cirA Promoter Disruption and SHV-12 Production Contribute to Cefiderocol Resistance in Carbapenem-Resistant Klebsiella pneumoniae.

Susceptibilities of cefiderocol, meropenem-xeruborbactam, cefepime-taniborbactam, aztreonam-avibactam, and sulbactam-durlobactam against imipenem-non-susceptible Gram-negative bacilli in Taiwan.

Antibiotic resistance, largely driven by plasmid-mediated genes, is a growing public-health threat, yet the presence of resistant bacteria in herbal cosmetic powders remains underexplored, creating a critical research gap in the safety of widely used natural products. This study aimed to isolate Escherichia coli from commonly used herbal cosmetic powders and evaluate the effectiveness of plasmid curing using sodium dodecyl sulfate (SDS) and heat-shock treatment. E. coli strains were isolated from Multani Mitti, Avaram Poo, Rose Powder, and Kasturi Manjal, confirmed through standard biochemical and Gram-staining techniques, and assessed for antibiotic resistance. Resistant isolates were subjected to plasmid curing, and plasmid loss was analyzed by alkaline lysis and agarose gel electrophoresis, followed by post-curing antibiotic susceptibility testing. The key findings revealed that several isolates carried plasmids associated with resistance, and SDS- and heat-shock-based curing resulted in partial or complete loss of plasmids, restoring antibiotic sensitivity in treated strains. These results demonstrate that herbal cosmetic powders can serve as overlooked reservoirs of plasmid-mediated antibiotic-resistant E. coli, and effective plasmid curing highlights the direct link between plasmid presence and drug resistance. The study emphasizes the need for stricter microbial quality control in herbal cosmetic manufacturing and provides baseline evidence that can guide regulatory policies and future antimicrobial resistance research.

Yersinia pestis caused the three plague pandemics that claimed more than two hundred million human lives. There is still no vaccine that meets all WHO requirements, and many researchers continue to develop plague vaccines using various technological platforms. For example, researchers led by Roy Curtiss 3rd have developed a new approach to achieve controlled, delayed attenuation of bacterial pathogens. Mutants generated using this method were superior in protecting Y. pestis-infected mice immunized with strains generated using traditional gene knockout. However, further studies are needed to determine the safety and efficacy of these delayed-attenuated strains in other mammalian species in order to extrapolate on humans the data obtained in accordance with the FDA Animal Rule. Three Y. pestis strains, a Δcrp mutant, a mutant with arabinose-dependent regulated crp expression (araC PBAD crp) or an araC PBAD crp mutant cured of plasmid pPst were derived from virulent wild-type strain 231. To evaluate the safety, outbred mice or guinea pigs were immunized subcutaneously with serial tenfold dilutions of mutated strains. For vaccine studies, immunized animals were subcutaneously challenged with 200 LD100 (lethal dose in all exposed subjects) of the wild-type Y. pestis strain. The challenge caused the death of 100% of naïve animals in controls. The Y. pestis strain 231Δcrp was nonlethal in mice at a dose of 107 CFs. The LD50 of the 231Δcrp strain in guinea pigs increased by at least 107-fold compared to that of the wild-type strain. The LD50s of the 231PBAD-crp mutant in mice and guinea pigs were approximately 104-fold and 107-fold higher than those of Y. pestis 231, respectively. The 231PBAD-crp(pPst¯) strain did not cause death in mice (LD50 > 107 CFU) and guinea pigs (LD50 > 109 CFU) when administered subcutaneously and was capable of inducing intense protective immunity in both species of laboratory animals. Our research has shown once again the necessity of balance between safety and effectiveness demonstrating the feasibility of further investigation of crp mutants as promising candidate plague vaccines.

Isolation, Characterization, Partial Purification and Plasmid Curing Analysis of Bacteriocin of Pediococcus acidilactici KX688798 Isolated from Fermented Vigna radiate

Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO2 as their sole carbon source with H2. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14-100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.

