Characterization of KPC-176, an S171P variant derived from KPC-2, associated with ceftazidime-avibactam resistance in ST11-KL64 Klebsiella pneumoniae.

Mucoid Pseudomonas aeruginosa (m.PAE) presents a major clinical challenge due to its high resistance to ceftazidime (CAZ), a cornerstone antibiotic. This resistance is orchestrated through three barriers, including the formation of an alginate-rich mucoid biofilm that limits drug penetration, upregulated efflux pumps such as MexAB-OprM that reduce intracellular CAZ accumulation, and expression of β-lactamases that hydrolyze CAZ. To address these barriers simultaneously, we engineered a multifunctional nanobooster termed A/C-pAg3PO4, which integrates a silver phosphate nanoparticle (NAg3PO4) core for bacterial disruption, a carboxyl-PEG coating (pAg3PO4) to improve biofilm penetration, and dual co-loading of avibactam (AVB) to inhibit β-lactamase and carbonyl cyanide m-chlorophenylhydrazone (CCCP) to block efflux pumps. In vitro, A/C-pAg3PO4 showed potent antibacterial efficacy, penetrated mucoid biofilms efficiently, and restored bacterial susceptibility via metabolic reprogramming. In murine models of acute pneumonia and lethal sepsis, it markedly reduced bacterial load, mitigated inflammatory damage, and achieved complete survival with no evident toxicity. Our work thus provides a promising strategy to overcome m.PAE resistance to CAZ by concurrently targeting its key defensive mechanisms.

Piperacillin-tazobactam (TZP) resistance in Klebsiella pneumoniae involves diverse mechanisms with unclear prevalence and phenotypic impact. To elucidate these mechanisms, we analyzed K. pneumoniae clinical isolates resistant to TZP but susceptible to cefotaxime and cefepime. Among 53 isolates, 14 were further studied by MIC testing for TZP, amoxicillin-clavulanic acid (AMC), and ceftazidime (CAZ). Short-read sequencing was performed for all 14 isolates and long-read sequencing for two. Core-genome MLST showed that all were unrelated. Two had a blaOXA-1 gene, one also carrying an ompK35 porin gene mutation; two others had the same mutation in the promoter of the chromosomal copy of blaSHV usually associated with overexpression. In the remaining 10, resistance correlated with plasmid-borne blaSHV-1 copies. Nine isolates carried blaSHV-1v1 in the same IS26 pseudocompound transposon (PTn), corresponding to PTnSHV-L and located on a conserved IncFIB(K)_1_Kpn3 plasmid in eight. The tenth isolate carried PTnSHV-L with a distinct blaSHV-1 variant on both an IncHI1B_1_pNDM-MAR plasmid and a high-copy-number Col-type plasmid. Read depth analysis confirmed that blaSHV copy number correlated with TZP, AMC, and CAZ MICs. Large-scale database screening identified related IncFIB(K)_1_Kpn3 plasmids, strongly associated with K. pneumoniae and frequently carrying a PTnSHV-L marker. Analysis of a K. pneumoniae genome data set confirmed the frequent co-occurrence of this plasmid and the PTnSHV-L marker in strains with multiple blaSHV copies. These findings suggest the emergence of an epidemic plasmid adapted to K. pneumoniae and driving TZP resistance through blaSHV-1 amplification, underscoring the need for genomic surveillance to detect amplification-based resistance overlooked by standard phenotypic or PCR assays.

Bacterial multidrug resistance represents a major global public health challenge, highlighting the need for alternative or complementary therapeutic strategies. This study evaluated the antibacterial effects of myrcene (MYR), camphene (CAM), their combination, and their association with ceftazidime (CAZ). Antibacterial activity was assessed using the broth microdilution method to determine the minimum inhibitory concentrations (MICs) against 14 strains. The individual and combined effects of MYR and CAM were evaluated, followed by the release of cytoplasmic material absorbing at 260 nm, checkerboard assay, and in vitro hemolysis assay. MYR inhibited the growth of Pseudomonas aeruginosa and Acinetobacter baumannii by more than 50%, while CAM showed inhibitory activity against Staphylococcus aureus. The combined use of MYR and CAM enhanced antibacterial effects, with increases ranging from 1.8% to 44.2% and 2.0% to 39.8% compared to their individual activities, respectively. Terpenes combined with CAZ lowered MIC in six strains, notably reducing A. baumannii resistance by eightfold. Cytoplasmic release indicated membrane involvement, and terpenes showed no hemolytic activity. The results indicate that terpenes, particularly in combination with CAZ, may enhance antibacterial activity against specific resistant strains, suggesting a promising adjunctive therapeutic strategy.

