Search Results (64)

Extended-spectrum β-lactamase genes in foodborne Salmonella enterica can disseminate through mobile multidrug-resistance platforms. IncHI2 plasmids are important resistance vehicles capable of carrying complex resistance regions and facilitating their horizontal transfer across diverse bacterial backgrounds, but the transfer and genomic organization of IncHI2 elements co-carrying bla_LAP-2 and bla_CTX-M-55 remain insufficiently characterized. This study investigated two multidrug-resistant foodborne isolates recovered in Shanghai in 2022: Salmonella Agona ST13 isolate Sal22C150 and Salmonella Havana ST1527 isolate Sal22P208. Antimicrobial susceptibility testing, whole-genome sequencing, conjugation, plasmid-retention analysis, comparative genomics, as well as strain- and plasmid-level phylogenetic analyses were performed. Both isolates exhibited broad antimicrobial resistance, including resistance to extended-spectrum cephalosporins. In both isolates, bla_LAP-2 and bla_CTX-M-55 co-transferred with the IncHI2 replicon to Escherichia coli J53 at frequencies of (4.95 ± 0.41) × 10⁻⁵ and (4.46 ± 0.42) × 10⁻⁶ transconjugants per donor cell, respectively. All tested plasmid markers remained detectable through 20 passages without antimicrobial selection. Complete assembly of Sal22P208 confirmed the location of the three β-lactamase genes on the 275,096 bp IncHI2 plasmid pSal22P208. The plasmid contained a conserved conjugative backbone and mosaic accessory regions carrying 15 antimicrobial-resistance determinants together with mercury- and tellurium-resistance loci. SNP-based analysis placed pSal22P208 within a closely related cluster containing six reference IncHI2 plasmids differing by fewer than 30 SNPs and recovered from Salmonella and E. coli of animal, food, and human origin, suggesting a broad distribution of this plasmid lineage across diverse bacterial and ecological backgrounds. Sal22P208 additionally contained a Tn3-associated chromosomal multidrug-resistance region between rpmJ and rpmE that shared extensive structural similarity with a region in Citrobacter braakii LBA3. These findings highlight the role of transferable IncHI2 resistance platforms in the horizontal dissemination and short-term post-transfer maintenance of linked resistance determinants, while chromosomally integrated resistance regions may provide an additional route for the accumulation and inheritance of multidrug resistance in foodborne Salmonella. Full article

Antimicrobial resistance (AMR) is a global public health concern, and animal food production contributes to its spread. This study compared antimicrobial resistance gene (ARG) profiles in Salmonella spp., Escherichia coli, and Enterococcus spp. isolated from chicken meat produced under conventional (CONV) and antibiotic-free (ATB-Free) systems using whole-genome sequencing of 207 phenotypically selected isolates. We hypothesized that the ATB-Free production would be associated with fewer resistance genes, but the observed patterns differed among the bacterial groups. In E. coli (41 genes; 61.0% shared between chains), the gene load per isolate was similar between the production systems (median of four in both chains). In Salmonella spp., only five ATB-Free isolates were available, precluding a meaningful comparison between the production systems; a rarefaction analysis showed that the lower gene count observed in this group was consistent with the sampling effort. In Enterococcus spp., the CONV isolates carried a higher gene load (median of two vs. one) and a higher proportion of enzymatic determinants (48.4% vs. 11.8%). This difference was concentrated in isolates carrying the ionophore resistance genes narA and narB (CONV 41.9% vs. ATB-Free 5.9%; p < 0.001), which were not associated with transferable vancomycin or high-level aminoglycoside resistance determinants in this dataset. The resistance determinants associated with the clinically important antimicrobial classes, including bla_CTX-M-8, quinolone resistance genes, and fosfomycin resistance genes, were detected in isolates from both production systems. Overall, the observed ARG profiles differed among the bacterial groups, indicating that the relationship between the production system and the resistome was not uniform. Since the sequenced isolates constituted a phenotypically selected subset, the observed ARG frequencies should not be interpreted as population-level prevalence estimates for either production system. Full article

