Rapid detection of KPC-producing Enterobacterales by using a modified Carba NP test with imipenem/relebactam.

INTRODUCTION: Here, we propose a novel modified Carba NP test for detecting KPC-producing Enterobacterales using imipenem/relebactam. MATERIAL AND METHODS: The test performance was evaluated in a random selection of 160 previously molecularly characterized clinical isolates carrying the 110 blaKPC, 1 blaGES, 12 blaVIM, 4 blaIMP, 3 blaNDM and 42 blaOXA-48-like genes. The proposed method relies on the detection of imipenem hydrolysis in an imipenem/relebactam antibiotic solution and subsequent visual interpretation by color change. RESULTS: All class A producing Enterobacterales (111/111) were detected using imipenem/relebactam as no visual appreciation of color change was perceived due to a nule hydrolysis of imipenem in the antibiotic solution. Overall, the assay showed 100% sensitivity (111/111) and specificity (69/69) for detecting class A KPC-producing Enterobacterales. DISCUSSION: The biochemical assay provides very reliable results for detecting KPC-producing Enterobacterales, with a turnaround time of less than 1 hour, minimum handling and no specialized equipment required.

Rapid detection of KPC-producing Enterobacterales by using a modified Carba NP test with imipenem/relebactam / Detección rápida de Enterobacterales productoras de KPC mediante una modificación del test Carba NP utilizando imipenem/relebactam

Introduction: Here, we propose a novel modified Carba NP test for detecting KPC-producing Enterobacterales using imipenem/relebactam. Material and methods: The test performance was evaluated in a random selection of 160 previously molecularly characterized clinical isolates carrying the 110 blaKPC, 1 blaGES, 12 blaVIM, 4 blaIMP, 3 blaNDM and 42 blaOXA-48-like genes. The proposed method relies on the detection of imipenem hydrolysis in an imipenem/relebactam antibiotic solution and subsequent visual interpretation by color change. Results: All class A producing Enterobacterales (111/111) were detected using imipenem/relebactam as no visual appreciation of color change was perceived due to a nule hydrolysis of imipenem in the antibiotic solution. Overall, the assay showed 100% sensitivity (111/111) and specificity (69/69) for detecting class A KPC-producing Enterobacterales. Discussion: The biochemical assay provides very reliable results for detecting KPC-producing Enterobacterales, with a turnaround time of less than 1 hour, minimum handling and no specialized equipment required.(AU)

Introducción: En este manuscrito proponemos un novedoso ensayo basado en el Carba NP test para la detección de Enterobacterales productoras de KPC utilizando imipenem/relebactam. Material y métodos: La evaluación se realizó en una selección aleatoria de 160 aislados clínicos previamente caracterizados molecularmente que portaban los genes 110 blaKPC, 1 blaGES, 12 blaVIM, 4 blaIMP, 3 blaNDM y 42 blaOXA-48-like. El método propuesto se basa en la detección de la hidrólisis de imipenem en una solución de imipenem/relebactam y posteriormente su interpretación mediante un cambio de color. Resultados: El ensayo tiene una sensibilidad (111/111) y una especificidad (69/69) del 100% para detectar Enterobacterales productoras de KPC, de clase A. Discusión: Este ensayo bioquímico proporciona resultados muy fiables para la detección de Enterobacterales productoras de KPC, con un tiempo de respuesta inferior a 1 h, además de una manipulación mínima y sin necesidad de ningún equipamiento especializado.(AU)

CRISPR-Cas, a Revolution in the Treatment and Study of ESKAPE Infections: Pre-Clinical Studies.

One of the biggest threats we face globally is the emergence of antimicrobial-resistant (AMR) bacteria, which runs in parallel with the lack in the development of new antimicrobials. Among these AMR bacteria pathogens belonging to the ESKAPE group can be highlighted (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) due to their profile of drug resistance and virulence. Therefore, innovative lines of treatment must be developed for these bacteria. In this review, we summarize the different strategies for the treatment and study of molecular mechanisms of AMR in the ESKAPE pathogens based on the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins' technologies: loss of plasmid or cellular viability, random mutation or gene deletion as well directed mutations that lead to a gene's loss of function.

Enhanced Antibacterial Activity of Repurposed Mitomycin C and Imipenem in Combination with the Lytic Phage vB_KpnM-VAC13 against Clinical Isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 ß-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD600) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 ß-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 ß-lactamase.

Rapid detection of KPC-producing Enterobacterales by using a modified Carba NP test with imipenem/relebactam.

INTRODUCTION: Here, we propose a novel modified Carba NP test for detecting KPC-producing Enterobacterales using imipenem/relebactam. MATERIAL AND METHODS: The test performance was evaluated in a random selection of 160 previously molecularly characterized clinical isolates carrying the 110 blaKPC, 1 blaGES, 12 blaVIM, 4 blaIMP, 3 blaNDM and 42 blaOXA-48-like genes. The proposed method relies on the detection of imipenem hydrolysis in an imipenem/relebactam antibiotic solution and subsequent visual interpretation by color change. RESULTS: All class A producing Enterobacterales (111/111) were detected using imipenem/relebactam as no visual appreciation of color change was perceived due to a nule hydrolysis of imipenem in the antibiotic solution. Overall, the assay showed 100% sensitivity (111/111) and specificity (69/69) for detecting class A KPC-producing Enterobacterales. DISCUSSION: The biochemical assay provides very reliable results for detecting KPC-producing Enterobacterales, with a turnaround time of less than 1 hour, minimum handling and no specialized equipment required.