The global rise in antimicrobial resistance (AMR) among environmental bacteria poses an escalating threat to public health, particularly in developing regions where antibiotic use in agriculture, aquaculture, and clinical settings remains poorly regulated. This study characterized antibiotic resistance patterns, virulence traits, and antibiotic resistance genes (ARGs) of Escherichia coli isolated from 24 major inland water bodies across Sri Lanka. A total of 120 isolates were recovered and subjected to susceptibility testing against seven clinically and agriculturally relevant antibiotics: amoxicillin (AMX), cloxacillin (CLOX), tetracycline (TET), oxytetracycline (OTC), piperacillin (PIP), cefotaxime (CTX), and ceftazidime (CAZ). Isolates were confirmed using standard biochemical tests and Gram staining, and minimum inhibitory concentrations (MICs) were determined via the agar dilution method (60 to 360µg/mL). Multiple antibiotic resistance (MAR) indices were calculated to assess prior antibiotic exposure at each sampling site. PCR assays detected resistance genes (tetA, tetM) and virulence genes (eae, stx1, stx2). Widespread resistance was observed against AMX (100%) and CLOX (100%), while most isolates remained susceptible to the third-generation cephalosporins CTX and CAZ, suggesting the absence of extended-spectrum β-lactamase activity. MAR indices exceeded 0.2 across all sites, indicating pervasive prior antibiotic exposure. Resistance genes tetA (8.3%) and tetM (6.0%) were detected at moderate prevalence, consistent with widespread TET use in agricultural and veterinary contexts. Virulence genes eae and stx2 were absent; nevertheless, stx1 was detected in 2.0% of isolates from Minneriya Canal and Mahakandarawa Tank, indicating the presence of Shiga toxin-producing E. coli (STEC) with pathogenic potential. These findings demonstrate that Sri Lankan inland water bodies serve as critical reservoirs for ARG dissemination and potentially pathogenic strains, posing direct risks to communities dependent on these waters for domestic use, irrigation, and food production. Furthermore, this study provides a replicable methodological framework for future AMR surveillance studies in environmental and freshwater contexts. Systematic AMR surveillance, enhanced wastewater management, and targeted public health interventions are urgently needed, in alignment with One Health priorities and the United Nations Sustainable Development Goals.

Antimicrobial resistance and genomic characterization of Escherichia coli isolated from mink in northern China.

Abstract Salmonella strains carrying the bla CTX-M-55 gene encoding the β-lactamase CTX-M-55 present markedly greater resistance to ceftazidime (CAZ) than those carrying the bla CTX-M-14 gene encoding CTX-M-14, but the structural basis remains unclear. In this study, we combined susceptibility testing, binding affinity measurement, molecular dynamics simulation, and site-directed point mutation to examine how CTX-M-55 enhances CAZ hydrolysis. Compared with CTX-M-14, CTX-M-55 conferred an eightfold higher minimum inhibitory concentration for CAZ and exhibited more than tenfold stronger CAZ binding affinity. Structural and simulation analyses revealed that CTX-M-55 possesses a larger and more hydrophobic active pocket that supports more rapid interactions with CAZ and forms a more stable binding environment, driven mainly by favorable van der Waals and solvation energies. Mutation analysis further revealed two functionally distinct classes of residues contributing to CAZ resistance. Asp-131 and Asn-132 represent functional determinants of CAZ hydrolysis in CTX-M-55, as their substitution disrupts the structure of the binding pocket and hinders the effective binding of the substrate. In contrast, Ser-272 acts as an optimized residue that strengthens the hydrolytic capacity of CTX-M-55 compared with that of CTX-M-14 by modulating CAZ accommodation within a stable pocket. Together, these results indicate that the stronger CAZ resistance of CTX-M-55 results from both a favorable pocket structure and specific residue effects, with Asp-131 and Asn-132 providing the basic hydrolysis capacity and Ser-272 playing an optimized role for CAZ. This work helps clarify the differences in CAZ resistance among the CTX-M family and points to pocket features that may be useful targets for inhibitor design.

