Search Results (167)

During the COVID-19 pandemic carbapenem-resistant Acinetobacter baumannii (CRAB) was found to be the major pathogen associated with ventilator-associated pneumonia in mechanically ventilated patients. This prompted us to analyze the post-pandemic mechanisms of carbapenem resistance, antibiotic resistance trends, and molecular epidemiology of CRAB in Croatia. In total, 94 CRAB isolates from two hospital centers, including outpatient settings, were investigated. Antimicrobial susceptibility testing was performed by broth microdilution. PCR was used to detect genes encoding carbapenemases of group A, B and D and extended-spectrum β-lactamases (ESBL). Randomly selected isolates were subjected to whole resistome analysis by Inter-array CarbaResist Kit and whole-genome sequencing (WGS). Phylogenetic tree and sequence types (STs) were retrieved from WGS. Plasmid incompatibility groups were determined by PCR-based replicon typing (PBRT). All isolates were extensively drug resistant (XDR), showing resistance to ceftazidime, cefepime, piperacillin–tazobactam, imipenem, meropenem, gentamicin, amikacin and ciprofloxacin, and 13% (n = 12) were also resistant to colistin. The Hodge and CIM test exhibited poor sensitivity with only 32 and 30% of isolates being identified as carbapenemase producers, respectively. PCR identified bla_OXA-23 as the dominant carbapenemase gene in both hospitals, found in 71% of the isolates (67/94). In an outpatient setting, bla_OXA-24/40 was dominant. bla_OXA-23 and bla_OXA-72 were the only allelic variants. The Inter-array CarbaResist Kit and whole-genome sequencing (WGS) identified a variety of aminoglycoside (armA, ant(3″)-IIa, aph(3″)-Ib, aph(6)-Id) and sulphonamide resistance (sul1 and sul2) genes. The representative bla_OXA-23-positive isolates belonged to ST2, while bla_OXA-72-positive isolates were allocated to ST492. These data show that there are different populations of XDR A. baumannii between hospital and outpatients. Full article

Chronic wounds represent a major complication of underlying conditions such as diabetes mellitus, arterial ischemia, surgical wound and burns. This study aimed at the phenotypic and molecular characterization of antimicrobial resistance for a selection of bacterial isolates, originating from wounds harvested from patients hospitalized in the Vascular Surgery and Plastic Surgery wards. The microbiological diagnosis of wound infections was established according to the laboratory’s working protocol. PCR screening of antibiotic resistance genes was performed using a real-time PCR, while the microtiter plate assay was used to determine the biofilm-forming capacity. Testing of biofilm susceptibility to meropenem and amikacin was performed on Calgary biofilm device. Of the 88 bacterial isolates studied, 78.40% were Gram-negative bacilli (GNB)—Klebsiella pneumoniae (K.P), Pseudomonas aeruginosa (P.A), Proteus mirabilis (P.M), Acinetobacter baumannii (A.B), while the remaining 21.60% were Gram-positive cocci (GPC)—Staphylococcus aureus (S.A). All A.B isolates and 92.59% of K.P were carriers of β-lactamase- and carbapenemase-encoding genes, while 57.89% of S. aureus isolates were carriers of mecA (methicillin-resistant). Strong biofilm-forming isolates (B+++) were more frequent in P.A than in K.P (p = 0.002) and P.M (p = 0.02), with a frequency comparable to that of A.B strains (p = 0.212). When analyzing the biofilm reaction to meropenem, a significantly lower susceptibility was detected in the biofilm for K.P isolates, compared to the planktonic ones. Most GNB have been extensively multidrug-resistant, particularly K.P and A.B. Isolates from chronic wounds are major biofilm-formers. A strong and statistically significant association has been identified in the case of K.P and P.M between the presence of resistance genes and the biofilm-forming capacity. These findings highlight the need for a customized therapeutic approach for each chronic wound, considering the mechanisms underlying treatment resistance. These include bacterial virulence factors and the wound microenvironment colonized by the biofilm and the relative contribution of each to the overall resistance profile. Full article