Antimicrobial resistance (AMR) poses a growing threat to global public health, progressively compromising the efficacy of available antimicrobials. Technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as promising tools for controlling resistant pathogens, offering high specificity and versatility. However, a comprehensive and systematic synthesis of CRISPR strategies applied to AMR remains limited. From February 12, 2025, we conducted a systematic review of the PubMed, Embase, and Scopus databases, using the following search strategy: Population (resistant bacteria or plasmid-mediated resistance), Intervention (CRISPR, including variants such as CRISPR-Cas9, Cas3, Cas12, Cas13, and CRISPR interference [CRISPRi]), and Outcomes (bacterial resensitization or plasmid curing). The CRISPR-Cas9 system was the most frequently employed (75.7%), with conjugation identified as the primary delivery method. We identified the advantages and limitations of each system, highlighting CRISPRi and CRISPR-Cas13a as alternatives to overcome the constraints of direct genome editing. Delivery efficiency remains a central challenge, although nanocarrier- and bacteriophage-based methodologies show promising potential. We also propose a decision map that guides the selection of the most appropriate CRISPR-Cas system and delivery strategy, considering factors such as therapeutic objective, gene location, methodology efficiency, application environment, and clinical feasibility. This review provides an updated and structured synthesis of CRISPR strategies applied to AMR, emphasizing their potential translational and clinical applications. The findings can inform the development of CRISPR-based therapeutics, guide the design of preclinical studies, and support future strategies for combating multidrug-resistant infections in clinical settings.

ZnSe nanoparticles (NPs) are employed in multiple fields, including environmental, electronic, and materials science. However, their biological features have not been extensively studied. Thus, this study focused on the biosynthesis of ZnSe nanoparticles using the aqueous extract of Stachys lavandulifolia as the unique origin. Moreover, the biogenic ZnSe NPs were examined using UV–Vis, FTIR, XRD, SEM-EDX, and TEM techniques. The bioactivity of ZnSe NPs was evaluated using various trials. The ZnSe NPs showed a distinct absorbance peak in the UV–visible spectrum at 304 nm. The FTIR revealed the potential functional groups linked to biomolecules participating in the synthesis of ZnSe NPs. SEM and TEM showed that the ZnSe NPs were spherical, with a size range of 10–30 nm. XRD revealed that the crystallite size of the NPs was 15.2 nm, and EDX indicated that their elemental compositions included selenium and zinc in a 40.9:37.0 ratio. The DPPH scavenging potential of ZnSe NPs and plant extract was found to have IC50 values of 16.8 and 35.7 mg/mL, respectively. Antibacterial and antibiofilm activity against three MDR pathogens exhibited two-fold potency against P. aeruginosa (NDM-1) compared to K. pneumonia (blakpc) and S. aureus (MRSA). Additionally, plasmid curing tests established resistance disruption in P. aeruginosa (hospital isolate) and K. pneumonia (blakpc). Cytotoxicity tests on the KB cancer and HF normal cell lines, based on IC50 values, showed that ZnSe NPs were equivalent to 23.43 µg/mL and 30.15 µg/mL, respectively. To conclude, the eco-friendly synthesis of ZnSe NPs can meet the essential requirements for therapeutic and preventive uses.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19525-2.

Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.