N-acetylglucosamine (GlcNAc) is an amino sugar that serves as a structural component, nutrient source, and signal molecule in bacteria. In this study, we sought to identify the genes involved in GlcNAc utilization in Stenotrophomonas maltophilia and to evaluate the impact of GlcNAc utilization on antibiotic susceptibility, oxidative stress tolerance, and swimming motility. The roles of the nagPIBAF operon, nagK, and nagA2 in GlcNAc utilization were investigated through mutant construction and growth assays. NagPsm and NagF mediated GlcNAc transport across the inner membrane, while NagK phosphorylated GlcNAc in the cytoplasm. NagA and NagA2 exhibited functional redundancy as N-acetylglucosamine-6-phosphate deacetylases, and NagB functioned as a glucosamine-6-phosphate deaminase. The nagPIBAF operon was repressed by NagI and derepressed in response to GlcNAc-6P. Antibiotic susceptibility and oxidative stress tolerance were assessed by E-test and menadione IC50, respectively. Among the clinical isolates tested, 71.4% (5/7) and 57.1% (4/7) of the strains showed increases in MIC values for colistin and ceftazidime (CAZ) of 1.3 to 2.6 times and 1.3 to 2 times, respectively. Furthermore, GlcNAc utilization enhanced swimming motility but had little effect on oxidative stress tolerance. Overall, the nagPIBAF operon, nagK, and nagA2 contribute to GlcNAc utilization, which in turn can increase resistance to CAZ and colistin and promote swimming motility in some clinical isolates of S. maltophilia.IMPORTANCEN-acetylglucosamine (GlcNAc) is widely used as a dietary supplement due to its proposed cartilage-protective and anti-inflammatory properties. In bacteria, however, GlcNAc functions as a structural component of peptidoglycan and lipopolysaccharide, as a signal molecule, and as a nutrient source. Stenotrophomonas maltophilia is a gram-negative opportunistic pathogen associated with nosocomial infections, particularly in cystic fibrosis (CF) patients. The abundance of amino sugars derived from mucin degradation is present in the CF lung. Utilization of GlcNAc can reprogram bacterial metabolism, leading to pleiotropic effects on physiology and stress tolerance. We were therefore interested in how bacteria adapt their physiology and stress tolerance when residing in GlcNAc-rich infection niches. Here, we investigated GlcNAc utilization and its impact on physiology and stress tolerance in S. maltophilia.

Biofilms formed by multidrug-resistant (MDR) Klebsiella spp. present a significant clinical challenge due to elevated antibiotic tolerance. Bacteriophages (phages) represent a promising alternative, particularly in combination with antibiotics, where phage-antibiotic synergy (PAS) can increase antibiofilm activity. Evaluating treatment efficacy in these complex structures requires real-time, noninvasive viability analysis. To address this, we used light-sheet fluorescence microscopy (LSFM), a high-resolution, minimally invasive approach, for dynamic tracking of PAS in intact biofilms. To our knowledge, this is the first in vitro application of LSFM for investigating PAS. We studied the combined activity of a virulent phage (vB_KpUKJ_2) and ceftazidime (CAZ) against an extended-spectrum β-lactamase-producing Klebsiella quasipneumoniae. In planktonic cultures, PAS was strongly affected in a dose-dependent manner. In mature biofilms, LSFM imaging revealed that high-dose phages (10⁸ PFU/mL) combined with CAZ at a 0.25 × minimum inhibitory concentration (MIC) induced a rapid and sustained reduction in viability over 24h. This regimen significantly outperformed mono-treatments (p < 0.01), demonstrating that phage coadministration can reduce the required antibiotic dose. Mechanistically, treatment resulted in phage-mediated degradation of α- and β-polysaccharides within the extracellular polymeric substance(EPS). Crucially, while phage mono-treatment led to the emergence of resistant mutants, the combination treatment fully suppressed resistance. Whole-genome sequencing revealed mutations in genes such as fhuA, purA, and rpoC, suggesting diverse resistance mechanisms linked to fitness trade-offs such as impaired biofilm formation. Our findings highlight a precision-guided strategy with translational potential for device-associated infections, providing a mechanistic and methodological foundation for optimizing PAS-based therapies.