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) from companion dogs is an emerging One Health concern because dogs may serve as asymptomatic reservoirs of multidrug-resistant (MDR) and potentially pathogenic bacteria that can be transmitted to humans. This study aimed to determine antimicrobial susceptibility profiles, characterize antimicrobial resistance and virulence associated genes, and identify nucleotide substitutions in resistance determinants of E. coli isolated from healthy dogs in Thailand. Fecal samples (n = 200; 100 samples per province) were collected from healthy dogs attending a total of 50 small animal clinics located in Nakhon Si Thammarat and Chiang Mai provinces between January and March 2026. E. coli isolates were identified by culture and polymerase chain reaction (PCR) confirmation. Antimicrobial susceptibility was evaluated against 12 antimicrobial agents using the disk diffusion method according to CLSI guidelines. Thirty randomly selected isolates were further screened for resistance and virulence-associated genes by PCR, and the tetA gene was sequenced to identify nucleotide substitutions. Differences in gene distributions between provinces were assessed using Fisher’s exact test. A total of 66 E. coli isolates were recovered. High resistance rates were observed for ampicillin (100%), piperacillin (84.85%), ceftriaxone (60.61%), tetracycline (56.06%), and aztreonam (46.97%). Multidrug resistance was identified in 47% of the isolates. The 30 isolates exhibiting the highest levels of antimicrobial resistance were selected for molecular characterization. Among these, the β-lactam resistance gene bla_TEM was significantly more prevalent in Nakhon Si Thammarat than in Chiang Mai (60% vs. 0%; p = 0.022). The virulence gene stx2 was also detected significantly more frequently in isolates from Nakhon Si Thammarat (93.33% vs. 26.67%; p < 0.001). Sequence analysis of tetA revealed multiple nucleotide substitutions in two isolates, suggesting ongoing genetic variation within tetracycline resistance determinants that may contribute to the evolution and persistence of antimicrobial resistance. These findings demonstrate that healthy dogs in Thailand can act as reservoirs of MDR and potentially virulent E. coli. The observed geographic variation in resistance and virulence gene distributions highlights the importance of antimicrobial stewardship and continuous molecular surveillance in companion animals within a One Health framework. Full article

Background/Objectives: Klebsiella (K.) pneumoniae is a significant pathogen in both humans and animals. However, data on its occurrence in animals in Germany remain limited. This study aimed to investigate the antimicrobial resistance (AMR) phenotypes, AMR genes, and virulence traits of animal-derived K. pneumoniae isolates from Germany. Methods: A total of 59 K. pneumoniae isolates obtained in 2023 from dogs, cats, horses, cattle, and chickens across 11 German federal states were analysed. Phenotypic antimicrobial susceptibility testing (AST) was performed, and whole-genome sequencing (WGS) was used for genomic characterisation, including detection of AMR genes, virulence-associated genes, sequence types (STs), and plasmid replicons. Results: Most isolates (78%) were susceptible to all tested antibiotics, while three isolates were classified as multidrug-resistant (MDR). Resistance was most frequently observed for piperacillin (n = 8) and trimethoprim/sulfamethoxazole (n = 4). Carbapenem resistance was detected in two isolates (one from a dog and one from a cat), and phenotypic colistin resistance in one dog isolate. WGS identified 96 AMR genes across isolates, with 20–42 AMR determinants per isolate, conferring resistance to β-lactams, aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, trimethoprim, and fosfomycin. Ten extended-spectrum β-lactamase (ESBL)-producing isolates carried genes including bla_CTX-M-15, bla_SHV-2, bla_SHV-27, bla_SHV-42, bla_SHV-106, and bla_TEM-158. Although fosA was detected in all isolates, only three exhibited phenotypic resistance to fosfomycin. A total of 52 STs were identified, including high-risk clones. One hypervirulent isolate (ST60) carrying hypervirulence-associated genes rmpA and iroB was detected. Plasmid replicons were present in 70% of isolates, while plasmid-associated AMR genes were identified in nine isolates. Conclusions: This study demonstrates the genomic diversity of K. pneumoniae identified in companion animals and highlights the presence of AMR and virulence determinants relevant to a One Health context. Full article