In 2023, the European Centre for Disease Prevention and Control surveillance report highlighted an increasing number of carbapenem-resistant Escherichia coli isolates carrying the less common bla_NDM-5 variant in Europe. The aim of this study was to investigate the molecular epidemiology of NDM-producing (New Delhi metallo-β-lactamase) E. coli isolates collected in Croatia over a one-year period. A total of 160 carbapenemase-producing E. coli isolates were reported through national surveillance in Croatia between March 2023 and March 2024. Whole-genome sequencing was performed on 22 NDM-producing E. coli isolates. Phylogenetic analysis identified 17 sequence types, indicating high diversity and polyclonal spread. High variability in resistome profiles and co-occurrence of resistance genes across multiple antimicrobial classes indicate multidrug resistance. The predominant bla_NDM variant was bla_NDM-1 (77.27%), followed by bla_NDM-5 (22.73%). Co-occurrence of bla_NDM with extended-spectrum β-lactamase (ESBL) encoding genes was detected in 12/22 isolates (54.55%). Plasmid analysis identified 22 different replicon types, with IncFII (54.54%) and IncA/C2 (45.45%) being the most frequent. Our findings provide insights into the molecular epidemiology of NDM-producing E. coli at the national level, highlighting the presence of the bla_NDM-5 variant. These results emphasize the need for genomic surveillance and strengthened infection control strategies to better understand and limit its spread. Full article

Klebsiella pneumoniae has been associated with reproductive infections in equines. The detection of β-lactam resistance determinants, especially extended-spectrum β-lactamase (ESBL) genes, within genomic regions linked to horizontal gene transfer (HGT), is of a particular concern. In this study, we characterize the whole-genome sequences (WGS) of three K. pneumoniae equine isolates harboring multiple antimicrobial resistance genes. Two isolates were recovered from uterine washes of mares: one with endometritis (YAH-KPEM1) and one clinically normal (YAH-KPSE1), and a third from the feces of a diarrheic foal (YAH-KPF132). WGS was performed using the Illumina MiSeq platform, and the reads were subsequently processed through hybrid assembly in Unicycler v0.5.1. Genome annotation was completed using PROKKA v1.14.5. Strain YAH-KPEM1 was classified as ST127, whereas YAH-KPSE1 and YAH-KPF132 belonged to ST1630 and ST224, respectively. Notably, K. pneumoniae ST1630 and ST224 have not been reported before in equines. All three genomes encoded multiple antimicrobial resistance (AMR) determinants, including two encoding ESBL genes (CTX-M-15), as well as virulence factors and regions associated with HGT. Additionally, two (YAH-KPEM1 and YAH-KPSE1) isolates were found to be multidrug resistant (MDR), harboring an IncFIB(K) plasmid replicon, and another isolate, YAH-KPF132, carried an IncFII replicon. The detection of AMR and virulence genes in equine Klebsiella isolates has important clinical implications for guiding antimicrobial selection and improving treatment success. Full article

