Search Results (621)

One of the most important adverse consequences of antibiotic use is the development of bacterial resistance. This study investigated the prevalence and antimicrobial resistance patterns of multidrug-resistant (MDR) Escherichia coli in a tertiary care hospital during 2013–2015 and 2024, together with trends in antibiotic consumption and the molecular characteristics of resistance genes in 2024 isolates. Identification and susceptibility testing were performed using the Vitek^® 2 system, antibiotic consumption was assessed according to WHO ATC/DDD methodology, and resistance genes were detected by PCR. No significant differences were observed in the total number of isolates, patients, or the proportion of E. coli isolates during 2013–2015. However, the isolation rate of MDR E. coli significantly declined from 36.9% to 30%. Total antibiotic consumption remained stable, with no correlation between consumption and MDR isolation rates, although ampicillin resistance increased significantly. In 2024, MDR E. coli accounted for 24.3% of isolates despite lower antibiotic consumption. Compared with 2015, resistance significantly increased to amoxicillin–clavulanic acid, cefotaxime, cefepime, ceftazidime, ciprofloxacin, and levofloxacin, while ceftriaxone resistance decreased. The blaCTX-M gene was detected in 53% of bloodstream isolates, indicating widespread dissemination of ESBL-producing E. coli and highlighting the need for continuous surveillance and antimicrobial stewardship. Full article

Background/Objectives: Hospital water and wastewater environments are potential reservoirs for antimicrobial resistance genes (ARGs), particularly in high-risk clinical units. This study examined site-specific ARG DNA detection patterns in wet and waste-associated sites of a haematology ward and an intensive care unit (ICU) to identify environmental hotspots that may inform targeted infection prevention and control (IPC) interventions. Methods: A descriptive point-prevalence environmental study was conducted in May 2025 in a tertiary acute-care hospital. Samples were collected after routine cleaning and during usual clinical activity. Real-time PCR assays were used to detect bla_TEM, bla_SHV, bla_CTX-M, bla_OXA-48-like, bla_VIM, bla_NDM, bla_IMP, bla_KPC, and mcr-1. Analytical summaries included samples meeting predefined quality-control criteria. Ct signals were categorised descriptively; Ct values ≤ 40 were considered positive, with Ct values 36–40 classified as very low molecular signals, whereas Ct values > 40 were considered negative. Results: The analytical dataset comprised 60 samples: 31 from haematology and 29 from ICU. At least one included ARG signal was detected in 40/60 samples (66.7%), including 23/31 haematology samples (74.2%) and 17/29 ICU samples (58.6%), bla_TEM (51.7%) and bla_SHV (43.3%) were most frequent. Sink drains showed the broadest multi-gene profiles, especially combinations of ESBL- and carbapenemase-associated genes. Haematology showed broader carbapenemase-associated diversity, whereas ICU detections clustered in patient-room sink drains and sluice-room wastewater infrastructure. Conclusions: ARG DNA signals were unevenly distributed, with sink drains representing most frequently positive sampling sites for multi-gene detection. This molecular, single-timepoint environmental study does not demonstrate viable organisms, colonisation, or transmission; however, point-prevalence environmental assessments may identify wet and wastewater-associated hotspots that can inform targeted IPC interventions against the spread of multidrug-resistant microorganisms. Full article

Background/Objectives: The emergence of plasmid-mediated mcr genes has enabled horizontal dissemination of resistance to colistin, a last-resort antibiotic against multidrug-resistant Enterobacterales. In Tunisia, genomic data on mcr-positive Escherichia coli are still limited. This study reports the genomic characterization of human clinical mcr-positive E. coli isolates from the Military Hospital of Tunis. Methods: Between August 2023 and March 2025, seven E. coli isolates with low-level colistin-resistance (MIC = 4–8 µg/mL) were collected from six patients. They were characterized by antibiotic susceptibility testing and WGS to determine resistome, MLST, genetic relatedness, and plasmid content. Results: The E. coli isolates belonged to diverse sequence types (STs), except for two isolates collected from the same patient 2.5 months apart, which were highly related. Overall, this pattern is consistent with a polyclonal spread. The mcr-1.1 gene was located on IncI2 (n = 5) or IncX4 (n = 2) plasmids, which exhibited high similarity both among themselves and in comparison with plasmids previously reported in human and livestock isolates. Most isolates were multidrug-resistant, harboring acquired resistance genes to multiple antibiotic classes, and chromosomal mutations conferring fluoroquinolone resistance. Three isolates additionally carried chromosomal insertions of the bla_CTX-M-55 gene. Resistance to cefiderocol was observed in one isolate and was associated with CirA and Fiu truncation. Conclusions: These findings highlight ongoing dissemination of mcr-1.1-positive E. coli isolates in Tunisia, primarily driven by plasmid transfer. Continuous genomic surveillance and One Health-oriented antibiotic stewardship are essential to limit the spread of colistin-resistance and the emergence of resistance to newer agents such as cefiderocol. Full article