Ceftazidime (CAZ) is a critically important broad-spectrum antibiotic that is widely used in clinical practice. However, the rapid emergence of bacterial resistance to CAZ poses a significant challenge in treating infections caused by multidrug-resistant pathogens. In this study, we employed a metabolism-reprogramming approach to characterize key features of laboratory-evolved CAZ-resistant Escherichia coli K12 and identified repressed glutamate metabolism as a reprogrammable target. Exogenous glutamate effectively resensitized both lab-evolved and clinically isolated multidrug-resistant E. coli strains to CAZ. The resensitization mechanism operates through two synergistic pathways driven by glutamate metabolic flux. First, glutamate conversion to inosine activates the inosine-CpxA/CpxR-OmpF regulatory axis, increasing outer membrane permeability. Second, glutamate entry into the pyruvate cycle restores the proton motive force (PMF), energizing the inner membrane. Together, increased outer membrane permeability and a restored PMF synergistically enhance intracellular accumulation of CAZ─by facilitating its entry through the widened OmpF porin and promoting its active uptake across the cytoplasmic membrane. This dual-mechanism strategy provides a novel two-pronged approach to overcoming CAZ resistance. Our findings underscore the potential of targeting bacterial metabolic pathways to restore susceptibility and extend the utility of existing antibiotics against resistant pathogens.

The alarming rise of multidrug-resistant (MDR) bacteria, particularly Salmonella spp., has prompted an urgent search for alternative and synergistic antimicrobial strategies. In this study, a novel, green, and multicomponent nanocomposite was synthesized by integrating zinc oxide nanoparticles (ZnO NPs), chitosan (CS), the β-lactam antibiotic ceftazidime (CAZ), and the antidiabetic agent metformin (MTF) straightforward and economical manner. Bacillus subtilis strain ATCC 6633 was used to biosynthesize ZnO NPs, acting as a reliable bio-nanofactory. Various characterization techniques such as FTIR, XRD, TEM, and zeta potential analysis verified the successful integration and structural integrity of the ZnO NPs within the CS nanocomposite containing CAZ and MTF (ZnO/CS/CAZ/MTF). The FTIR spectra confirmed the presence of proteins that act as binding and supportive agents during the biosynthesis process. The produced nanomaterials have a significant positive surface charge of +28.61 mV, which enhances their stability. The particle sizes of the NPs ranged from 9.93 to 17.44nm. The nanocomposite exhibited strong antibacterial activity against MDR Salmonella enterica subsp., enterica serovar Typhi ATCC 19214, showing a significantly increased inhibition zone of 42mm and a greatly reduced minimum inhibitory concentration (MIC) value of 8µg/ml, compared to the separate components. The minimum bactericidal concentration (MBC) value was found to be consistent with the MIC result, emphasizing the potent bactericidal action of the prepared nanocomposite. In silico molecular docking further supported these findings by revealing favorable interactions between the nanocomposite constituents and the outer membrane proteins (OMPs) of Salmonella enterica serovar Typhimurium (PDB ID: 4W4M) and S. typhi (PDB ID: 3UU2). Key interactions included hydrogen bonding, ionic forces, and metal coordination with critical residues. Cytotoxicity assessment using WI-38 lung fibroblast cells revealed an IC₅₀ of 84.26µg/ml, indicating acceptable preliminary biocompatibility. The present study demonstrates the novelty of a ZnO-based multicomponent nanocomposite that uniquely integrates CAZ, MTF, and CS. This novel formulation exhibited synergistic antibacterial effects against multidrug-resistant Salmonella enterica alongside acceptable in vitro safety. The findings underscore the potential of microbially synthesized nanocomposites as promising candidates for combating antibiotic-resistant bacterial infections and support further preclinical investigations.

Photocatalytic oxidation is a well-established and promising technique for degrading organic contaminants into harmless end products. While antibiotic degradation using photocatalysis has been widely studied, much of the research typically focuses on contaminant degradation in isolated conditions. This study investigates the efficiency of a photocatalyst to degrade an antibiotic in the presence of a real-world contaminant background. Specifically, the performance of a semiconductor-composite, Bismuth Titanate-TiO2 coated onto recycled glass chips was studied - for the degradation of ceftazidime (CFZD), a commonly used antibiotic. To evaluate the photocatalyst's effectiveness, CFZD degradation was tested in deionised water, a humic acid solution, and a real-world hospital wastewater matrix. Quantification before and after treatment was performed using UV-Vis Spectroscopy, High-Performance Liquid Chromatography (HPLC), Mass Spectrometry, and Chemical Oxygen Demand analysis. Based on HPLC results, the photocatalyst was able to degrade CFZD by up to 87% within 2 hours, with the degradation efficiency dropping to 82% in the presence of humic acid. Of all the quantification methods, HPLC provided the most accurate results, as expected; however, quantifying CFZD degradation in the hospital wastewater matrix was challenging even by HPLC due to interference from other contaminants and high pollutant loads. This is the first study to demonstrate the removal of CFZD, a key antibiotic, using a bismuth titanate photocatalyst immobilised on recycled glass chips, while also highlighting the challenges of quantifying pharmaceuticals in matrices with dissolved organic matter using different analytical methods. The study discusses the results, future development trends, challenges, and offers a prospective outlook.

