Carbapenemase Variants Research Articles

Evaluation of a novel chromogenic screening method for detection of carbapenem-resistant Escherichia coli.

Introduction. Early detection of carbapenem-resistant Escherichia coli (CREco), categorized as a critical priority pathogen by the World Health Organization (WHO), is crucial in optimizing therapeutic options and to thwart outbreaks in clinical settings.Gap statement. The need of the hour is a diagnostic method that can detect carbapenem resistance conferred by intrinsic or acquired carbapenem resistance mechanisms or both.Aim. The study investigates the performance of a novel screening chromogenic method for detection of CREco.Methodology. Carbapenem-susceptible (n=23) and non-susceptible (n=90) E. coli were used to investigate the efficiency of the blue chromogenic test. All of the isolates were received from a tertiary referral hospital in Silchar, India and subjected to the blue chromogenic test and observed for colour change. A colour change from colourless to blue is interpreted as a positive result. The test results were further compared with available methods for detection of carbapenem resistance conferred by carbapenemase production or other carbapenem resistance mechanisms.Results. The blue chromogenic test generated 100 % (CI: 95.98-100 %) sensitive and 100 % (CI: 85.75-100 %) specific results for the detection of CREco with no false-positive or false-negative results. Within 3 h after incubation, the test detects all CREco with carbapenemase activity. Additionally, the blue chromogenic test also positively detected E. coli harbouring carbapenemase variants and with efflux and porin activity, compared to other phenotypic-based approaches.Conclusion. The study highlights a novel method that is highly sensitive and specific, inexpensive, rapid and user-friendly for the detection of CREco. With the surge and expansion of CREco, this sensitive, specific, user-friendly and inexpensive method can be used in laboratories with limited facilities for early detection of CREco, thereby improving infection control along with averting future outbreaks.

Klebsiella pneumoniae carbapenemase variants: the new threat to global public health.

SUMMARYKlebsiella pneumoniae carbapenemase (KPC) variants, which refer to the substitution, insertion, or deletion of amino acid sequence compared to wild blaKPC type, have reduced utility of ceftazidime-avibactam (CZA), a pioneer antimicrobial agent in treating carbapenem-resistant Enterobacterales infections. So far, more than 150 blaKPC variants have been reported worldwide, and most of the new variants were discovered in the past 3 years, which calls for public alarm. The KPC variant protein enhances the affinity to ceftazidime and weakens the affinity to avibactam by changing the KPC structure, thereby mediating bacterial resistance to CZA. At present, there are still no guidelines or expert consensus to make recommendations for the diagnosis and treatment of infections caused by KPC variants. In addition, meropenem-vaborbactam, imipenem-relebactam, and other new β-lactam-β-lactamase inhibitor combinations have little discussion on KPC variants. This review aims to discuss the clinical characteristics, risk factors, epidemiological characteristics, antimicrobial susceptibility profiles, methods for detecting blaKPC variants, treatment options, and future perspectives of blaKPC variants worldwide to alert this new great public health threat.

Epidemiology of Resistance Determinants Identified in Meropenem-Nonsusceptible Enterobacterales Collected as Part of a Global Surveillance Study, 2018 to 2019.

The objective of this study was to describe the frequency of resistance determinants in meropenem-nonsusceptible (MEM-NS) Enterobacterales isolates collected in 2018 and 2019 as a part of the ATLAS global surveillance program. Among a total of 39,368 Enterobacterales isolates collected in 2018 and 2019, 5.7% were MEM-NS (MIC ≥2 μg/mL). Among the different regions, the proportion of MEM-NS isolates ranged from 1.9% (North America) to 8.4% (Asia/Pacific). The majority of MEM-NS isolates collected were of the species Klebsiella pneumoniae (71.5%). Among the MEM-NS Enterobacterales isolates collected, metallo-β-lactamases (MBL) were identified in 36.7%, KPC in 25.5%, and OXA-48-like in 24.1%. The predominance of resistance mechanisms among MEM-NS isolates varied by region: MBLs were dominant in isolates collected in Africa and Middle East (AfME, 49%) and Asia/Pacific (59.4%), OXA-48-like carbapenemases were predominant in Europe (30%), and KPC in Latin America (51.9%) and North America (53.6%). NDM β-lactamases accounted for the majority of MBLs identified (88.4%). Of the 38 carbapenemase variants identified, NDM-1 (68.7%), KPC-2 (54.6%), OXA-48 (54.3%), and VIM-1 (76.1%) were the common variants within their respective families. Among the MEM-NS isolates, 7.9% co-carried two carbapenemases. Notably, the proportion of MEM-NS Enterobacterales increased from 4.9% in 2018 to 6.4% in 2019. The results of this study show a continuation of the trend of increasing carbapenem-resistance within clinical Enterobacterales with mechanisms of resistance varying across different regions. The existential threat to public health posed by the continued spread of nearly untreatable pathogens requires a multifaceted approach to prevent the collapse of modern medicine.