Background/Objectives: Plasmids are key vehicles for the dissemination of antimicrobial resistance (AMR), yet their contribution to the global resistome architecture of Escherichia coli remains poorly resolved. This study aimed to quantify how plasmid backbones shape the distribution, mobility, and stabilization of resistance genes across diverse phylogenetic backgrounds. Methods: We analyze 9700 high-quality genomes spanning major phylogroups and sequence types. Plasmidome reconstruction was integrated with lineage-resolved antimicrobial resistance gene (ARG) mapping to characterize plasmid–ARG associations and evolutionary patterns. Results: Although most antimicrobial resistance genes (ARGs) are chromosomal, plasmids disproportionately encode clinically important determinants including bla_NDM-5, mcr-1.1, and multiple bla_CTX-M alleles that show strong, recurrent associations with a restricted set of backbone families, most notably IncX3, IncX4, IncI, and IncF. These conserved plasmid–gene modules recur across phylogenetic backgrounds and continental scales. We identify a marked divergence in evolutionary strategies: generalist phylogroups (A, B1, D) maintain plasmid-rich and highly diverse resistomes, whereas globally dominant Extraintestinal Pathogenic E. coli (ExPEC) clones such as ST131 and ST410 exhibit reduced plasmid dependency and frequent chromosomal integration of extended-spectrum β-lactamase (ESBL) genes, particularly bla_CTX-M-15, consistent with a shift toward vertically stabilized resistomes. By integrating plasmidome reconstruction with lineage-resolved ARG mapping, this study delivers the most extensive plasmid-focused resistome analysis to date, revealing highly modular plasmid–ARG networks structured around a small number of high-risk backbone types. These backbones account for the majority of globally relevant ARGs, including 64.6% of bla_NDM-5 and 76.4% of mcr-1.1 detections. Conclusions: Together, our findings establish plasmid lineages rather than individual genes or clones as central units of AMR dissemination and critical targets for future genomic surveillance and intervention strategies. Full article

Objective: The main aim of the study was to evaluate the role of wild fishes inhabiting in three anthropogenic-impacted Bays in Chile as reservoirs of antimicrobial resistance genes (ARGs). Methods: A total of 245 antimicrobial-resistant isolates were isolated from fish captured in the Coquimbo (142 isolates), Concepción (44 isolates), and Puerto Montt (59 isolates) Bays, and were identified by 16S rRNA gene sequence analysis, Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, and the carriage of genes encoding for resistance to main antimicrobial classes, such as β-lactams, amphenicols, tetracyclines, and sulfonamides by PCR (Polymerase Chain Reaction). Results: A predominance of the Pseudomonas (37.04%), Vibrio (14.40%), and Shewanella (13.99%) genera. Antimicrobial-resistant isolates were tested for susceptibility to 12 antimicrobials by an agar disk diffusion method, showing highest resistance to streptomycin (82.4%), amoxicillin (67.4%), and furazolidone (63.3%), and lowest resistance to ciprofloxacin (3.7%), meropenem (22.5%), and oxytetracycline (29.8%) and exhibiting a high occurrence of the multi-drug resistance phenotype (76.9%). Furthermore, an important number of isolates recovered from sampled fish species carried plasmids (53.5%), floR gene (36.7%), and tet genes (19.2%), whereas the detection of sul genes and class 1-integron was rare. As an overall result, 10.6% of isolates carried at least one bla gene, encoding an extended-spectrum-β-lactamase, with a high predominance of the bla_CTX-M1 gene (23 isolates), whereas 14 out of 245 isolates (5.7%) were positive for the carriage of carbapenemases encoding genes, which both groups exhibited the β-lactam resistance phenotype. Conclusions: The wide distribution of ARG-carrying bacteria in wild fishes from all sampled Bays provides evidence that wild fish are important reservoirs and drivers of spread of ARGs in the marine environment, prompting the need of a continuous surveillance of these genes in wild fishes inhabiting anthropic impacted coastal marine environments in Chile. Full article

