Search Results (168)

Carbapenem resistance is considered one of the greatest current threats to public health, particularly in the management of infections in clinical settings. Carbapenem resistance in bacteria is mainly due to mechanisms such as the production of carbapenemases (such as the imipenemase IMP, or other enzymes like VIM, NDM, and KPC), that can be detected by several laboratory tests, including immunochromatography and automated real-time PCR (qPCR). Methods: As part of local studies to monitor carbapenem-resistant bacteria in Costa Rica, two cases were initially identified with inconsistent IMP detection results. A possible gene drop-out in the automated qPCR test was suggested based on the negative result, contrasting with the positive result by immunochromatography and whole-genome sequencing. We hypothesized that molecular testing could be optimized through the development of tailored assays to improve the detection of IMP genes. Thus, using IMP gene sequences from the local isolates and regional sequences in databases, primers were redesigned to extend the detection of IMP alleles of regional relevance. Results: The tailored qPCR was applied to a local collection of 119 carbapenem-resistant isolates. The genomes of all 14 positive cases were sequenced, verifying the results of the custom qPCR, despite the negative results of the automated testing. Conclusions: Guided by whole-genome sequencing, it was possible to extend the molecular detection of IMP alleles circulating in Latin America using a tailored qPCR to overcome IMP gene drop-out and false-negative results in an automated qPCR. Full article

Parasitoid venom significantly influences host physiology and development. Our previous research identified high levels of insulin-binding protein IMP-L2 in the venom of Scleroderma guani. IMP-L2 may inhibit the insulin/insulin-like growth factor signaling (IIS) cascade by competitively binding insulin-like peptides (ILPs) with insulin receptor (InR). However, how to regulate IIS transduction is unclear. We speculate that venom-derived IMP-L2 may bind ILPs to inhibit IIS transduction. Consequently, we investigated the regulation of the IIS/TOR pathway by venom-derived IMP-L2. An expression analysis of IIS/TOR pathway genes across various developmental stages of Tenebrio molitor demonstrated that this pathway governs the entire developmental process. By examining gene expression before and after parasitism, we determined that S. guani predominantly inhibits TOR pathway signaling in T. molitor post-parasitism. Bioinformatics and expression analyses revealed that IMP-L2 is critically involved in Hymenoptera insects, exhibiting high expression in the venom apparatus, and is upregulated in response to S. guani parasitism factors. Additionally, recombinant IMP-L2 was produced via eukaryotic expression. Finally, the recombinant IMP-L2 was found to inhibit the TOR and IIS/TOR signaling pathways at early (6 h) and late (24 h) stages post-injection. Knockdown of IMP-L2 in S. guani parasitized T. molitor pupae, resulting in accelerated death of T. molitor. During parasitism, S. guani may suppress host growth and development by modulating the IIS/TOR signaling pathway through venom-derived IMP-L2, potentially affecting host lifespan. Full article

Background: Antimicrobial resistance (AMR) is a growing global health concern, driven in part by the environmental release of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs). Aquatic systems, particularly those exposed to urban, agricultural, and industrial activity, are recognized as hotspots for AMR evolution and transmission. In Chile, the Aconcagua River—subject to multiple anthropogenic pressures—offers a representative model for studying the environmental dimensions of AMR. Methods: Thirteen surface water samples were collected along the Aconcagua River basin in a single-day campaign to avoid temporal bias. Samples were filtered through 0.22 μm membranes and cultured on MacConkey agar, either unsupplemented or supplemented with ceftazidime (CAZ) or ciprofloxacin (CIP). Isolates were purified and identified using MALDI-TOF mass spectrometry. Antibiotic susceptibility was evaluated using the Kirby–Bauer disk diffusion method in accordance with CLSI guidelines. Carbapenemase activity was assessed using the Blue-Carba test, and PCR was employed for the detection of the bla_VIM, bla_KPC, bla_NDM, and bla_IMP genes. Results: A total of 104 bacterial morphotypes were isolated; 80 were identified at the species level, 5 were identified at the genus level, and 19 could not be taxonomically assigned using MALDI-TOF. Pseudomonas (40 isolates) and Aeromonas (25) were the predominant genera. No growth was observed on CIP plates, while 24 isolates were recovered from CAZ-supplemented media, 87.5% of which were resistant to aztreonam. Five isolates exhibited resistance to carbapenems; two tested positive for carbapenemase activity and carried the bla_VIM gene. Conclusions: Our results confirm the presence of clinically significant resistance mechanisms, including bla_VIM, in environmental Pseudomonas spp. from the Aconcagua River. These findings highlight the need for environmental AMR surveillance and reinforce the importance of adopting a One Health approach to antimicrobial stewardship and wastewater regulation. Full article