Abstract Background Tebipenem pivoxil hydrobromide (TBP) (formerly SPR994) is in clinical development as the potential first oral broad-spectrum carbapenem agent in the US for the treatment of complicated urinary tract infections (cUTI) and acute pyelonephritis (AP). This study reports on the in vitro activity of TBP and comparator agents against molecularly characterized Enterobacterales isolates recovered from UTI and bloodstream infections (BSI) in the US, including ESBL and carbapenemase (CP) producing isolates. Methods A total of 3,523 Enterobacterales isolates collected from the US in 2023 were included. (UTI, 74.2% (2,614), BSI, 25.8% (909)). Isolates were tested for susceptibility (S) by CLSI reference broth microdilution method. E. coli and K. pneumoniae with aztreonam (ATM), ceftazidime (CAZ), or ceftriaxone (CRO) MICs of ≥2 µg/mL, and P. mirabilis with cefpodoxime (CPD) or CAZ MICs of ≥2 µg/mL were classified as ESBL phenotype. Isolates with MIC ≥2 µg/mL for imipenem (IMI) and/or meropenem (MER), or ≥1 µg/mL for ertapenem (ERT), were categorized as carbapenem-nonsusceptible (CNSE) phenotype. Isolates that met these criteria were screened for plasmid-mediated AmpC (pAmpC), ESBL, and CP genes. Results A total of 14.1% (496/3,523) of isolates were identified with an ESBL phenotype and 13.3% (471/3,523) were ESBL, CSE phenotype (Table). Of the latter, 91.7% (432/471) carried ESBL and/or pAmpC genes. TBP had MIC50/90 of 0.015/0.03 µg/mL against this subset, with those for IMI (MIC50/90, ≤0.12/0.5 µg/mL), MER (MIC50/90, 0.03/0.06 µg/mL) and ERT (MIC50/90, 0.03/0.12 µg/mL). The S to other comparators was below 81%. The CNSE phenotype accounted for only 1.6% (56/3,523) of isolates, and 39.3% (22/56) carried CP. TBP displayed MIC50/90 of 1/ &amp;gt;8 µg/mL. IMI (MIC50/90, 2/ &amp;gt;8 µg/mL), MER (MIC50/90, 1/ &amp;gt;32 µg/mL) and ERT (MIC50/90, 2/ &amp;gt;2 µg/mL) were active against 48%, 61% and 9% of the CNSE subset, respectively, while the S of oral comparators was ≤48%. Conclusion TBP displayed MICs similar to those for overall isolates against ESBL-producing Enterobacterales isolates from UTIs and BSIs in US medical centers. These results indicate that TBP has activity comparable to IV carbapenems and has the potential for use as oral treatment option for cUTI and AP. Disclosures Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Mariana Castanheira, PhD, Melinta Therapeutics: Advisor/Consultant|Melinta Therapeutics: Grant/Research Support Didem Torumkuney, PhD, GSK: Stocks/Bonds (Public Company) Ian A. Critchley, PhD, Spero Therapeutics: Stocks/Bonds (Public Company)

Enterotoxigenic Escherichia coli (ETEC)-induced neonatal calf diarrhea (NCD) causes significant economic losses to the cattle industry; therefore, understanding its antibiotic resistance is crucial for developing targeted prevention and treatment strategies. However, reports on antibiotic resistance in bovine ETEC are currently limited. This study conducted whole-genome sequencing (WGS) and antimicrobial susceptibility testing on ETEC F5- and F5-F41-positive strains isolated from neonatal calf diarrhea samples in Inner Mongolia, China. The results showed that both ETEC F5- and ETEC F5-F41-positive strains are multidrug-resistant, containing ceftriaxone (CRO), ceftazidime (CAZ)) and ciprofloxacin (CIP), which are listed as the highest priority critically important antimicrobial (HP-CIAs) by the World Health Organization (WHO). Combined analysis using the Comprehensive Antibiotic Resistance Database (CARD) and ResFinder 4.1 predictive analysis revealed that the chromosomes and plasmids of the 2 ETEC-positive strains contained 11 classes of antibiotic resistance genes, with the top 3 categories in terms of the number of resistance genes being aminoglycosides, β-lactamases, and chloramphenicols. In addition, various bacterial efflux pumps, including RND, MFS, SMR, and the ABC efflux pump family, were detected. A total of 74 antimicrobial resistance genes were identified in the 2 strains, belonging to 5 categories of drug resistance mechanisms; the antimicrobial resistance phenotype was consistent with the genotype. This study provides a reference for the prevention and treatment of diarrhea caused by ETEC.