A community of bacteria that are permanently adhered to a surface and covered with extracellular polymeric substances (EPS) and have heightened resistance to host cellular and chemical reactions is referred to as a biofilm in microbiology. One or consortium of microorganisms affixed to various surfaces make up biofilms. This study analyzed the biochemical and molecular characterization of biofilm producing Escherichia coli isolated from environmental specimens within Ekpoma and its environs to separate the ambient E. coli specimens, identify the biofilm-forming capacity or capacities, and show the antibiotic resistance and biofilm-forming genes. In ambient samples, Escherichia coli was recovered with a prevalence of 112. Biofilm generation was demonstrated using two techniques: the microtitre plate method and the Congo red agar (CRA) method. This resulted in a 90% re-producibility. There were a lot of weak formers (41.1%) among the environmental isolates (20.5%). The results of the statistical analysis indicated that there was no significant difference between the two approaches, with the p-value being 0.167369 and p≥0.05. Prior to biofilm formation, antibiotic resistance was modest (65.2%), but it increased after biofilm formation. Plasmid-encoded fimbriae H (fim h) and aggregation genes [(agg)3IV] for biofilm formation for strong, moderate, and weak formers alone from both isolate sources were constituted by molecular characterization test. Multidrug resistance of routinely used antibiotics in their typical routine dosages was made possible by the inclusion of plasmids for biofilm development. The existence of antibiotic resistance genes for aminoglycosides and quinolones served as additional evidence of this. These can be investigated further with the goal of preventing the formation of biofilm by infection-associated cells. Acrydine orange (AO) dye at a concentration of 75 ul/g was used to evaluate biofilm-forming isolates for plasmid curing. The findings revealed a decrease in antibiotic resistance from 87.5% to 6.3% and biofilm development from roughly 90% to 9.9%. Plasmid-mediated antibiotic resistance is a major public health concern and a serious threat to chemotherapy and medicine worldwide.

Plasmid-encoded antibiotic resistance transcends bacterial genus and species, as plasmid-encoded resistance has been observed clinically for various Gram-negative and Gram-positive pathogenic bacteria. Plasmid curing is the treatment of cells with a substance that interferes with plasmid replication. The present study was to investigate the plasmid curing activities of natural compounds over multidrug-resistant bacteria. E. coli strains known to have pUC19 and F plasmid were selected to assess the plasmid-curing efficacy of the compounds. The plasmid curing was done by overnight incubating the culture with the test compounds. Chloroform extracts of Tridax procumbens (Dagadi pala) and ethanol extract of Vitex negundo (Nirgudi) showed promising curing activity. Plasmid isolation and the replica plate method confirmed the elimination of the plasmid. Eliminating the plasmid using the natural compound makes it vulnerable to any antibiotic without drug resistance.

Asymptomatic bacteriuria (ASB) is the presence of actively multiplying bacteria in urine of any individual without any visible signs or symptoms of urinary tract infection (UTI) such as painful urination, frequent urination, fever, abdominal pain, cloudy or strong smelling urine. In this research on antimicrobial activities of Andrographis paniculata against profiled multi drug resistant clinical isolates from asymptomatic bacteriuria, seventy (70) clinical isolates collected from apparently healthy female students of Renaissance University Enugu were used. The isolates were identified and biochemical tests were carried out according to standard methods. The antibiotic susceptibility tests of the isolates was done using the single disk diffusion method according Kirby-Bauer. The leaves of Andrographis paniculata were extracted by cold maceration using methanol as the solvent and their phytochemical analysis done. The antimicrobial susceptibility pattern of the extract against the clinical isolates were determined using the agar disk diffusion technique. The minimum inhibitory concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were done using standard method. The plasmid profiling of the 16 multidrug isolates that were resistant to the extract and ciprofloxacin (positive control) was done using standard method. Furthermore, plasmid curing was done using sodium dodecyl sulpfate (SDS). Their antibacterial susceptibility testing of the resistant isolates were repeated with the leaf extract to confirm their curing. The result of the study showed that 70 identified isolates occurred in the frequency of 15 K. pneumoniae, 10 S. aureus, 16 E. coli, 5 E. fecalis, 2 P. mirabilis, 4 P. aeruginosa, 7 E. aerogenes, 1 S. saprophyticus, 2 S. epidermis, 8 E. cloacae. The antibiotic susceptibility result showed that the isolates showed varying degrees of resistance to Nitrofurantoin, Gentamicin, Cefotaxime, Ofloxacin, Ampiclox, Cefixime, Ceftriaxone Sulbactam, Amoxicillin clavulanate, Nalidixic acid, Cefuroxime, Levofloxacin, Imipenem, The phytochemical analysis of the leaves showed that they contain bioactive compounds such as tannins, alkaloids, saponins, steroids, flavonoids, terpenoids, proteins, carbohydrates, reducing sugars, anthocyanin, and phenols. The extract of A. paniculata had the highest invitro antimicrobial activity against K. pneumoniae (31.60±0.85mm at 100mg/ml) while the least antimicrobial activity was on S. aureus (4.83±0.00 mm at 50mg/ml). The minimum inhibitory concentration of the extract of A. paniculata showed inhibition of the bacterial isolates at 3.125mg/ml and 12.5mg/ml for strains of E. cloacae and P. aeruginosa. The minimum bactericidal concentration of A.paniculata was 6.25mg/ml against strains E. cloacae. Plasmid profile gel images of 16 selected bacterial isolates that were resistant to extract and ciprofloxacin showed 10 samples out of 16 selected bacterial isolates have mild resistant genes in them. The result of the repeated antimicrobial susceptibility after curing with SDS showed that 8 of the bacterial resistant isolates became susceptible to the extract and or ciprofloxacin to which they previously showed resistance prior to curing while the rest of the selected bacterial isolates were not affected by the curing agent as they still maintained their resistance and this showed that their resistance could be plasmid mediated. The result in our findings showed the relationship of plasmids with antibiotic resistance and that methanol extract of A. paniculata has broad-spectrum antimicrobial activity and can serve as natural therapeutic agent against asymptomatic bacteriuria pathogens.