In hospital environments, pathogenic bacteria spread easily and acquire virulence and antibiotic resistance genes. The aim of the study was an evaluation of the genetic diversity of 109 K. pneumoniae isolates recovered from patients of a district hospital in central Poland. The frequencies of genes coding for β-lactamases, efflux pumps, and virulence factors were determined. Genotyping of the isolates was performed with ERIC (Enterobacterial Repetitive Intergenic Consensus) and REP (Repetitive Element Sequence Based) PCR techniques, with 21 and 19 genotypes being identified, respectively. The bla_SHV-1 (92.7%), bla_CTX-M group 1 (83.5%), bla_TEM-1 (28.4%), bla_NDM-1 (16.5%), bla_VEB-1 (11.0%), bla_CTX-M group 9 (3.7%), bla_KPC (1.8%), bla_IMP, bla_OXA-48, bla_CTX-M group 2, bla_CTX-M groups 8, and 25/26 (0% each) and efflux pumps: AcrAB (100%), tolC (93.6%), and mdtk (60.5%), and virulence genes coding: urease subunit ureA (94.5%) endotoxins wabG (92.7%) and uge (64.2%), and siderophore iucB (3.7%) were detected. The bla_SHV-1, bla_CTX-M group 1, mdtk, tolC, AcrAB (16.5%); bla_SHV-1, bla_CTX-M group 1, tolC, AcrAB (15.6%), and bla_SHV-1, bla_CTX-M group 1, bla_NDM-1, mdtk, tolC, AcrAB (11.9%) were the most common resistance patterns. The distribution of resistance and virulence genes varied more between hospital wards than between different clinical materials. Hospital’s antibiotic-resistant and virulent K. pneumoniae, able to spread among humans, animals, and in the environment, pose a significant threat to public health. Full article

This systematic review assessed the global epidemiology of metallo-β-lactamase (MBL)-producing Acinetobacter clinical isolates and the associated antimicrobial resistance. A total of 475 relevant articles from the Cochrane Library, Google Scholar, PubMed, Scopus, and Web of Science were identified and screened as potentially eligible articles. Data from 85 articles were extracted for the analysis. Most reports on MBL-producing Acinetobacter clinical isolates originated from Asia [68/85 (80%) studies] and Africa [14/85 (16.5%) studies]. There were also scarce reports from Europe and America. The bla_VIM (in 31 studies), bla_IMP (in 29 studies), and bla_NDM (in 21 studies) genes were the most commonly identified genes. In 22 out of 28 (78.6%) studies with comparable data, the proportions of MBL-producing pathogens detected using phenotypic methods were numerically higher than those using genotypic methods. MBL-producing Acinetobacter isolates showed high resistance (up to 100%) to several antibiotic classes, including carbapenems, cephalosporins, fluoroquinolones, and monobactams. However, they showed low resistance to colistin [ranging from 0% (in six studies) to 14.3% (in one study)] and to tigecycline [0% (in three studies)]. No risk of bias assessment was conducted. The findings emphasize the global spread of MBL-producing Acinetobacter and the need for enhanced antimicrobial stewardship, infection control measures, and surveillance. Full article

Phytoplasmas of the X-disease group (16SrIII) are economically significant pathogens in South America, causing severe crop losses. Traditional classification based on the 16S rRNA gene has limitations in resolving closely related strains, prompting the exploration of alternative markers. This study focuses on the immunodominant membrane proteins imp and idpA, which exhibit high variability and play crucial roles in host–pathogen interactions. Through molecular characterization of imp and idpA genes in 16SrIII subgroups, we identified significant genetic diversity and distinct evolutionary pressures. The imp gene, under positive selection, showed high variability in its hydrophilic extracellular domain, suggesting adaptation to host immune responses. In contrast, idpA exhibited strong negative selection, indicating functional conservation. Phylogenetic analyses revealed that imp and idpA provide higher resolution than the 16S rRNA gene, enabling finer differentiation within subgroups. These findings highlight the potential of imp and idpA as complementary markers for phytoplasma classification and diagnostics. Full article

