 100% (38/38), 92.1% (35/38), 89.5% (34/38), and 31.6% (12/38) susceptible to ceftazidime–avibactam, meropenem–vaborbactam, imipenem–relebactam, and ceftolozane–tazobactam, respectively. None of the CRE strains were identical by whole genome sequencing. At this health system, CRE were mediated by diverse mechanisms with predictable susceptibility to newer β-lactamase inhibitors.

Frequency of colistin and fosfomycin resistance in carbapenem-resistant Enterobacteriaceae from a tertiary care hospital in Karachi.

IntroductionManagement of infections with carbapenem-resistant Enterobacteriaceae (CRE) is challenging. In recent times, agents such as colistin and fosfomycin have been used in combination with other antibiotics to treat such infections. In this study, we aim to seek frequency of colistin and fosfomycin resistance in CRE from Pakistan.MethodsThis study was conducted at clinical laboratories, Aga Khan University Hospital. In total, 251 CRE were included in the study. Colistin minimum inhibitory concentrations (MICs) were performed using broth microdilution (BMD) method and VITEK® 2 system, whereas fosfomycin susceptibility was performed using Kirby–Bauer method. MIC50 and MIC90 were calculated for colistin and agreement between VITEK and BMD was also calculated.ResultsOut of 251 strains colistin MIC of ≥4 µg/mL was seen in 40 (15.9%). Of these strains 20 (50%) were Klebsiella pneumoniae. Colistin MIC50 and MIC90 were found to be 0.5 and 16 µg/mL, respectively. BMD and VITEK 2 showed 100% categorical agreement. Essential agreement was 88.5% with kappa score 0.733 indicating strong agreement between VITEK and BMD. 31 out of 251 (12.3%) CREs were resistant to fosfomycin.ConclusionStudy shows frequency of colistin and fosfomycin resistance to be 15.9% and 12.3%, respectively. In countries where rate of CREs is high, emerging resistance against these last resort antibiotics is alarming as it leaves clinicians with almost no options to manage such multidrug resistant and extensively drug resistant infections.

Identification of Carbapenemase-mediated Resistance among Enterobacteriaceae Bloodstream Isolates: A Molecular Study from India

Acquired resistance in carbapenem-resistant Enterobacteriaceae (CRE) conferred by carbapenemases is a major concern worldwide. Consecutive, non-duplicate isolates of Escherichia coli (EC) and Klebsiella pneumoniae from clinically diagnosed bloodstream infections were screened for the presence of carbapenem resistance by standard disk-diffusion method and minimum inhibitory concentration breakpoints using the Clinical and Laboratory Standards Institute guidelines. Carbapenemase-encoding genes were amplified by polymerase chain reaction. Of 387 isolates (214 K. pneumoniae, 173 EC) tested, 93 (24.03%) were found to be CRE. Of these, 71 (76.3%) were positive for at least one tested carbapenemase gene. The frequency of carbapenemase genes was New Delhi metallo-β-lactamse-1 (65.6%), oxacillinase (OXA)-48 (24.7%), OXA-181 (23.6%), Verona integron-encoded metallo-β-lactamase (6.4%) and K. pneumoniae carbapenemase (2.1%). Our study identified presence of carbapenemases in a large proportion of CRE isolates. Delineation of resistance mechanisms is important in view of future therapeutics concerned with the treatment of CRE and for aiding control efforts by surveillance and infection control interventions.

Multicenter Clinical and Molecular Epidemiological Analysis of Bacteremia Due to Carbapenem-Resistant Enterobacteriaceae (CRE) in the CRE Epicenter of the United States.

Although the New York/New Jersey (NY/NJ) area is an epicenter for carbapenem-resistant Enterobacteriaceae (CRE), there are few multicenter studies of CRE from this region. We characterized patients with CRE bacteremia in 2013 at eight NY/NJ medical centers and determined the prevalence of carbapenem resistance among Enterobacteriaceae bloodstream isolates and CRE resistance mechanisms, genetic backgrounds, capsular types (cps), and antimicrobial susceptibilities. Of 121 patients with CRE bacteremia, 50% had cancer or had undergone transplantation. The prevalences of carbapenem resistance among Klebsiella pneumoniae, Enterobacter spp., and Escherichia coli bacteremias were 9.7%, 2.2%, and 0.1%, respectively. Ninety percent of CRE were K. pneumoniae and 92% produced K. pneumoniae carbapenemase (KPC-3, 48%; KPC-2, 44%). Two CRE produced NDM-1 and OXA-48 carbapenemases. Sequence type 258 (ST258) predominated among KPC-producing K. pneumoniae (KPC-Kp). The wzi154 allele, corresponding to cps-2, was present in 93% of KPC-3-Kp, whereas KPC-2-Kp had greater cps diversity. Ninety-nine percent of CRE were ceftazidime-avibactam (CAZ-AVI)-susceptible, although 42% of KPC-3-Kp had an CAZ-AVI MIC of ≥4/4 μg/ml. There was a median of 47 h from bacteremia onset until active antimicrobial therapy, 38% of patients had septic shock, and 49% died within 30 days. KPC-3-Kp bacteremia (adjusted odds ratio [aOR], 2.58; P = 0.045), cancer (aOR, 3.61, P = 0.01), and bacteremia onset in the intensive care unit (aOR, 3.79; P = 0.03) were independently associated with mortality. Active empirical therapy and combination therapy were not associated with survival. Despite a decade of experience with CRE, patients with CRE bacteremia have protracted delays in appropriate therapies and high mortality rates, highlighting the need for rapid diagnostics and evaluation of new therapeutics.

Treatment of sepsis: What is the antibiotic choice in bacteremia due to carbapenem resistant Enterobacteriaceae?

Sepsis is one of the major challenges of today. Although gram-positive bacteria related infections are more prevalent in hospital setting, the highest mortality rate is associated with gram-negative microorganisms especially Enterobacteriaceae. Enterobacteriaceae, including Escherichia coli, Klebsiella spp., Proteus spp., Enterobacter spp. and Serratia spp. Resistance to β-lactams in Enterobacteriaceae is primarily attributed to the production of B-lactamase enzymes with subsequent antibiotic hydrolysis and to a lesser extent by alteration of efflux pump or porins expression. Carbapenem resistant Enterobacteriaceae (CRE) and Acinetobacter baumannii are the most notorious pathogens due to the high incidence of morbidity and mortality especially in the immunocompromised patients in the intensive care unit. The most appropriate antimicrobial therapy to treat CRE is still controversial. Combination therapy is preferred over monotherapy due to its broad-spectrum coverage of micro-organisms, due to its synergetic effect and to prevent development of further resistance. Current suggested therapies for CRE resistance as well as promising antibiotics that are currently under investigation for winning the war against the emerging CRE resistance are reviewed and discussed.

What's Lurking Around the Corner: Identifying Novel Carbapenem-Resistant Enterobacteriaceae Resistance Mechanisms in the Los Angeles County Healthcare Community, 2015

What's Lurking Around the Corner: Identifying Novel Carbapenem-Resistant Enterobacteriaceae Resistance Mechanisms in the Los Angeles County Healthcare Community, 2015