The skin of turtles, particularly aquatic species, can harbor a diverse range of bacteria, including Citrobacter species, which are recognized as causative agents of Septicemic Cutaneous Ulcerative Disease. Consequently, turtles may act as reservoirs of pathogenic and multidrug-resistant bacteria, posing a potential public health concern. This case-based study investigated the presence of Citrobacter spp. in a loggerhead sea turtle (Caretta caretta) housed at the Livorno Aquarium, Italy. Nine swabs were collected from skin lesions (plastron, carapace, nuchal mass), the oral cavity, and the cloaca. The isolated strains were identified by MALDI-TOF MS and tested for their susceptibility to 12 antimicrobials, belonging to eight antimicrobial classes, by the disc diffusion method. Isolates were investigated genotypically for extended-spectrum-β-lactamase (ESBL) bla_CTX−M, bla_TEM, bla_SHV, bla_PER, and metallo-β-lactamase (MBL) bla_IMP, bla_OXA−48, bla_VIM, bla_NDM, bla_GES genes. Biofilm production ability was also evaluated. Fifteen Citrobacter spp. strains were recovered from the analyzed samples. Complete resistance was recorded for ampicillin, followed by high levels of resistance to imipenem, tetracycline and piperacillin-tazobactam. Worryingly, 86.7% were classified as multidrug-resistant. The most common ESBL-genotype combination was bla_SHV and bla_PER genes (60%), while the most frequently detected MBL gene was bla_NDM (46.7%), followed by bla_GES (40%). Most isolates were classified as weak biofilm producers (80%). The findings of this study demonstrate the presence of Citrobacter spp., an opportunistic pathogen, with a notable prevalence of multidrug-resistant strains carrying beta-lactamase-encoding genes, in a loggerhead sea turtle in Italy, across both lesioned and healthy anatomical sites. Full article

Background/Objectives: This study assessed the risk associated with third-generation cephalosporin- and fluoroquinolone-resistant Salmonella from pork consumption by integrating phenotypic resistance profiles with genetic data to characterize the risks and transmission pathways. Methods: Salmonella were isolated from raw pork meat samples (n = 793) collected from fresh markets and hypermarkets across Bangkok during 2021–2022, of which 150 were extended-spectrum β-lactamase (ESBL)-producing and 31 were fluoroquinolone-resistant isolates. Phenotypic and genotypic resistance profiles were characterized. Quantitative antimicrobial resistance risk assessment (AMR RA) was conducted using a dose–response model. Results: Salmonella spp. was detected in 42.75% of pork samples, with a higher prevalence in fresh markets (75.5%) than in hypermarket samples and with concentrations ranging from 1.3 to 180 MPN/g. Twenty-eight percent of isolates were ESBL producers, with ciprofloxacin and levofloxacin resistance observed in 5.3% and 3.0%, respectively. The bla_CTX-M55 genes were located on conjugative plasmids. Whole genome sequencing revealed both vertical and horizontal gene transfer. IncHI2/N and IncC plasmids shared conserved backbones and resistance gene architectures, indicating horizontal dissemination of resistance genes. Phylogenomics suggested possible clonal transmission among pigs, pork, and humans. AMR RA estimated 88,194 annual illness cases per 100,000 people from ESBL-producing Salmonella and 61,877 from ciprofloxacin-resistant strain, compared with 95,328 cases predicted by QMRA from Salmonella contamination. Cooking pork at ≥64 °C for 3 min eliminated the risk in all scenarios. Sensitivity analysis identified initial contamination level and cooking temperature as key determinants. Conclusions: Raw pork meat consumption represents the highest risk, which can be mitigated by thorough cooking (>64 °C, ≥3 min), while integrating genomic data enhances AMR hazard identification, source attribution, and exposure assessment. Therefore, promoting well-cooked meat consumption and safe cooking practices, alongside the use of AMR genetic data to inform targeted interventions, is recommended. Full article