Pingliang Red Cattle, a renowned geographical indication product in China, is distinguished by its superior meat quality, yet the scientific basis for its unique attributes remains underexplored. This study integrated metabolomic and transcriptomic analyses to elucidate the biochemical and physiological factors underlying the enhanced flavor, color stability, and tenderness of Pingliang Red Cattle beef compared to Qinchuan and Simmental cattle. Metabolomic profiling revealed significantly elevated levels of inosine monophosphate (IMP, 2.86–3.96× higher) and glutathione (GSH, 2.42–5.43× higher) in Pingliang Red Cattle, contributing to intense umami flavor and prolonged meat color retention. Notably, ergothioneine (EGT), a potent antioxidant, was identified for the first time in Pingliang Red Cattle beef, with concentrations 2.55× and 4.25× higher than in Qinchuan and Simmental, respectively. Transcriptomic analysis highlighted the upregulation of 21 tenderness-related genes (e.g., FABP3, PRDX6, CAST) and key enzymes in purine and glutathione metabolism pathways (e.g., PDE4D, ADSL, GGT1), correlating with meat tenderness and the improved meat quality. Additionally, Pingliang Red Cattle’s natural forage-rich diet and low-density rearing practices were critical in enhancing these traits. These findings provide a scientific foundation for Pingliang Red Cattle’s premium quality, offering actionable insights for GI product branding, quality optimization, and market competitiveness. The multi-omics approach established here serves as a paradigm for quality assessment and improvement of other GI agricultural products, bridging traditional reputation with molecular evidence. Full article

Introduction/Objectives: Antibiotic resistance makes the treatment of pneumonia challenging. Effective management depends on accurate diagnostic techniques to identify resistance genes and customize drugs. This study primarily aimed to identify antibiotic resistance genes in respiratory samples from patients with pneumonia using polymerase chain reaction (PCR) to determine the prevalence of specific resistance genes and analyze clinical factors contributing to antibiotic resistance, as well as to provide actionable insights into resistance patterns in Jordan and support efforts to improve pneumonia management. Methods: This retrospective observational study included 114 patients who were diagnosed with pneumonia. Clinical data, including prior antibiotic exposure and treatment history, were collected. PCR diagnostics were used to detect resistance genes in respiratory samples. In this study, we evaluated 14 antibiotic resistance genes in pneumonia pathogens, highlighting their diverse resistance mechanisms. Results: Mec A was the most frequently detected gene, appearing in 87 samples (77.3%). Additionally, Tem in 80 samples (70.2%), Oxa-48-like in 15 samples (13.2%), and Ctx-M-1 in 38 samples (33.3%) were among the most commonly detected genes. In contrast, Oxa-40-like (7.0%), Vim (8.8%), and Imp (4.4%) genes exhibited a lower prevalence. The Oxa-51-like gene showed the only significant association with ertapenem resistance (p-value = 0.046). Further analysis revealed statistically significant associations between Mec A and methicillin resistance (p < 0.001), underscoring its critical role. However, other genes, such as Oxa-40-like and Oxa-48-like, showed no significant correlation with the antibiotic resistance patterns of imipenem and meropenem (p > 0.05). Conclusions: This study demonstrates the utility of PCR-based diagnostics for detecting resistance genes and highlights the critical clinical factors associated with antibiotic resistance in patients with pneumonia. These findings underscore the importance of integrating molecular diagnostics into routine care to improve treatment outcomes and combat the growing threat of antibiotic resistance in Jordan. This highlights PCR’s value in guiding effective treatment strategies and addressing multidrug-resistant pneumonia. Full article

This article methodically reveals how, in woody plants (poplar), the interaction between N and P coordinates root structure and nutrient absorption through a complex hormone signaling network. This study bridges a significant gap in our knowledge of nutrient interaction networks. The results demonstrate that NO₃⁻ significantly enhances the gene expression and enzymatic activity of organic acid synthases (MDH, PEPC) and APs. Furthermore, it synergizes with IAA/ABA signals to refine root structure, enhancing the surface area for P absorption. In low Pi availability environments, NO₃⁻ further promotes P recycling by simultaneously boosting the levels of Pi transport proteins (notably, the PHO family), facilitating myo-inositol phosphate metabolism (via IMP3/ITPK1-mediated PP-InsPs degradation), and augmenting IAA/SA signals. Pi induces the activity of N assimilation enzymes (GS/GOGAT/GDH), facilitating nitrogen metabolism. However, in the absence of N, it leads to a metabolic imbalance characterized by high enzymatic activity but low efficiency. Alternatively, adequate N availability allows Pi to improve root robustness and N assimilation efficiency, mediated by IAA/GA accumulation and ABA signaling (e.g., SNRK2/ABF). We propose the existence of an intricate network in poplar, orchestrated by transcriptional cascades, metabolic regulation, and hormonal synergism. Key modules such as SPX-PHR, NLA, HHO2, and MYB59 are likely central to this network’s function. These findings offer a foundational framework for the development of molecular breeding and precise fertilization strategies, enhancing the efficient use of N and P in forestry. Full article