The present study was performed to determine the antibiotic resistance genes (ARGs), virulence determinants, and mobile genetic elements in multidrug-resistant Escherichia coli CUK-76 isolated from wastewater in Himachal Pradesh, India. Whole genome sequencing was performed using the Illumina Miseq system, and the draft genome sequence was assembled by Unicycler v0.5.1 and annotated by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP v6.10). The bioinformatics-based prediction analysis was performed using ResFinder v4.7.2 and CARD v4.0.1 (antibiotic resistance genes), VirulenceFinder v2.0, VFDB and MGEFinder v1.0.3 (virulence determinants), PlasmidFinder v2.0.1 (plasmid sequences), MLST v2.0 (sequence type), ISFinder and TnCentral v2.0 (insertion sequences and transposons), PathogenFinder2 v0.6.0 (pathogenicity), RAST (subsystems category) and Phigaro (prophage sequences). The draft genome of E. coli CUK-76 strain comprised 4,607,136 bp with a GC content of 51%. Genome annotation revealed 4498 genes of which 4289 were protein-coding genes, 78 RNA genes, and 131 pseudogenes. It was related to sequence type ST949 and its predicted resistome consisted of bla_CTX-M-15, bla_TEM-1B (class A β-lactamase genes), bla_EC-14 (class C β-lactamase gene), aph(6)-Id, aph(3″)-Ib, qnrS1, sul2, tet(A), and dfrA14 genes. Additionally, multiple virulence genes, two plasmid sequences viz. IncFIB(K) and IncFIB(AP001918), insertion sequences, transposons and prophage sequences were detected. The genomic dataset of this strain will be a valuable resource for comparative genomic studies on E. coli. Full article

Antimicrobial resistance (AMR) is a major One Health challenge driven by antimicrobial misuse in human medicine, veterinary practice, animal production, and polluted environments. Migratory birds move among wetlands, farms, wastewater-affected habitats, landfills, and coastal ecosystems and may acquire and redistribute antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) across ecological and political boundaries. This perspective synthesizes evidence on exposure sources, bacterial reservoirs, resistance determinants, cross-species interfaces, and surveillance priorities while explicitly distinguishing four claims: detection or carriage, persistence in individual birds, redistribution along migratory routes, and onward transmission to recipient hosts or environments. Published studies report multidrug-resistant Escherichia coli, Klebsiella pneumoniae, Salmonella spp., Enterococcus spp., and Campylobacter spp., with determinants including blaCTX-M, blaTEM, blaNDM, mcr, tet, sul, and qnr genes. The eight evidence groups summarized here constitute an illustrative, non-comprehensive selection; they are predominantly observational surveys or screenings, and none reconstructs a complete source–bird–destination–recipient transmission chain. Taxon-specific ecology modifies exposure: gulls and storks frequently exploit refuse and wastewater, waterfowl and shorebirds connect aquatic habitats, whereas passerines often reflect more local point-source contamination. Current evidence therefore supports migratory birds primarily as mobile sentinels and opportunistic carriers of anthropogenic AMR, while acknowledging possible natural or ancestral resistance in avian-associated microbiota. Future surveillance should combine longitudinal sampling, baseline cohorts such as pre-migratory nestlings, paired bird–water–soil–sediment sampling, whole-genome sequencing, plasmid profiling, telemetry, environmental DNA, wastewater-based epidemiology, and interoperable veterinary reporting. Practical mitigation requires antimicrobial stewardship, wastewater and landfill control, farm biosecurity, and coordinated veterinary, environmental, and public-health action. Full article

Extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) is a priority antimicrobial-resistant pathogen with the capacity to persist and disseminate across environmental and food animal production systems. This study conducted genomic surveillance of ESBL-K. pneumoniae recovered from municipal wastewater treatment plants and food animal production environments in Fayetteville, North Carolina. A total of 449 wastewater and livestock farm environment samples were analyzed. K. pneumoniae was recovered from 162 (36.1%) samples, including 68 (15.1%) ESBL-producing isolates. Antimicrobial susceptibility testing showed that 77.9% of ESBL-producing isolates were multidrug-resistant. Polymerase chain reaction identified bla_CTX-M-1, bla_SHV, and bla_ACT as the predominant resistance genes. Whole-genome sequencing of selected isolates identified diverse antimicrobial resistance determinants, virulence-associated genes, and plasmid replicons, with IncFIB(K) being the predominant plasmid type. Phylogenetic analysis demonstrated close genetic relatedness between wastewater and farm environment isolates, with some differing by only 0–6 single-nucleotide polymorphisms, suggesting possible dissemination between environmental reservoirs. These findings highlight municipal wastewater and food animal production environments as important reservoirs of ESBL-K. pneumoniae and reinforce the value of integrated genomic surveillance within a One Health framework to monitor the emergence and spread of antimicrobial resistance. Full article

Background: Aztreonam–avibactam (ATM-AVI) is an important therapeutic option for infections caused by metallo-β-lactamase-producing Enterobacterales. However, the emergence of resistance may limit its clinical effectiveness. Here, we report two high-level ATM-AVI-resistant Escherichia coli isolates recovered from critically ill patients in a tertiary hospital in Guangzhou, both of whom had prior exposure to broad-spectrum antimicrobial agents. Methods: Antimicrobial susceptibility testing was performed by broth microdilution. Whole-genome sequencing (WGS) was conducted using the Illumina NovaSeq and Oxford Nanopore Technologies platforms for hybrid assembly. Resistome analysis, multilocus sequence typing (MLST), plasmid replicon typing, and phylogenomic analysis were performed. Comparative genomics with global ST410 isolates was used to investigate the evolutionary origin. Results: Antimicrobial susceptibility testing revealed high-level resistance to ATM-AVI. Hybrid whole-genome sequencing showed that both isolates belonged to the globally disseminated high-risk ST410 lineage and carried bla_NDM-5, CTX-M-type extended-spectrum β-lactamase genes, and an identical four-amino-acid insertion in penicillin-binding protein 3 (ftsI_I334IYRIK). Both isolates exhibited highly conserved chromosomal backbones, differed by only 29 core-genome single-nucleotide polymorphisms, and clustered within a China-associated ST410 clade. Notably, their key resistance determinants were located on distinct plasmid backgrounds: bla_NDM-5 was found on a predicted conjugative IncFIB/IncFIC plasmid in one isolate and on an IncX1 element with potential mobilization in the other. Conclusions: These findings provide important clinical evidence of high-level ATM-AVI resistance in Escherichia coli ST410 isolates in China and highlight the emergence of ATM-AVI resistance-associated determinants within a high-risk genomic background. Active phenotypic and genomic surveillance is warranted as ATM-AVI enters broader clinical use. Full article

Background: Antimicrobial resistance mediated by ESBL-producing Klebsiella pneumoniae is an emerging concern in both human and veterinary medicine, with companion animals increasingly considered relevant within the One Health framework. This study aimed to investigate the fecal carriage of ESBL-producing K. pneumoniae complex in cats from Italy and to characterize the strains by the phenotypic and genetic profile of ESBL production, virulent pathotypes, antibiotic resistance profile and biofilm production. Methods: Fecal samples collected from cats admitted to the Veterinary Teaching Hospital of Milan (Italy) in 2020–2026 were bacteriologically and genetically analyzed. Results: All the Klebsiella pneumoniae strains isolated [4/200 (2%, 95% CI: 0.06–3.94%)] were ESBL-producing K. pneumoniae complex isolates harboring bla_CTX-M-15, bla_SHV, and bla_TEM genes. The isolates were detected with higher presence in cats with diarrhea and were found only in cats treated with antibiotics and hospitalized. All four ESBL-producing isolates were classified as the classical K. pneumoniae pathotype based on the negative string test results, the absence of reliable virulence genes used for pathotype identification (peg-344, iucA, rmpA and rmpA2), and the lack of K1 and K2 serotypes, despite the detection of terB and irp2 virulence genes in one and two isolates, respectively. All four ESBL-producing K. pneumoniae complexes were classified as multidrug-resistant, with resistance mainly observed to β-lactams, fluoroquinolones, quinolones and folate antagonists. All four ESBL-producing K. pneumoniae complexes demonstrated biofilm-forming abilities, with two isolates showing weak adhesion, one moderate adhesion, and one strong adhesion. Conclusions: The detection of ESBL genes together with the MDR pattern, biofilm-forming capacity and selected virulence determinants suggests the potential epidemiological relevance of cats in the dissemination of antimicrobial-resistant K. pneumoniae complexes, underscoring the need for strengthened surveillance and prevention strategies in veterinary settings to provide information to pet cat owners and children who may interact with stray cats, in full implementation of the One Health approach. Full article