Direct detection of OXA-48-like carbapenemase variants with and without co-expression of an extended-spectrum β-lactamase from bacterial cell lysates using mass spectrometry

IntroductionAntibiotic-resistant Gram-negative bacteria are of a growing concern globally, especially those producing enzymes conferring resistance. OXA-48-like carbapenemases hydrolyze most β-lactam antibiotics, with typically low-level hydrolysis of carbapenems, but have limited effect on broad-spectrum cephalosporins. These are frequently co-expressed with extended spectrum β-lactamases, especially CTX-M-15, which typically shows high level resistance to broad-spectrum cephalosporins, yet is carbapenem susceptible. The combined resistance profile makes the need for successful detection of these specific resistance determinants imperative for effective antibiotic therapy. ObjectivesThe objective of this study is to detect and identify OXA-48-like and CTX-M-15 enzymes using mass spectrometry, and to subsequently develop a method for detection of both enzyme types in combination with liquid chromatography. MethodsCells grown in either broth or on agar were harvested, lysed, and, in some cases buffer-exchanged. Lysates produced from bacterial cells were separated and analyzed via liquid chromatography with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). ResultsThe intact proteins of OXA-48, OXA-181, and OXA-232 (collectively OXA-48-like herein) and CTX-M-15 were characterized and detected. Acceptance criteria based on sequence-informative fragments from each protein group were established as confirmatory markers for the presence of the protein(s). A total of 25 isolates were successfully tested for OXA-48 like (2), CTX-M-15 (3), or expression of both (7) enzymes. Thirteen isolates served as negative controls. ConclusionsHere we present a method for the direct and independent detection of both OXA-48-like carbapenemases and CTX-M-15 β-lactamases using LC-MS/MS. The added sensitivity of MS/MS allows for simultaneous detection of at least two co-eluting, co-isolated and co-fragmented proteins from a single mass spectrum.

PREVALENCE OF ANTIMICROBIAL RESISTANCE AND MOLECULAR CHARACTERIZATION OF CARBAPENEMASE ENCODING GENE IN ESCHERICHIA COLI ISOLATES IN HOSPITAL OF HUE UNIVERSITY OF MEDICINE AND PHARMACY

Background: Currently, the existence of Extended-spectrum beta-lactamases (ESBL) and carbapenemase genes as well as genetic diversity has led to the rapid spread of resistance between E. coli species and the emergence of bacteria with pan-resistance. Objectives: To investigate the prevalence of antimicrobial resistance, prevalence of ESBL-producing E. coli, and carbapenemase-coding gene in E. coli isolates at Hospital of Hue University of Medicine and Pharmacy. Materials and method: The susceptibility to 13 antimicrobials of 246 E. coli isolates was detemined by the Kirby-Bauer method. ESBL-producing potential E. coli isolates were confirmed phenotypic by the Double Disc Synergy Test (DDST) and Combination Disc Test (CDT). Using multiplex PCR to detect genes encoding carbapenemase belonging to class B (Metallo β-lactamase), class D (oxacillinase), and blaKPC gene. Sequencing of carbapenemase genes were used to identify carbapenemase variants. Results: 91.46% of E. coli strains were identified as multidrug-resistant; most of them resistant to penicillins, fluoroquinolone, sulfonamide; high frequency of resistance to 3rd generation cephalosporin, chloramphenicol, gentamycin. The high isolation rate of ESBL-producing E. coli (50%) was detected. The resistance to carbapenems was largely mediated by the expression of acquired carbapenemases: blaKPC-2, blaNDM-4, and blaOXA-48. This study first reported blaNDM-4-harboring E. coli isolates in Vietnam. Conclusion: The high prevalence of multidrug-resistant and ESBL-producing E. coli, as well as the coexistence of ESBLs and carbapenemase genes, could seriously limit options for clinical treatment.