Salmonella species isolated from chicken meat pose an increasing threat to public health. According to ECDC data, salmonellosis cases have shown a significant upward trend in many European countries between 2019 and 2023, almost reaching pre-pandemic levels. EFSA reported 77,486 confirmed human cases in the EU in 2023. This corresponds to a notification rate of 18 cases per 100,000 people, compared to 15.4 cases per 100,000 in 2022. This study evaluated the prevalence of Salmonella spp., antimicrobial resistance (AMR) profiles, and the effectiveness of natural biological preservatives in raw chicken meat obtained from retail outlets in Southeast Turkey. Among 100 samples analyzed according to ISO 6579-1:2017, suspicious colonies were detected after selective enrichment in XLD and n = 3 isolates were confirmed to be Salmonella enterica subsp. enterica serovar Infantis by real-time PCR. Disk diffusion tests performed in accordance with EUCAST showed that all isolates were resistant to beta-lactam, tetracycline, trimethoprim, sulfonomid and aminoglycoside groups. All isolates were classified as multidrug-resistant. PCR detected blaTEM-1 (all isolates), aphA1-IAB (all isolates), aadA1 (two isolates), and sul1 (all isolates), while tetA/tetB genes were not detected. Among the natural compounds tested, carvacrol showed the strongest antimicrobial activity (MIC 1.56 µL/mL; MBC 3.125–6.25 µL/mL; inhibition zones 32–35 mm). Eugenol showed moderate effects with higher MIC/MBC values (3.125–6.25 µL/mL/12.25 µL/mL), while α-terpineol was effective only at higher concentrations. These findings are consistent with the global increase in Salmonella Infantis and AMR, supporting carvacrol followed by eugenol and α-terpineol as promising natural alternatives for controlling MDR Salmonella spp. in food safety applications. Full article

Closing the yield gap in smallholder farming systems requires precise identification of key limiting factors. This study addresses this need by applying boundary line analysis (BLA) to diagnose primary soil constraints to winter wheat (Triticum aestivum L.) yield across 95 smallholder farms in the Loess Plateau of China. The BLA approach effectively delineates optimum nutrient ranges amidst inherent field variability, offering a novel methodological advantage for heterogeneous agricultural landscapes. The results showed that, regarding variability, the coefficients of variation for productive spike number and grain yield were considerably greater than those for kernels per spike and thousand-kernel weight. Soil available phosphorus showed the highest coefficient of variation (67.1%), 1.8–2.2 times greater than that of soil organic matter, alkali-hydrolyzed nitrogen, and available potassium. Boundary line models identified significant (p < 0.05) parabolic relationships, defining optimal ranges of 18.5–21.7 g kg⁻¹ for soil organic matter, 10.4–49.0 mg kg⁻¹ for alkali-hydrolyzed nitrogen, 40.5–61.6 mg kg⁻¹ for available phosphorus, and 218.3–284.1 mg kg⁻¹ for available potassium. Crucially, maintaining soil organic matter and available phosphorus within their respective optimal ranges was fundamental for maximizing yield. These findings provide a scientific basis for site-specific nutrient management and offer direct implications for designing targeted agricultural extension services and fertilization policies to enhance productivity in smallholder systems. Full article