The spread of antimicrobial-resistant bacteria in aquatic environments is a public health concern, especially in areas with anthropogenic activity. Hydroelectric reservoirs may act as interfaces facilitating the dissemination of resistance. Escherichia coli is used as an indicator of environmental contamination and antimicrobial resistance. To investigate the occurrence of E. coli in reservoirs of the Urubupungá Hydroelectric Complex (Paraná River basin, Brazil) and to characterize antimicrobial resistance profiles and ESBL/AmpC-related genes. Methods: Water samples were collected during rainy and dry seasons (2022–2024). A total of 200 E. coli isolates were analyzed by broth microdilution (EUCAST). ESBL and AmpC production were phenotypically assessed, and resistance genes (blaCTX-M, blaTEM, blaSHV, and ampC) were detected by PCR. 26% of isolates showed resistance to at least one antimicrobial, mainly tetracycline (18%), amoxicillin (15%), and ampicillin (15%). All isolates were susceptible to imipenem, meropenem, and norfloxacin. Among the investigated resistance markers, ampC was the most frequently detected gene, whereas blaCTX-M was the predominant ESBL-associated determinant. Significant associations were observed between blaCTX-M and resistance to cefotaxime, ceftazidime, and cefepime, whereas the presence of ampC was significantly associated with cefoxitin resistance. Significant associations were observed between β-lactam resistance and ESBL genes. Hydroelectric reservoirs under anthropogenic influence may serve as environmental reservoirs of antimicrobial-resistant E. coli, particularly ESBL producers. These findings highlight the role of aquatic environments in the dissemination of clinically relevant resistance genes. Full article

Enterobacteriaceae are a diverse family of Gram-negative, rod-shaped, facultatively anaerobic bacteria that serve as important indicators of food safety. Many species within this family, including common foodborne pathogens, are major contributors to both community- and hospital-acquired infections. The widespread use of antibiotics in agriculture has facilitated the emergence and dissemination of antimicrobial-resistant bacteria in food products, livestock, and the environment. This study investigated the prevalence and genomic characteristics of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolated from food sources in Saudi Arabia. A total of 160 isolates preserved in the Saudi Food and Drug Authority (SFDA) biobank were screened for antimicrobial resistance using indicator β-lactam antibiotics according to CLSI guidelines. Phenotypic screening indicated that Escherichia coli O145, E. coli O157, Klebsiella pneumoniae, and Cronobacter sakazakii isolates were not ESBL producers. In contrast, ESBL production was detected among Salmonella spp. isolates, and 13 confirmed ESBL-producing Salmonella isolates were subsequently subjected to whole-genome sequencing (WGS). Genomic analysis identified the ESBL gene blaCTX-M-65, along with multiple antimicrobial resistance determinants, virulence-associated genes, and plasmid replicons, predominantly belonging to the IncFIB and IncX families. These findings provide important insights into the occurrence of ESBL-producing Enterobacteriaceae in food sources and highlight the value of integrating phenotypic and genomic approaches for antimicrobial resistance surveillance within food safety systems. Full article