Carbapenemase Producing Gram-Negative Bacteria in Tunisia: History of Thirteen Years of Challenge.

The wide spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacteria (CR-GNB), constitutes a major public health threat worldwide, owing to the limited therapeutic options. This review will describe and uncover the Tunisian experience in the challenge against carbapenem resistance. Indeed, we illuminate on the dissemination of CR-GNB in different hospitals, animals, and other natural environments in this country. We resumed the different carbapenemase variants detected from various bacterial species and mapped their regional distribution, basing on Tunisian published data during a period extended from 2006, the date of its first description in Tunisia, to February 2019. We also resumed the different mobile genetic elements implicated in their dissemination. This review shows that the majority of the research reports focused in the north and the coastal cities in spite of the fact that KPC and IMP carbapenemases were uncommonly detected in our country. However, VIM, NDM-1, and OXA-48 enzymes were usually reported with the predominance of OXA-48 among Enterobacteriaceae. Furthermore, OXA-23, OXA-51, and OXA-58 carbapenemases constituted the main mechanism conferring carbapenem resistance among Acinetobacter baumannii in Tunisia. Collaborative efforts and raising awareness of the threat of antibiotic resistance are required in order to minimize the spread of multidrug-resistant bacteria.

Structure-based functional fitness analyses of carbapenemase variants identified among pathogenic carbapenem-resistant Gram-negative bacteria.

Carbapenemase-mediated carbapenem resistance is a major public health concerns worldwide. In the present study, prevalence of circulating carbapenemases was estimated among carbapenem-resistant clinical isolates using PCR and sequencing. Diameters of zone of inhibition (ZDs) were compared for imipenem, meropenem and ertapenem among single carbapenemase producing isolates. Structure-based functional fitness of those carbapenemases was predicted through several in silico analyses. Approximately, 63.76% isolates demonstrated carbapenem resistance, of which 39.13% harboured carbapenemases like blaNDM-1 (33.23%), blaNDM-1-like (0.31%), blaVIM-2 (4.35%), blaKPC-2 (4.04%), blaOXA-181 (6.85%), blaOXA-23 (16.50%), blaOXA-69 (3.88%), blaOXA-66 (2.91%) and blaOXA-104 (1.94%). Omega values indicated selection pressure over blaOXA-69, blaOXA-66 and blaOXA-104. Protein structural dynamics predicted NDM-1 and KPC-2 to have the highest and least flexibility, indicating differences in β-lactam binding and catalytic efficiency. Increased requirement of free folding energy, improved solvent accessibility and decreased melting temperatures among NDM-1-like, OXA-181, OXA-66, OXA-69 and OXA-104 predicted functional improvement over their ancestral variants. NDM-1-like carbapenemases demonstrated improvement in binding stability, affinity and catalysis of meropenem than that of NDM-1. Catalytic activity of imipenem was predicted to improve among OXA-181, which could be correlated with more than 1.5 folds smaller ZDs around imipenem disc, than that of meropenem/ertapenem, among OXA-181 producing isolates. However, OXA-66 indicated greater binding stability and affinity for imipenem and meropenem. This study indicated structural/functional convergence as well as divergence among several carbapenemase variants and provided useful insights into carbapenemase-mediated carbapenem resistance that might help in identifying appropriate treatment regimen for bacterial infections.

Nosocomial transmission of carbapenem-resistant Klebsiella pneumoniae in an Italian university hospital: a molecular epidemiological study

To describe the phenotypic and genotypic profiles of Klebsiella pneumoniae carbapenemase-producing K.pneumoniae (KPC-Kp) strains isolated from patients with invasive infections at an Italian university hospital in order to assess the epidemiological trend. An observational prospective study was undertaken at the University Hospital of Sassari, Italy to detect KPC-Kp strains in patients with invasive bacteraemia. Isolates were identified phenotypically; carbapenemase production was assessed using phenotypic and genotypic methods. Sequencing of blaKPC genes, pulsed-field gel electrophoresis and multi-locus sequence typing were performed. During the period 2015-2017, 46 cases of invasive infection with K.pneumoniae were recorded. Two-thirds (67.4%) of the patients were male, and the mean age was 69.4 years. Most patients had at least one comorbidity (56.5%) and/or had been hospitalized previously (70.5%), 81.8% had current or recent medical device use, and 85.4% had recent antibiotic exposure. The mortality rate was 52.3%. A multi-drug-resistant pattern (including carbapenems, fluoroquinolones, third-/fourth-generation cephalosporins) was shown for all K.pneumoniae isolates. KPC-3 and -2 were produced by all strains. The most common sequence types were 512 (91.3%) and 101 (8.7%), grouped into three clusters (A, A1 and B). A high incidence of KPC-Kp in patients with invasive infections was recorded at an Italian university hospital compared with the incidence measured before 2015. This study confirmed the importance of the KPC-3 carbapenemase variant, as reported by other Italian studies. High mortality and comorbidity rates appear to be associated with KPC-Kp infection.