Background/Objectives: This study was aimed to investigate the safety profile of traditional Montenegrin Njeguški cheese by quantifying genes associated with resistance to clinically important antibiotics. Methods: Samples of Njeguški cheese were sourced from three artisan producers in Montenegro, identified as A, B, and C, with three individual batches selected per producer. Quantitative PCR (qPCR) was performed on bacterial DNA extracted directly from samples to detect genes encoding resistance to macrolide–lincosamide–streptogramin B (MLS_B) [erm(A), erm(B), erm(C)], vancomycin (vanA, vanB), tetracyclines [tet(M), tet(O), tet(S), tet(K), tet(W)], β-lactams (mecA, blaZ), aminoglycosides [aac (6′)-Ie aph (2″)-Ia], and carbapenems (bla_KPC, bla_OXA-48, bla_NDM-1, bla_GES, and bla_VIM). Results: Among the MLS_B resistance genes, erm(B) was detected in all samples, erm(C) was present only in those from producer B, while erm(A) was found exclusively in batch 3 from producer C. Tetracycline resistance genes were widely distributed, except for tet(O), which was absent in batch 3 from producers A and B. Regarding β-lactam resistance, both blaZ and mecA were consistently detected across all samples, with statistically significant differences observed between producers. None of the samples tested positive for vancomycin resistance genes or the aminoglycoside resistance gene, regardless of producer. Among the carbapenemase genes analyzed, bla_NDM-1 was the only one detected, found in most samples from producers B and C. Conclusions: This research provides the first risk assessment of artisanal and commercial Njeguški cheese regarding antimicrobial resistance genes. The findings offer valuable insights to enhance the microbiological safety of traditional Montenegrin cheeses, supporting consumer confidence in local and international markets. Full article

Sternal bursitis is an underexplored lesion in poultry, often overlooked in microbiological diagnostics. In this study, we characterized 36 Escherichia coli isolates recovered from sternal bursitis in broiler chickens, combining phenotypic antimicrobial susceptibility testing, PCR-based screening, and whole genome sequencing (WGS). The genetic analysis revealed a diverse population spanning 15 sequence types, including ST155, ST201, and ST58. Resistance to tetracycline and ciprofloxacin was common, and several isolates carried genes encoding β-lactamases, including blaTEM-1B. Chromosomal mutations associated with quinolone and fosfomycin resistance (e.g., gyrA p.S83L, glpT_E448K) were also identified. WGS revealed a high number of virulence-associated genes per isolate (58–96), notably those linked to adhesion (fim, ecp clusters), secretion systems (T6SS), and iron acquisition (ent, fep, fes), suggesting strong pathogenic potential. Many isolates harbored virulence markers typical of ExPEC/APEC, such as iss, ompT, and traT, even in the absence of multidrug resistance. Our findings suggest that E. coli from sternal bursitis may act as reservoirs of resistance and virulence traits relevant to animal and public health. This highlights the need for including such lesions in genomic surveillance programs and reinforces the importance of integrated One Health approaches. Full article

Pseudomonas aeruginosa poses a significant global concern in human and veterinary medicine due to its resistance to multiple antimicrobials. Limited research has been carried out on rifampicin-resistant P. aeruginosa, particularly in food-producing animals such as camels. Therefore, the purpose of this study was to investigate the occurrence of rifampicin- and multidrug-resistant P. aeruginosa in apparently healthy camels. Nasal swabs and tissue samples were collected from one hundred apparently healthy slaughtered camels, and they were subjected to bacteriological isolation and identification of P. aeruginosa. Antimicrobial susceptibility testing was performed, followed by phenotypic and genotypic detection of ESBL-producing P. aeruginosa isolates. Twenty-two P. aeruginosa strains were investigated for the rpoB gene, including rifampicin-resistant isolates. P. aeruginosa was found in 16% (16/100) of the investigated apparently healthy slaughtered camels. P. aeruginosa was confirmed in sixteen and six isolates from nasal swabs and tissue samples, respectively, by pigment production on cetrimide agar. The most predominant beta-lactamase-encoding gene in twenty-two ESBL-producing isolates was bla_PER (40.9%), followed by bla_CTX-M (36.4%), bla_TEM (31.8%), and bla_SHV (27.3%). Multidrug resistance was identified in 54.5% (12/22) of P. aeruginosa isolates. The rpoB gene was detected in 11 (50%) out of 22 P. aeruginosa strains, with eleven positive isolates being regarded as rifampicin-resistant. Furthermore, phylogenetic analysis of a rifampicin- and multidrug-resistant P. aeruginosa rpoB gene sequence revealed a genetic relatedness to P. aeruginosa strains retrieved from human clinical cases. In conclusion, this study provides a snapshot on the occurrence of rifampicin- and multidrug-resistant P. aeruginosa among apparently healthy camels. In line with a possible risk of animal-to-human transfer, further molecular studies on rifampicin-resistant P. aeruginosa in animals are required to better understand and combat this serious zoonotic pathogen. Full article