Extraintestinal pathogenic Escherichia coli (ExPEC) causes severe infections in humans and animals, yet bovine isolates remain poorly characterized. Here, we report the first complete genome of a bovine ExPEC strain, BN01 (serotype O101:H9-ST10-phylogroup A), isolated from calf meningoencephalitis. Unlike classical ExPEC that typically belong to B2/D phylogroups and O1/O2/O18 serotypes, BN01 represents the A-ST10-O101 sublineage that has emerged as predominant among bovine ExPEC populations. The genome comprises a chromosome encoding 197 virulence factors, with cdiA uniquely identified in BN01 compared to six other representative ExPEC genomes—a contact-dependent growth inhibition system, and three distinct plasmids: a conjugative ESBL carrier (blaCTX-M-164, IncI1), a bovine-associated multidrug resistance island (IncY), and a mobilization-ready vector (IncFII). Animal virulence assays demonstrated that intraperitoneal challenge with E. coli BN01 caused 80% mortality (8/10 mice) within the observation period and yielded an LD₅₀ of 10^6.3 CFU/mouse. These findings demonstrate that high-risk ExPEC can occur in phylogroups typically associated with commensal strains, expanding the conventional understanding of phylogroup–virulence associations. They highlight the need to assess the zoonotic potential of livestock-associated atypical lineages and support integrated genomic surveillance under the One Health framework. Full article

Urinary tract infections are among the most common bacterial infections encountered in clinical practice, with Escherichia coli representing the dominant urinary pathogen. Increasing detection of multidrug-resistant and extended-spectrum beta-lactamase (ESBL)-producing uropathogens has narrowed empirical treatment options and renewed interest in fosfomycin. However, local long-term surveillance data on fosfomycin susceptibility remain limited in Pakistan. This study evaluated temporal changes in major urinary isolate categories and fosfomycin susceptibility patterns within a diagnostic laboratory network in Pakistan from 2013 to 2025. An exploratory molecular sub-analysis was also performed to assess selected resistance-associated transcript patterns in archived fosfomycin-susceptible and fosfomycin-resistant isolates. A retrospective laboratory-based, isolate-level analysis was conducted using anonymized urine culture records. The source database included 34,230 urine sample records, from which eligible culture-positive urinary isolates with required organism classification and fosfomycin susceptibility data were included in the final analytical dataset. Analyses were performed across predefined mutually exclusive study intervals. Organism categories included non-ESBL E. coli, ESBL-producing E. coli, laboratory-coded ESBL E. coli 24 variant, Klebsiella spp., and Enterococcus spp. The ESBL E. coli 24 variant was treated as a laboratory reporting category, not as a genomically confirmed clone or sequence type. Fosfomycin resistance was evaluated using interval-based comparisons and odds ratios. A selected subset of 24 archived isolates, including fosfomycin-susceptible and fosfomycin-resistant E. coli and Klebsiella pneumoniae, was analyzed by RT-qPCR for glpT, uhpT, murA, fosA, fosA3, and blaCTX-M transcript abundance. The final isolate-level analytical dataset included 17,978 eligible urinary isolates. Among urine records with available sex data, female-associated records represented the majority throughout the study period, but this finding reflects laboratory record distribution rather than patient-level UTI prevalence. E. coli remained the predominant urinary isolate category. Non-ESBL E. coli declined across study intervals, whereas ESBL-associated E. coli categories represented a larger proportion of isolates in later years. The laboratory-coded ESBL E. coli 24 variant increased in later intervals, although this finding requires cautious interpretation because confirmatory molecular typing was not performed. Fosfomycin resistance showed a non-linear temporal pattern: resistance decreased from the early to the middle interval and then increased markedly to 23.8% during 2021–2025, while susceptibility declined to 60.6% in the same interval. Compared with the middle interval, isolates from 2021–2025 had higher odds of fosfomycin resistance (OR = 3.64, 95% CI: 3.23–4.12; p < 0.001). In the exploratory molecular subset, resistant isolates showed lower transcript abundance of selected uptake-associated genes, particularly glpT and uhpT, and higher expression of selected fosfomycin- and ESBL-associated genes, including fosA, fosA3, and blaCTX-M. These findings represent transcriptional associations in selected isolates and do not establish definitive resistance mechanisms. Urinary isolates in this diagnostic-network dataset showed a temporal shift toward greater representation of laboratory-reported ESBL-associated E. coli categories and a marked increase in fosfomycin resistance during 2021–2025. The findings support continued local surveillance of urinary pathogens and periodic reassessment of fosfomycin susceptibility for antimicrobial-stewardship guidance. The molecular findings should be interpreted as exploratory transcriptional observations because they were based on a small selected isolate subset and were not supported by genomic, mutational, uptake, or functional validation. Full article