Development of the CRISPR/Cas9 system for genome editing in Riemerella anatipestifer

Gene deletion is a valuable tool for phenotypic characterization in bacteriology, but in mycobacteria, generating deletion mutants remains a cumbersome and time-consuming process. Here, we present a modification to the widely used recombineering method in mycobacteria. By expressing the recombineering-promoting proteins from a single-copy episomal plasmid, we could use a constitutive promotor. This approach eliminates the need for induction prior to electroporation of the targeting substrate (linear DNA fragment comprised of homologous regions to the gene of interest, flanking a selection marker), and shortens the subsequent plasmid curing process. We successfully demonstrated this method in Mycobacterium tuberculosis, M. marinum, and M. abscessus.IMPORTANCEAlthough several techniques exist for generating gene-deletion mutants in mycobacteria, these procedures remain limited to laboratories more specialized in molecular biology. Here we present a very simplified procedure, which is a modification on a well-tried technique. Our proposed procedure makes genetic manipulation in mycobacteria more accessible to a greater number of researchers throughout the world, including those with less advanced molecular biology expertizse.

Antimicrobial resistance (AMR) is a global issue; however, in lower resource settings, uncontrolled measures and uncontrolled use of antibiotics in human, animal, and agricultural practices have increased their prevalence in developing countries. Various mechanisms have been implicated to explain the AMR, like the circulation of the plasmid carrying antibiotic resistance genes (ARG), mutation in target genes (intrinsic and plasmid), overexpression of efflux pumps, underexpression of porins, etc. Various therapeutic strategies used to combat AMR exist, such as nonantibiotic approaches (vaccinations or immunotherapy, nano-derived treatments, and bacteriophage therapy), Anti-plasmid and plasmid curing approaches, combinatorial approaches (combination of antibiotics as well as a combination of two different approaches), and plant-based therapeutics. In this focused review, we have discussed the potential use of bacteriophage-based therapy to combat AMR and biofilm formation through multifaceted ways, including lysis of the drug-resistant bacteria, targeting the pili of AMR plasmids conjugation systems, and use of phage-derived lytic proteins. Phages can also be used to decontaminate surfaces in healthcare settings, prevent bacterial contamination in food (meat and dairy), and control bacterial populations in environmental settings, such as water and soil. Therefore, the bacteriophages-based approach served as a dual sword and could not only prevent the spread of infectious diseases but also manage the AMR.