Acinetobacter baumannii (A. baumannii), a very common pathogen, poses a significant public health threat due to its antibiotic resistance and long survival in healthcare environments. Both A. baumannii and carbapenem-resistant A. baumannii (CRAB) can spread through the air, increasing infection risks. Therefore, monitoring their presence in the air is of great significance, especially in hospitals. Herein, we developed a Chelex-100-LAMP-CRISPR/Cas12a (CLC) platform including DNA release and nucleic acid test. Combined with a wet cyclone sampler, the platform can detect airborne A. baumannii and its most common carbapenem-resistant gene, bla_OXA-23, within 70 min. This CLC platform has also been proven to have a detection limit of 6 × 10² CFU of CRAB per test through simulated air samples. Moreover, this platform was also used to test five actual air samples from a tertiary hospital, and the results achieved perfect concordance with sequencing data, validating the platform’s accuracy and reliability. Therefore, the CLC platform showed great potential for the rapid, on-site detection of airborne A. baumannii and its carbapenem-resistant gene bla_OXA-23, offering a valuable tool for infection control in healthcare environments. Full article

Avian colibacillosis caused by avian pathogenic Escherichia coli (APEC) strains is a bacterial disease responsible for enormous economic losses in the poultry industry, due to high mortality rates in farms, antibiotic therapy costs, and seizures at slaughterhouses. The aim of this study was to characterize the serogroups and molecular features of extended spectrum β-lactamase (ESBL)-producing APEC isolates recovered from 248 liver samples of 215 broilers and 33 turkeys with colibacillosis lesions in northeast Algeria. For this, microbiological tests were carried out, according to the recommended standards: E. coli isolates were recovered using standard microbiological protocols, and identification was carried out by MALDI-TOF MS. Serogrouping was performed using a rapid agglutination slide and the antisera of three O somatic groups (O1, O2, O78). Antimicrobial susceptibility was determined by the disk diffusion method. PCR assays and sequencing were used to detect antimicrobial resistance genes, integrons, phylogrouping, and MLST. Conjugation experiments were also conducted to determine the transferability of the retrieved ESBL-encoding genes. Overall, 211 (85.1%) APEC isolates were collected (one per positive sample), and 164 (77.7%) of them were typable. The O2 and O1 serogroups were the most detected (46.1% in broiler typable isolates and 61.5% in turkey typable isolates). Seventeen APEC isolates were ESBL-producers and harbored the following genes (number of isolates): bla_CTX-M-1 (14), bla_CTX-M-15 (2), and bla_SHV-12 (1). They belonged to phylogroups D (10 isolates), B1 (6 isolates), and B2 (1 isolate). The MLST of 13 ESBL producers revealed seven STs: ST23, ST38, ST48, ST117, ST131, ST1146, and ST5087. The ESBL-encoding genes were transferred by conjugation among 15 ESBL-producing isolates, and transconjugants acquired either the IncK or IncI1 plasmids. Concerted efforts from all poultry actors are needed to establish surveillance monitoring strategies to mitigate the spread of ESBL-producing isolates implicated in avian colibacillosis. Full article