Activity of imipenem/relebactam against a Spanish nationwide collection of carbapenemase-producing Enterobacterales.

BACKGROUND: Imipenem/relebactam is a novel carbapenem/ß-lactamase inhibitor combination, developed to act against carbapenemase-producing Enterobacterales (CPE). OBJECTIVES: To assess the in vitro activity of imipenem/relebactam against a Spanish nationwide collection of CPE by testing the susceptibility of these isolates to 16 widely used antimicrobials and to determine the underlying ß-lactam resistance mechanisms involved and the molecular epidemiology of carbapenemases in Spain. MATERIALS AND METHODS: Clinical CPE isolates (n = 401) collected for 2 months from 24 hospitals in Spain were tested. MIC50, MIC90 and susceptibility/resistance rates were interpreted in accordance with the EUCAST guidelines. ß-Lactam resistance mechanisms and molecular epidemiology were characterized by WGS. RESULTS: For all isolates, high rates of susceptibility to colistin (86.5%; MIC50/90 = 0.12/8 mg/L), imipenem/relebactam (85.8%; MIC50/90 = 0.5/4 mg/L) and ceftazidime/avibactam (83.8%, MIC50/90 = 1/≥256 mg/L) were observed. The subgroups of isolates producing OXA-48-like (n = 305, 75.1%) and KPC-like enzymes (n = 44, 10.8%) were highly susceptible to ceftazidime/avibactam (97.7%, MIC50/90 = 1/2 mg/L) and imipenem/relebactam (100.0%, MIC50/90 = ≤0.25/1 mg/L), respectively.The most widely disseminated high-risk clones of carbapenemase-producing Klebsiella pneumoniae across Spain were found to be ST11, ST147, ST392 and ST15 (mostly associated with OXA-48) and ST258/512 (in all cases producing KPC). CONCLUSIONS: Imipenem/relebactam, colistin and ceftazidime/avibactam were the most active antimicrobials against all CPEs. Imipenem/relebactam is a valuable addition to the antimicrobial arsenal used in the fight against CPE, particularly against KPC-producing isolates, which in all cases were susceptible to this combination.

Viral Related Tools against SARS-CoV-2.

At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure.

Strategies to Combat Multidrug-Resistant and Persistent Infectious Diseases.

Antibiotic failure is one of the most worrying health problems worldwide. We are currently facing an international crisis with several problematic facets: new antibiotics are no longer being discovered, resistance mechanisms are occurring in almost all clinical isolates of bacteria, and recurrent infections caused by persistent bacteria are hampering the successful treatment of infections. In this context, new anti-infectious strategies against multidrug-resistant (MDR) and persistent bacteria, as well as the rescue of Food and Drug Administration (FDA)-approved compounds (drug repurposing), are being explored. Among the highlighted new anti-infectious strategies, in this review, we focus on antimicrobial peptides, anti-virulence compounds, phage therapy, and new molecules. As drugs that are being repurposed, we highlight anti-inflammatory compounds, anti-psychotics, anti-helminthics, anti-cancerous drugs, and statins.

Multiplex Real-Time PCR-shortTUB Assay for Detection of the Mycobacterium tuberculosis Complex in Smear-Negative Clinical Samples with Low Mycobacterial Loads.

Tuberculosis (TB) remains a major health problem worldwide. Control of TB requires rapid, accurate diagnosis of active disease. However, extrapulmonary TB is very difficult to diagnose because the clinical specimens have very low bacterial loads. Several molecular methods involving direct detection of the Mycobacterium tuberculosis complex (MTBC) have emerged in recent years. Real-time PCR amplification simultaneously combines the amplification and detection of candidate sequences by using fluorescent probes (mainly TaqMan or Molecular Beacons) in automated systems. The multiplex real-time PCR-short assay is performed using locked nucleic acid (LNA) probes (length, 8 to 9 nucleotides) in combination with CodUNG to detect multiple pathogens in clinical samples. In this study, we evaluated the performance of this novel multiplex assay for detecting the MTBC in comparison with that of the conventional culture-based method. The multiplex real-time PCR-shortTUB assay targets two genes, whiB3 (redox-responsive transcriptional regulator) and pstS1 (phosphate-specific transporter), yielding limits of detection (LOD) of 10 copies and 100 copies, respectively, and amplification efficiencies of 92% and 99.7%, respectively. A total of 94 extrapulmonary samples and pulmonary samples with low mycobacterial loads (all smear negative; 75 MTBC culture positive) were analyzed using the test, yielding an overall sensitivity of 88% and a specificity of 95%. For pleural fluid and tissues/biopsy specimens, the sensitivity was 83% and 85%, respectively. In summary, this technique could be implemented in routine clinical microbiology testing to reduce the overall turnaround time for MTBC detection and may therefore be a useful tool for the diagnosis of extrapulmonary tuberculosis and diagnosis using pulmonary samples with low mycobacterial loads.