The presence of resistant plasmids (R-plasmids) in microorganisms allows them to evade antibiotics, complicating infection treatment. Disinfectants, though essential in infection control, can become contaminated, contributing to antimicrobial resistance in hospital environments. This study investigated plasmid-mediated antibiotic resistance in bacteria isolated from used disinfectants across four hospitals by evaluating their susceptibility before and after plasmid curing. A total of 100 disinfectant samples (both used and unused diluted forms) were collected, with 21% found contaminated, 86% of which were Gram-negative bacteria. The most frequent isolates included Pseudomonas aeruginosa (24%), Klebsiella pneumoniae (19%), Escherichia coli (14%), Proteus vulgaris (14%), Salmonella typhi (14%), and Staphylococcus aureus (14%). Antibiotic susceptibility tests showed that 81% of isolates were sensitive to all 17 antibiotics tested, while 19% exhibited multidrug resistance, particularly Pseudomonas, Klebsiella, Salmonella, and E. coli. Ampicillin showed the highest resistance, while ciprofloxacin and levofloxacin retained effectiveness. Plasmid curing revealed partial plasmid-mediated resistance in key bacteria. Before curing, Salmonella typhi, Klebsiella pneumoniae, Pseudomonas aeruginosa, and E. coli resisted 15, 9, 8, and 7 antibiotics, respectively (100%). After curing, resistance reduced to 67% in Salmonella, 78% in Klebsiella, 88% in Pseudomonas, and 57% in E. coli, indicating loss of resistance to specific antibiotics. This suggests that resistance to erythromycin, amoxicillin, norfloxacin, and septrin in Salmonella; norfloxacin and septrin in Klebsiella; septrin in Pseudomonas; and norfloxacin, chloramphenicol, and septrin in E. coli was plasmid mediated.

This study investigated the bacteriological quality of Archachatina papyracea (land snail) sold in Benin Metropolis, Edo State, Nigeria. A total of 50 snail samples were purchased from vendors across five different markets within Benin metropolis and analyzed using standard microbiological procedures. Bacterial isolates were identified using cultural and PCR based techniques. Antibiotic susceptibility was done by disk diffusion method. Plasmid detection was carried out by PCR method while plasmid curing was achieved using acridine orange. Results revealed high microbial loads, with total heterotrophic bacterial counts ranging from 18.10 × 10³ to 21.30 × 10³ cfu/g, coliform counts of 11.50 × 10³ to 16.80 × 10³ cfu/g, Salmonella/Shigella counts of 9.60 × 10³ to 11.50 × 10³ cfu/g, and Vibrio counts of 3.70 × 10³ to 10.20 × 10³ cfu/g. Fifteen bacterial species were identified, including Escherichia coli (15.8%), Staphylococcus aureus (10.4%), Klebsiella oxytoca (7.3%), and Salmonella enterica (2.8%). Antibiotic susceptibility testing showed that while some isolates were sensitive to septrin, ciprofloxacin, and streptomycin, others exhibited resistance to commonly used antibiotics such as augmentin, erythromycin, and ampicillin. Plasmid profiling revealed that resistance in some isolates was plasmid-mediated. The presence of enteric and opportunistic pathogens in A. papyracea suggests significant public health risks if the snails are not properly handled and thoroughly cooked before consumption.