Background: Mapping the local etiology and susceptibility of common pathogens causing complicated urinary tract infection (cUTI) is important for promoting evidence-based antimicrobial prescribing. Evaluating the prevalence of extended-spectrum beta-lactamase (ESBL), AmpC beta-lactamase (AmpC), and carbapenemase-producing Enterobacterales (CPEs) is equally important as it informs treatment guidelines and empiric management. Whole genome sequencing (WGS) enhances antimicrobial resistance (AMR) surveillance by complementing phenotypic antimicrobial susceptibility testing, offering deeper insights into resistance mechanisms, transmissions, and evolutions. Integrating it into routine AMR monitoring can significantly improve global efforts to combat antimicrobial resistance. Methods: Antimicrobial susceptibility profiles of isolates from cUTI were collected from patients presenting with Sultan Qaboos University Hospital, Muscat and Suhar Hospital, Suhar, Oman. Automated systems as well as manual methods were used for detection of ESBL, AmpC, and CPE. ESBLs, AmpC β-lactamases, and CPEs were further detected by manual methods: double-disk synergy test for ESBL; disk approximation assay and D69C AmpC detection set for AmpC, and mCIM and KPC/IMP/NDM/VIM/OXA-48 Combo test kit for CPE. WGS was carried out in 11 FOX-resistant E. coli and (22 carbapenem-resistant K. pneumoniae) isolates with varying susceptibilities to identify circulating clades, AMR genes, and plasmids. Bioinformatic analysis was performed using online tools. Results: The susceptibility patterns of E. coli from cUTI were as follows: nitrofurantoin (96%), fosfomycin (100%), fluoroquinolones (44%), aminoglycosides (93%), piperacillin-tazobactam (95%), and carbapenems (98%). In comparison, susceptibility rates of K. pneumoniae were far lower: nitrofurantoin (38%), fosfomycin (89%), aminoglycosides (82%), piperacillin-tazobactam (72%), and carbapenems (83%). K. pneumoniae, however, was more susceptible to fluoroquinolones at 47% in comparison to E. coli. The prevalence of ESBL among E. coli and K. pneumoniae was 37.2% and CRE was 6.2% while the estimated prevalence of AmpC was 5.4%. It was observed that E. coli was the predominant ESBL and AmpC producer, while K. pneumoniae was the major carbapenem-resistant Enterobacterales (CREs) producer. No predominant multi-locus sequence typing (MLST) lineage was observed in AmpC-producing E. coli with nine E. coli MLST lineages being identified from eleven isolates: ST-10, ST-69, ST-77, ST-131, ST-156, ST-167, ST-361, ST-1125, and ST-2520. On the other hand, a less diverse MLST spectrum (ST-2096, ST-231, ST-147, ST-1770, and ST-111) was observed in the CRE K. pneumoniae. Among the five MLST lineages, ST-2096 (twelve isolates) and ST-147 (seven isolates) predominated. WGS revealed that DHA-1 was the predominant plasmid-mediated AmpC gene in E. coli, while OXA-232 and NDM-5 were the most common carbapenemase genes in K. pneumoniae. All E. coli DHA-1-positive isolates co-harbored the quinolone resistance gene qnrB4 and the sulfonamide resistance gene sul1 while no aminoglycoside resistance genes were detected. The majority of CPE CRE K. pneumoniae carried other β-lactamase genes, such as blaCTX-M-15, blaSHV, and blaTEM; all co-harbored the quinolone resistance gene OqxAB; and 77% carried the aminoglycoside resistance gene armA. Conclusions: Our results suggest that fosfomycin is an excellent empiric choice for treating complicated cystitis caused by both E. coli and K. pneumoniae, while nitrofurantoin is an appropriate choice for E. coli cystitis but not for K. pneumoniae. Aminoglycosides and piperacillin-tazobactam are excellent intravenous alternatives that spare carbapenems. DHA-1 was the predominant AmpC in E. coli, while OXA-232 and NDM-5 were the predominant carbapenemases in K. pneumoniae. In AmpC-producing E. coli, no MLST predominated, suggesting a significant flux in E. coli with lack of stable clades in this region. In contrast, ST-2096 and ST-147 predominated in CRE Klebsiella pneumoniae, suggesting a stable circulation of these in Oman. WGS profiling provides a deeper understanding of the genetic basis of resistance and enhances surveillance and offers comprehensive insights into pathogen evolution and transmission patterns. Full article