Background/objectives: A rise in infections associated with carbapenem-resistant Providencia species (CRPS) has been observed worldwide. This study presents a genomic analysis of CRPS isolates from four hospitals in Croatia and the outpatient setting, in order to determine the extent of the spread of CRPS in Croatia. In the present study, we applied a combination of phenotypic characterization and molecular analysis of resistance traits to determine the mechanisms and the routes of spread of CRPS. Material and methods: The antibiotic susceptibility testing was performed using disk-diffusion and broth dilution methods. The nature of extended-spectrum β-lactamases (ESBLs), carbapenemases, and fluoroquinolone resistance determinants was investigated by polymerase chain reaction (PCR). In order to obtain an insight into the whole resistome, selected isolates were subjected to the Interarray Genotyping Kit CarbaResist and whole genome sequencing (WGS). Results: In total, 30 isolates were collected from four centers, located in different geographic regions of Croatia. There was uniform resistance to piperacillin-tazobactam, cefuroxime, expanded-spectrum cephalosporins (ESCs), imipenem, ertapenem, meropenem, and ciprofloxacin. Immunochromatographic testing and PCR revealed OXA-48 and NDM carbapenemase in 15 isolates, respectively. Phenotypic tests for ESBLs were positive in all OXA-48 and one NDM-positive organism (16 isolates). The isolates were categorized as extensively drug-resistant (XDR). OXA-48-producing isolates were susceptible only to ceftazidime-avibactam, whereas NDM producers were susceptible to cefiderocol and, in the majority of cases, also to amikacin. WGS identified a plethora of genes encoding resistance to aminoglycosides, such as aadA1 and aadA2, (aph(3″)-Ib and aph(6)-Id, sulfonamides sul1 and sul2, trimethoprim dfrA1, dfrA10, and dfrA12, tetracyclines tet(A) and tet(B), and chloramphenicol catA3 and catA5. Conclusions: Providencia spp., in spite of being a rare pathogen, should be included in the surveillance studies across the medical centers in Croatia. Full article

Escherichia coli is an important human pathogen that is able to cause a variety of infections, which can result in diarrhoea, urinary tract infections, sepsis, and even meningitis, depending on the pathotype of the infecting strain. Like many Gram-negative bacteria, E. coli is becoming increasingly resistant to many frontline antibiotics, including third-generation cephalosporins and carbapenems, which are often considered the antibiotics of last resort for these infections. This is particularly the case in Egypt, where multidrug-resistant (MDR) E. coli is highly prevalent. However, in spite of this, few Egyptian MDR E. coli strains have been fully characterised by genome sequencing. Here, we present the genome sequences of ten highly MDR E. coli strains, which were isolated from children who presented with diarrhoea at the Outpatients Clinic of Assiut University Children’s Hospital in Assiut, Egypt. We report that they carry multiple antimicrobial resistance genes, which includes extended spectrum β-lactamase genes, as well as bla_NDM and bla_OXA carbapenemase genes, likely encoded on IncX3 and IncF plasmids. Many of these strains were also found to be high-risk extra-intestinal pathogenic E. coli (ExPEC) clones belonging to sequence types ST167, ST410, and ST617. Thus, their presence in the Egyptian paediatric population is particularly worrying, and this highlights the need for increased surveillance of high-priority pathogens in this part of the world. Full article

Emerging antimicrobial resistance (AMR) in companion animals represents a global health concern as they serve as potential reservoirs for multidrug-resistant (MDR) bacteria, which can be transmitted to humans. Herein, we provide comprehensive surveillance data on resistance patterns in veterinary hospital settings, focusing on urinary tract infection. A total of 23 bacterial strains were isolated from urine specimens of hospitalized companion animals suspected of urinary tract infections (UTIs) between 2022 and 2024. 16S rRNA sequencing analysis revealed that Escherichia coli (47.8%), Klebsiella pneumoniae (21.7%), and Pseudomonas aeruginosa (8.7%) were predominant uropathogens. Minimum inhibitory concentration and minimum bactericidal concentration tests were employed to analyze AMR patterns across different classes of antibiotics. Moreover, antimicrobial susceptibility test exhibited 73.91% MDR according to the standard definition given by the Clinical and Laboratory Standards Institute (CLSI) M100 guidelines. Most Gram-negative bacteria have been shown to be resistant to beta-lactam antibiotics, especially carbapenems. Notably, an E. coli strain was confirmed to possess the bla_NDM-1 gene encoding the carbapenemase New Delhi metallo-β-lactamase. These findings support the implementation of targeted infection control measures and evidence-based treatment protocols to preserve antimicrobial efficacy in companion animal medicine to minimize potential public health risks through the One Health approach. 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: Infections caused by the multidrug-resistant pathogen Mycobacterium abscessus (Mab) are notoriously difficult to treat. The novel β-lactamase inhibitor durlobactam, in combination with β-lactams, shows potent bactericidal activity against Mab, but the potential for acquired resistance remains a clinical concern. Objectives: To identify and characterize mechanisms of acquired resistance to durlobactam in Mab. Methods: In vitro single-step resistance selection was performed by plating wild-type Mab ATCC 19977 and by transcriptional silencing using a CRISPR interference (CRISPRi) system. Minimum inhibitory concentrations (MICs) were determined by both an agar-based method and broth microdilution. Results: Whole-genome sequencing of durlobactam-resistant mutants identified loss-of-function mutations in ponA1, a gene encoding a class A penicillin-binding protein involved in cell wall synthesis. Targeted deletion of ponA1 (ΔponA1) and CRISPRi-mediated knockdown of ponA1 expression both recapitulated the resistance phenotype, resulting in a significant increase in the durlobactam MIC on solid agar media. Strikingly, broth microdilution MICs remained largely unaffected. Conclusions: Inactivation of the peptidoglycan synthase PonA1 is a novel mechanism of resistance to durlobactam in Mab that is phenotypically expressed only during growth on solid surfaces. This finding identifies a specific genetic pathway for resistance and highlights that standard broth-based susceptibility testing could miss clinically relevant resistance mechanisms. Full article