Male infertility is simply the male’s inability to achieve pregnancy in a fertile female following at least 12 months of regular, unprotected sexual intercourse. There are several reasons for male infertility to occur, including both reversible and irreversible conditions. Various microorganisms, mainly bacteria, viruses, and parasites, can infect the male reproductive system and induce a series of inflammatory responses that impair male fertility. This study explores the antibiotic resistance patterns of Acinetobacter radioresistens, Burkholderia diffusa and Acinetobacter junii, focusing on plasmid-mediated resistance and the efficacy of plasmid curing agents. A total of 196 semen samples were collected from patients attending Chukwuemeka Odumegwu Ojukwu University Teaching Hospital (COOUTH), Awka. The specimens were analyzed using the basic semen parameters for semen analysis and culture. Molecular identification, plasmid profiling and plasmid curing were also carried out for identification of isolated bacteria and detection of plasmid resistant genes. From the results, bacteriospermia was seen in 65.32% of the samples. Burkholderia diffusa (39.84%) had the highest frequency of occurrence, followed by Acinetobacter radioresistens (32.81%) and lastly, Acinetobacter junii (27.34%). Antibiotic susceptibility testing revealed high resistance patterns, particularly in Acinetobacter radioresistens, which exhibited resistance to seven antibiotics out of ten tested. Most of the isolates were susceptible to ciprofloxacin and ofloxacin. It was also observed that the Multiple Antibiotics Resistance (MAR) index was greatly reduced in all the isolates, following treatment with 3 different curing agents (sodium dodecyl sulphate, ethidium bromide and acridine orange). Acridine orange was the best curing agent for Acinetobacter radioresistens (MAR reduced from 0.70 to 0.10). Sodium dodecyl sulphate and ethidium bromide were best curing agents for Burkholderia diffusa and Acinetobacter radioresistens respectively. Following the treatments, plasmid was implicated as the cause of the resistance among most of the isolates. The study highlights a high prevalence of bacteriospermia, particularly in sexually active age groups, which may contribute to male infertility. The findings also emphasize the role of plasmids in antibiotic resistance and the potential for plasmid-curing agents to restore antibiotic sensitivity. These results call for enhanced screening for reproductive infections, targeted antibiotic therapy, and further research into alternative antimicrobial strategies.

Abstract The rise of antibiotic‐resistant bacteria, driven by antibiotic misuse, is a major global health threat. Addressing this issue requires understanding resistance mechanisms and developing innovative solutions. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐associated systems (Cas), a genome‐editing tool derived from prokaryotic defense mechanisms, offers precise targeting of antibiotic‐resistant genes. By reprogramming CRISPR‐Cas, bacteria can be killed or resensitized to antibiotics through plasmid curing. However, clinical applications face challenges, particularly in delivering CRISPR‐Cas components effectively. Nanotechnology has emerged as a promising approach for targeted delivery to tissues and cells. This paper explores the molecular mechanisms of antibiotic resistance, emphasizing the structure and function of CRISPR‐Cas systems and their delivery mechanisms. It highlights the use of nanoparticles (NPs) and nanoscale carriers to deliver CRISPR‐Cas components, reviewing recent studies that combine NPs and CRISPR to target resistance genes. Additionally, the paper discusses current challenges and future prospects in this field, underscoring the potential of CRISPR‐Cas and nanotechnology to combat antibiotic resistance.

Acinetobacter baumannii is a nosocomial pathogen associated with various infections, including urinary tract infections (UTIs). In the course of an infection, A. baumannii is known to rapidly become resistant to antibiotic therapy, but much less is known about possible adaptation without antibiotic pressure. Through a retrospective study, we investigated within-host genetic diversity during a subclinical 5-year UTI in an animal-patient after withdrawal of colistin treatment. We conducted whole-genome sequencing and phenotypic assays on 17 clonally related isolates from the Sequence Type 25 lineage. Phylogenomic analysis revealed their proximity with animal and human strains from the same country suggesting zoonotic transmission (France). In this case study, the clonally related strains presented variations in genome sizes and nucleotide sequences. Over the course of the infection, A. baumannii underwent genome reduction through insertion sequence (IS) recombination, phage excision or plasmid curing. Alongside this global genome reduction, we observed an expansion of IS17, initially located on the endogenous large plasmid. Genetic variations were mainly located in biofilm formation and metabolism genes. We observed repeated variations affecting three biofilm genes and two adhesion operons associated with weak biofilm-forming capacity. Conversely, only two metabolic genes were recurrently affected, and phenotypic assays indicated a rather stable metabolism profile between the isolates suggesting minor adaptations to its host. Lastly, an overall decreased antibiotic resistance - expected in the absence of antibiotic treatment - contrasted with a conserved colistin resistance due to a pmrB mutation among the isolates.