‘Candidatus Phytoplasma prunorum’ has been associated with severe disease in Prunus spp., which are commodities of economic importance in the USA. The introduction and establishment of ‘Ca. P. prunorum’ in the USA could result in huge economic losses, thus creating a need for validated diagnostic tools, which are the cornerstone of successful surveillance, quarantine, and eradication measures. Whole-genome comparisons led to the identification of a diagnostic marker gene specific to ‘Ca. P. prunorum’ (PE639). The PE639 assay was duplexed with an 18S rDNA plant internal control and compared to modified 23S (phytoplasmas) and imp (‘Ca. P. mali’) assays. The PE639 assay produced congruent results to 23S and imp assays for all metrics, demonstrating high linearity, repeatability, intermediate precision, and reproducibility. The limit of detection was comparable for all assays tested, and all demonstrated 100% analytical specificity, selectivity, and diagnostic specificity for their respective target species. Assays metrics were consistent across two platforms, the ABI QuantStudio™ 5 and Bio-Rad CFX96™ OPUS. A synthetic gBlocks™ control was designed and validated to work with all assays, as well as conventional PCR assays targeting 16S rDNA and tuf genes. These validated assays and synthetic control represent beneficial tools that support efforts to protect USA agriculture and facilitate safe trade. Full article

Background: The alarming increase in carbapenemase-producing Enterobacterales is a matter of grave public health concern. The most ubiquitous carbapenemases, Klebsiella pneumoniae carbapenemase (KPC)-, New Delhi metallo-β-lactamase (NDM)-, and oxacillinase (OXA-48)-like enzymes, belong to the Ambler molecular classes A, B, and D, respectively. KPC- and OXA-48-like enzymes have a serine-based hydrolytic mechanism, while NDMs are metallo-β-lactamases that contain zinc in the active site. For the judicious use of reserve drugs and promoting antimicrobial stewardship, timely detection of carbapenemases is essential. While molecular tools are the gold standard for the detection of these enzymes, many laboratories have limited access to them. This study focused on evaluating in-house tools and commercial phenotypic tests for the detection of OXA-48-, KPC-, and NDM-like enzymes in K. pneumoniae, the predominant extremely drug-resistant pathogen in Oman. Methods: In total, 80 GeneXpert/PCR-confirmed (40 OXA-48 and 20 KPC and NDM each) and 37 whole-genome-sequenced (25 OXA-232 and 6 KPC-2, plus NDM-1 and NDM-5) K. pneumoniae were subjected to screening by temocillin (30 μg disk) (MAST Diagnostica, Germany) and D71C (MASTDISCS^®). Isolates resistant to temocillin (<11 mm) and D71C were subjected to four tests: an in-house tool (OXA-48 disk test) and three commercial phenotypic tests: (i) the MASTDISCS^® Combi (D72C) (MAST Group Ltd., Bootle, UK); (ii) the MASTDISCS^® Combi (D73C) (MAST Group Ltd., UK); and (iii) an immunochromatographic assay (ICT), which is the KPC/IMP/NDM/VIM/OXA-48 Combo test kit (Medomics, China), for the detection of OXA-48-, KPC-, and NDM-like carbapenemases. Results: Temocillin exhibited good sensitivity and specificity (100% and 97.50%) compared to D71C (70% and 100%). Among the confirmatory tests, the in-house OXA-48 disk test had 92.50% sensitivity and 100% specificity, while the commercial MAST DISC tests D72C, D73C, and ICT had 97.50%, 95.00%, and 100% sensitivity and 100%, 91.67%, and 95% specificity, respectively. Conclusions: The temocillin disk test is a good screening tool. With high sensitivity and specificity, ease of performance, short turnaround time, and low cost, we recommend the ICT format for routine diagnostic use. In resource-constrained centers, the OXA-48 disk test is an excellent alternative with high sensitivity and specificity. Full article