Background: Infections caused by antimicrobial-resistant bacteria are difficult to treat and increase the risk of death in animals. This report describes a fatal case of diarrhea in a horse that, despite intensive treatment including surgery and broad-spectrum antimicrobials (ceftiofur and amikacin), experienced a worsening of its condition and subsequent death. Methods: A fecal swab sample was subjected to microbiological culture for the identification of bacteria and assessment of their phenotypical antimicrobial susceptibility profiles using the disk-diffusion and broth microdilution methods. The double-disk synergy test, polymerase chain reactions for the detection of genes encoding extended-spectrum β-lactamases, and whole-genome sequence-based analysis were also performed. Results: Strains of Salmonella enterica and Escherichia coli were isolated, with the E. coli strain DSL-HVUVV-2025 presenting resistance to a third-generation cephalosporin. Accordingly, the bla_CTX-M-2 gene was identified in the DSL-HVUVV-2025 strain, which was submitted to whole-genome sequencing. Genomic analysis showed several antimicrobial resistance determinants, as well as virulence genes, including those associated with the enteroaggregative pathotype. The bla_CTX-M-2 gene was surrounded by an ISCR1 element and embedded in a complex class 1 integron that is part of the Tn7337 transposon. Strain DSL-HVUVV-2025 belonged to a novel sequence type. Conclusions: This case highlights the importance of monitoring antimicrobial resistance and performing genomic characterization of bacteria involved in equine diarrhea to guide effective clinical management in veterinary hospitals. It also reinforces the role of horses as potential carriers of WHO critical priority pathogens and the need for responsible antimicrobial use. Full article

Wastewater treatment plants (WWTPs) are recognized hotspots for the convergence and dissemination of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) into the environment. Among ARB, carbapenem-resistant Enterobacterales (CR-E) and extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae (ESBL-Ec/Kp) are of particular concern due to their clinical relevance. We characterized 30 CR-E and 176 ESBL-Ec/Kp isolates (two of them were both ESBL-producing and carbapenem-resistant) recovered from influent, intermediate, effluent, sludge, and downstream river samples collected from two WWTPs in northern Spain. Isolates were evaluated for resistance phenotypes against 12 antimicrobials, and β-lactamase-encoding genes were assessed by PCR and sequencing. Notably, among CR-E isolates, bla_KPC-2 was the most prevalent (93%), followed by bla_OXA-48-like, detected in two isolates from non-treated and pasteurized sludge; both isolates also carried bla_CTX-M-15, a finding not previously reported specifically in sludge samples. Among ESBL-Ec/Kp, a broad diversity of ESBL genes was identified, including bla_{CTX-M group 1} (variants 1, 3, 15, 32, 55), bla_{CTX-M group 9} (variants 14, 27, 65, 97), bla_SHV-12 and bla_TEM-169. The most prevalent ESBL gene was bla_CTX-M-15 (48.3%), followed by bla_CTX-M-14, bla_CTX-M-32, and bla_SHV-12, detected in 10.8%, 8.5%, and 6.8% of isolates, respectively. CR-E and ESBL-Ec/Kp were found in all sample types and were still detectable at terminal stages, indicating persistence throughout treatment. These findings support the need to improve and optimize current wastewater treatment methods and underscore the importance of integrating culture-based and molecular methods into routine WWTP monitoring for early detection of microbiological hazards, although further research is still needed. Full article