Detection of Antibiotic Resistance Determinants and Their Transmissibility among Clinically Isolated Carbapenem-Resistant Escherichia coli from South India

Objectives: The aim of this study was to analyze the prevalence of the CTX-M, TEM, SHV, VIM, NDM, and OXA genes in carbapenemase-producing Escherichia coli and their transmissibility at a tertiary care hospital in south India. Materials and Methods: Twenty-one carbapenem-resistant E. coli (carbapenem-resistant Enterobacteriaceae; CRE) were collected from the Sri Sathya Sai Institute of Higher Medical Sciences (Puttaparthi India). Resistance to antibiotics was analyzed by Vitek-2, and the identity of the isolates was confirmed by 16S rDNA sequencing. RAPD and enterobacterial repetitive intergenic consensus (ERIC)-PCR were performed for molecular typing. Metallo-β-lactamase production was confirmed by a double disc synergy test. The presence of the extended-spectrum β-lactamases CTX-M, TEM, and SHV and of the carbapenemases NDM, VIM, and OXA was determined by PCR. Carbapenemase variants were further confirmed by sequencing. The transmissibility of the genes was tested by conjugation. Results: Twelve of the 21 (57%) carbapenem-resistant E. coli isolates were community acquired, indicating the spread of CRE in environmental samples. TEM and NDM-5 were found to be the major β-lactamases produced by the pathogens. OXA-181 was found in 5 of the isolates. All 21 isolates were found to harbor more than one of the tested β-lactamases, and all of the isolates were found to have the capacity to participate in conjugation; 15 of the transconjugants were found to have acquired the tested β-lactamases, substantiating their ability to be transferred to other strains of bacteria. Conclusion: Monitoring of community-acquired carbapenem-resistant bacteria is very important as the association of resistance determinants with mobile genetic elements would present a serious clinical challenge.

Emergence of OXA-48 carbapenemase-producing Enterobacter cloacae ST89 infection in Poland

The utility of carbapenems, which are considered 'last-line' agents, is being diminished by the growing incidence of various resistance mechanisms in bacteria. We aimed to investigate the molecular mechanism of carbapenem resistance in Enterobacter cloacae recovered from a 76-year-old patient who had undergone coronary artery bypass grafting and repair of the mitral and tricuspid valves. Interestingly, the patient had no prior history of hospital admission abroad. The Carba-NP test II and synergy testing were performed to confirm carbapenemase activity. PCR was used to detect carbapenemase-encoding genes. Nucleotide and amino acid sequence analysis was performed to identify OXA-48 variants. Moreover, we performed multilocus sequence typing (MLST) of multidrug-resistant (MDR) E. cloacae. We detected no significant increase in zone diameter around disks with inhibitors. However, the Carba-NP test II revealed carbapenemase activity in all isolates. All isolates showed the presence of the exact OXA-48 carbapenemase variant. Furthermore, MLST analysis revealed that the MDR E. cloacae isolates belonged to ST89. We report a case of infection caused by a unique carbapenem-resistant E. cloacae ST89 producing OXA-48 carbapenemase. Interestingly, these pathogens developed resistance to other 'last-resort' agents, namely colistin and tigecycline. There is a crucial need for surveillance programs aimed at screening for carbapenemase-producing Gram-negative bacteria, especially in patients transferred from high-incidence areas.

Emergence of clinical Klebsiella pneumoniae producing OXA-232 carbapenemase in Singapore.

We report the emergence of OXA-232, a newly described OXA-48-like carbapenemase variant, in Southeast Asia. Molecular characterization of eight Klebsiella pneumoniae obtained from local and foreign patients reveals clonality of the isolates. blaOXA-232 was located on a non-conjugative plasmid of 6141 base pairs (GenBank accession number JX423831.1).