Multidrug-resistant (MDR) Klebsiella pneumoniae is a critical cause of nosocomial infections associated with high mortality. Of particular concern is the genomic convergence of multidrug resistance and hypervirulence (MDR-hvKP), which represents a severe public health threat. This study characterised the resistome, virulome, and population structure of seven clinical MDR K. pneumoniae isolates collected between 2024 and 2025 across four Peruvian regions (Moquegua, Ucayali, Loreto, and Lima) using whole-genome sequencing (WGS). Genomic analysis identified six distinct sequence types, with the high-risk clone ST307 being the most prevalent (2/7 isolates), consistently associated with the KL102 capsular locus. Remarkably, one isolate from Loreto (oph_54) exhibited a convergent MDR-hvKP genomic profile (ST218, KL57, virulence score 4), carrying yersiniabactin, salmochelin (iro), and aerobactin (iuc) loci alongside multiple resistance determinants. The isolates presented a robust resistome dominated by the extended-spectrum beta-lactamase gene bla_CTX-M-15 (5/7) and bla_OXA-1, coupled with aac(6′)-Ib-cr and fosA6 genes. Plasmid analysis revealed a predominance of IncFIB(K) replicons. Although exploratory, these findings demonstrate the regional presence of high-risk ST307 lineages and the emergence of convergent MDR-hvKP genomic profiles within the analysed Peruvian hospitals. Immediate and continuous genomic surveillance is urgently required to monitor the potential spread of these highly dangerous, MDR bacterial lineages within these healthcare environments. Full article

Diarrheal disease remains a significant public health concern in Bhutan; however, the genomic epidemiology of the circulating diarrhoeagenic Escherichia coli (DEC) strain remains poorly understood. This study characterized the genomic diversity, antimicrobial resistance (AMR), and virulence determinants of DEC isolates using whole-genome sequencing (WGS). DEC isolates recovered from stool samples and collected through Bhutan’s National Diarrheal Disease Surveillance sentinel hospitals during 2023 were identified by a multiplex polymerase chain reaction, tested for antimicrobial susceptibility using the Kirby–Bauer disc diffusion method, and sequenced on the Illumina MiSeq platform. Genomes were analyzed using the Bohra pipeline to determine pathotypes, phylogeny, multilocus sequence types, serotypes, virulence factors, and AMR genes. Of the 29 DEC isolates, 27 were confirmed by WGS and enteropathogenic E. coli (37.0%) and enteroaggregative E. coli (33.3%) were the predominant pathotypes. Isolates exhibited extensive genetic diversity, representing phylogroups A and B1 and 22 serotypes. Phenotypic resistance to β-lactams was common, with 25.9% of isolates carrying bla_CTX-M-15. Virulence profiling identified diverse adhesins, toxins, iron acquisition systems, and type III secretion system components. DEC isolates in Bhutan comprise a genetically diverse population with a concerning convergence of virulence determinants and multidrug resistance. The findings underscore the strengthening of sustained genomic surveillance to monitor AMR and genomic epidemiology of bacterial pathogens. Full article

This study presents the first comprehensive molecular characterization of Escherichia coli producing extended-spectrum beta-lactamases (ESBL-Ec) in surface waters in Albania, focusing on the Shkumbini river. Antimicrobial resistance (AMR) in aquatic ecosystems poses a significant threat to public health, yet data from Albania remain scarce. Thirty water samples were collected from six locations near Elbasan between September 2022 and February 2024. Following the WHO Tricycle protocol, 52 ESBL-Ec isolates were recovered and characterized for antimicrobial susceptibility, biofilm formation, resistance genotypes and clonal relatedness via pulsed-field gel electrophoresis (PFGE). ESBL-Ec was detected in 80% of the samples analyzed, with 94.2% of the isolates classified as multidrug-resistant (MDR). High resistance frequencies were observed for ampicillin (98.1%) and cefotaxime (86.5%), while 7.7% of the isolates displayed colistin resistance associated with the mcr-3 gene. The bla_CTX-M-1 genotype was the most prevalent (57.7%), and almost half of the isolates harbored multiple ESBL genes. Phylogroup A (46.2%) predominated, followed by the high-risk extraintestinal lineages B2 (23.1%) and D (11.5%). PFGE revealed high genetic heterogeneity, with 51 distinct pulsotypes indicating multiple sources of contamination, such as untreated municipal, agricultural and industrial waste. Additionally, 55.8% of the isolates were capable of forming biofilms. These results highlight the critical role of the Shkumbini river as a reservoir for highly resistant pathogens and emphasize the urgent need for integrated environmental surveillance and improved wastewater management in Albania. Full article