Background: Antimicrobial resistance (AMR) is a major global health threat, particularly in surgical site infections (SSIs), where multidrug-resistant (MDR) pathogens complicate treatment. Objective: This study aimed to identify antimicrobial resistance genes and assess their prevalence in bacterial species causing SSIs in Lebanon. Materials and Methods: The present research is a multicenter and prospective study that included patients who developed SSIs after surgery in seven hospitals, within the period of January 2024–September 2024. Bacterial isolates from wound swabs or tissue samples were identified using standard microbiological methods. Antimicrobial susceptibility was tested by disk diffusion, and resistance genes were detected by PCR. Data were analyzed using Statistical Package for the Social Sciences (SPSS). Results: Among 6933 surgical patients, 63 developed SSIs (0.91%; 95% CI [0.70–1.15]). Gram-negative bacteria predominated (73%), mainly Escherichia coli and Pseudomonas aeruginosa, while Gram-positive isolates accounted for 27%, mostly Staphylococcus aureus. MDR was observed in 71% of Gram-positive and 61% of Gram-negative isolates. The most frequent genes were mecA in S. aureus (100%) and coagulase-negative staphylococci (83.3%); bla_CTX-M in E. coli, Klebsiella pneumoniae, and Enterobacter cloacae (100%); and bla_NDM in E. cloacae (100%) and Acinetobacter baumannii (60%). bla_KPC was less common, and no isolates carried Imipenemase (IMP), Verona integron-encoded metallo-β-lactamase (VIM), and Oxacillinase-48-like β-lactamase (OXA-48). Conclusions: This study highlights the high prevalence of antibiotic resistance in agents causing SSIs in Lebanese hospitals. Resistance genes, particularly mecA, bla_CTX-M, and blaNDM, were highly prevalent in SSI pathogens, underscoring the urgent need for surveillance and judicious antibiotic use in Lebanese hospitals. Full article

This study investigated the molecular and phenotypic characteristics of antimicrobial resistance in Escherichia coli isolates recovered from the ceca of healthy broilers in Tunisia. A total of 111 E. coli isolates were obtained from chicken samples collected at slaughterhouses and cultured on cefotaxime-supplemented MacConkey agar. All isolates exhibited a multidrug-resistant (MDR) phenotype, and 72.1% were confirmed as extended-spectrum β-lactamase (ESBL) producers. The most frequent β-lactamase gene was bla_CTX-M-G1, followed by bla_TEM and bla_SHV. Carbapenem resistance genes (bla_OXA-48 and bla_IMP) were detected in 12.6% and 6.3% of isolates, respectively, while six isolates harbored the colistin resistance gene mcr-1. Among the tested virulence genes, fimH, traT, and iutA were the most prevalent, detected in over 70% of isolates. Class 1 integrons were present in 83% of isolates, and class 2 integrons in 39.6%, with gene cassettes encoding resistance to trimethoprim (dfrA) and streptomycin (aadA). These findings highlight the widespread presence of MDR and ESBL-producing E. coli strains with virulence traits and integrons in poultry, underscoring the risk of transmission to humans. This study provides essential data supporting the implementation of integrated surveillance strategies in line with the One Health approach. Full article