Adenylosuccinate synthetase (AdSS), encoded by the ADE12 gene in yeast Saccharomyces cerevisiae, plays a critical role in purine biosynthesis, catalyzing the conversion of inosine 5′-monophosphate (IMP) and aspartic acid to adenylosuccinate, a substrate for the following adenosine 5′-monophosphate (AMP) synthesis step. Mutants lacking AdSS activity exhibit a range of pleiotropic phenotypes: slow growth, poor spore germination, accumulation, and secretion of inosine and hypoxanthine. We report new phenotypes of ade12 mutants and explain their molecular mechanisms. A GC-MS analysis showed that ade12 mutants have highly altered metabolite profiles: the accumulation of IMP leads to an impaired cellular energy metabolism, resulting in a dysregulation of key processes—the metabolism of nucleotides, carbohydrates, and amino acids. These metabolic perturbations explain the cell division arrest observed in ade12 yeast strains. A slowed replication in ade12 mutants, because of the insufficient availability of energy, nucleotides, and proteins, leads to the error-prone DNA polymerase ζ-dependent elevation of spontaneous mutagenesis, connecting multiple roles of AdSS in metabolism with genome stability control. Full article

Outbreaks involving carbapenemase-producing enterobacteria (CPE) have become a common occurrence in healthcare settings. While clonal dissemination is firmly established as a cause for these outbreaks, horizontal gene transfers (HGTs) between different species of Enterobacterales found in clinical and environmental isolates are less so. To gather evidence backing up this hypothesis, a review covering the 2013–2024 period was performed. HGTs between different species of clinical and environmental Enterobacterales were identified in thirteen papers, half of those published within the last three years. A combination of short- and long-read whole genome sequencing (WGS) was predominantly used to identify mobile genetic elements and plasmids. The more frequently reported carbapenemases were KPCs, followed by NDMs and IMPs. Predictably, broad-host-range plasmids were responsible for over 50% of HGTs, with the IncA/C group being in the lead. Klebsiella pneumoniae and Enterobacter cloacae complexes were the most frequent species identified in clinical samples, while Citrobacter freundii dominated environmental ones. Drains and pipework frequently constituted CPE reservoirs in protracted outbreaks, alternating epidemic outbursts with silent phases. Including WGS in a systematic environmental surveillance would help in swiftly identifying those CPE reservoirs and possibly help better control plasmid outbursts by allowing the implementation of adequate infection prevention and control measures. Full article

Background/Objectives: Carbapenem-resistant Enterobacterales, largely due to carbapenemase production, are significant public health threats, which compromise treatment with key β-lactam antibiotics. Early detection is essential for guiding therapy and controlling spread. This study describes the design, optimisation and validation of a multiplex real-time PCR for the screening of the most frequent carbapenemases in our area. Methods: Primers and probes targeted at genes encoding carbapenemases bla_KPC, bla_IMP, bla_VIM, bla_NDM and bla_OXA-48-group were designed and adapted for the development, and in silico and experimental validation of a single-tube real-time PCR. Results: A good linear correlation between the fluorescence values in the real-time PCR and the log₁₀ of bacterial concentration of each carbapenemase-containing bacterial suspension was observed (R² > 0.98). The limit of detection was 2–15, 16–256, 42–184, 4–42, 42–226 CFU/reaction of VIM-, IMP-, NDM-, KPC- and OXA-48-carbapenemase-containing bacteria, respectively. Intra-assay coefficient of variation for the mean Ct values ranged from 0.99% for OXA-48 to 3.34% for KPC. Inter-assay variability remained below 7%. Real-time PCR tested on bacterial isolates yielded 100% sensitivity and specificity. Analysis of rectal swabs using extracted DNA and a DNA extraction-free protocol showed good concordance with culture-based phenotypic methods. Additionally, the molecular method could detect all targets, except for one sample where only the DNA extraction-free protocol detected NDM. Conclusions: The assay offers a rapid, sensitive and specific method for the screening of major carbapenemase genes, providing an effective tool for surveillance and infection control in clinical settings. The DNA extraction-free protocol converts this method into a good alternative for screening in 24/7 clinical laboratories. Further multiplexing to target other resistance genes, on demand, could add potential benefits to this laboratory-developed method. Full article