The growing and unprecedented water crisis leads to the need to find alternative water resources, and the reuse of treated urban wastewater is an excellent approach. Accordingly, in this work, the disinfection of a secondary effluent (W) discharged from a wastewater treatment plant (WWTP) by hydrogen peroxide combined with radiation (H₂O₂+UV/visible) was studied with the aim of obtaining treated water that can be reused. Firstly, the effect of hydrogen peroxide alone, radiation per se and the combined H₂O₂+UV/Visible process in the inactivation of enterobacteria were assessed. It was found that the oxidant alone is not efficient; the maximum inactivation is achieved when the oxidant and radiation are used simultaneously. For the first time, the effect of some operational parameters, namely the hydrogen peroxide concentration (between 50 and 125 mg/L), initial pH (from 5.0 to 7.0), temperature (between 15 and 25 °C), and radiation intensity (100 to 500 W/m²), on the efficiency of the disinfection process was assessed. When the process was carried out under the best operating conditions found ([H₂O₂] = 75 mg/L, pH = 5.0, T = 25 °C, and UV/visible light with I = 500 W/m²), total enterobacteria and total heterotrophs were inactivated and the abundance of the 16S rRNA, bla_TEM, qnrS, and intl1 genes was reduced. The cultivable microorganisms grew again after 3 days of storing the treated wastewater (TW), making it impossible to reuse such effluent after storage. Therefore, the potential capacity of a diverse bacterial community present in river water to inhibit the regrowth of potentially harmful bacteria present in the urban secondary wastewater after the application of the treatment process was also evaluated. To the authors’ knowledge, this has never been studied before. For this purpose, the TW was diluted with river water (R) at a volumetric percentage of 50/50—sample R+TW. It was found that, after storage, only the total heterotrophs grew, while the abundance of the targeted genes remained practically constant. The R+TW sample after storage met the legal limits for reuse in urban and agricultural applications. The results of this study suggest that the combination of the H₂O₂+UV/visible radiation treatment with dilution of the final treated effluent with natural surface water can contribute to reducing the burden of water scarcity. Full article

Background/Objectives: Infections caused by multidrug-resistant (MDR)bacteria pose a significant public health threat by worsening patient outcomes, contributing to hospital outbreaks, and increasing health and economic burdens. Advanced genomic tools enhance the detection of resistance genes, virulence factors, and high-risk clones, thus improving the management of MDR infections. In the Autonomous Community of Aragon, the diversity and incidence of carbapenemase-producing Enterobacteriaceae (CPE) have increased during the last years. This study analyses CPE trends at a tertiary hospital in Spain from 2021 to 2023, aiming to optimize personalized medicine. Methods: CPE isolates were the first isolate per patient, year, species, and carbapenemase from January 2021 to December 2023. Additional metadata were collected from the laboratory’s information system. Antibiotic susceptibility testing was performed by broth microdilution. Whole-genome sequencing (WGS) was performed using Illumina short reads. De novo assembly was used to generate draft genomes in order to determine their complete taxonomic classification, resistome, plasmidome, sequence type (ST), core–genome multilocus sequence typing (cgMLST), and phylogenetic relationships using a suite of bioinformatics tools and in-house scripts. Results: Between 2021 and 2023, 0.4% out of 38,145 Enterobacteriaceae isolates were CPE. The CPE rate tripled in 2022 and doubled again in 2023. The most common species was Klebsiella pneumoniae (51.8%) and the most common carbapenemase was bla_OXA-48. WGS revealed concordant species identification and the carbapenemase distribution in detail. Resistance rates to critical antibiotics, such as carbapenems, were variable, but in most cases were above 70%. Genetic diversity was observed in WGS and phylogenetic analyses, with plasmids often mediating carbapenemase dissemination. Conclusions: The increasing rate of CPE in healthcare settings highlights a critical public health challenge, with limited treatment options. Genomic characterization is essential to understanding resistance mechanisms, aiding therapy, limiting outbreaks, and improving precision medicine. Full article