Salmonella enterica, particularly non-typhoidal serovars (NTS), is a leading cause of foodborne illness, with invasive infections posing high mortality risks in developing countries. Fluoroquinolones and third-generation cephalosporins, such as ceftazidime (CAZ), are used to treat severe infections, yet they are facing concerning rates of antimicrobial resistance. Furthermore, recalcitrant and/or persistent infections are often linked to persister cells, a phenotype that enables cells to survive in the presence of high concentrations of antibiotics. Although persisters are associated with chronic infections, their interactions with the human immune system, particularly serum resistance and opsonophagocytosis, are not well understood. Here, three NTS isolates from the food protein chain (S45, S48, and 4SA(2)) were used. Persister cells were selected by exposure to CAZ concentration 100 times higher than the minimum inhibitory concentration and then assessed for serum resistance, opsonophagocytosis, and intracellular survival in primary human macrophages. The isolates exhibited heterogeneous persister fractions (1.06%-39.55% survival after 72h of CAZ exposure). Persisters exhibited equal or greater serum resistance than regular cells. Isolate 4SA(2) proliferated in 100% human serum, with persister-derived cells showing higher growth rates. Following opsonization, serum-resistant persisters of all isolates were phagocytosed at significantly higher rates than serum-resistant regular cells. Intracellular survival varied: S45 persisters proliferated post-internalization; S48 persisters and regulars were eradicated; 4SA(2) showed no phenotype difference. Complement enhanced the intracellular survival of S45 but not S48 or 4SA(2). Despite having different intracellular outcomes, Salmonella persisters showed higher levels of opsonophagocytosis and serum resistance. These findings suggest that cell surface modifications may facilitate host cell uptake and contribute to antimicrobial treatment failure and long-term infection. The phenotypic diversity among isolates underscores the importance of considering persister heterogeneity and host-pathogen immune interactions in order to understand recalcitrant infection dynamics and design more effective therapeutic strategies.

The amount of lipopolysaccharide (LPS) released by Escherichia coli and Klebsiella pneumoniae isolated from bovine clinical mastitis and cultured with marbofloxacin (MBFX), cefazolin (CEZ), ceftazidime (CAZ), and imipenem was quantified. The relationships between antimicrobial type, concentration, and bacterial count in relation to the amount of LPS released were evaluated. Bacterial morphology, which is closely associated with LPS release, was also investigated. At an initial inoculum of 105 CFU/mL, the amount of LPS released after 4 hr of incubation with MBFX and CEZ was significantly lower than that for the negative control (NC) group at concentrations higher than 5× the minimum inhibitory concentration (MIC) for E. coli and higher than 50× MIC for K. pneumoniae. These findings suggest that exposure to high-dose antimicrobials results in reduced LPS release associated with bacterial growth and lysis. Conversely, at an initial inoculum of 108 CFU/mL, incubation with MBFX and CEZ at concentrations of 50× MIC and 250× MIC for 4 hr resulted in an amount of LPS released that was comparable to that for the NC group for both E. coli and K. pneumoniae. Both MBFX and CEZ also induced bacterial filamentation, but the morphological changes were less pronounced than those induced by CAZ and were associated with lower LPS release. These suggest that early administration of high concentrations of antimicrobials at low bacterial loads may mitigate the adverse effects associated with LPS release in cases of mastitis caused by E. coli and K. pneumoniae.

The rapid emergence of multidrug‐resistant (MDR) pathogens, particularly in hospital wastewater, poses a serious threat to public health and emphasizes the need for alternative antimicrobial strategies. In this study, Enterococcus hirae, an environmentally derived strain, was used for the first time in the extracellular green synthesis of zinc oxide nanoparticles (ZnO NPs) and copper oxide/zinc oxide nanoparticles (CuO/ZnO NPs). The nanoparticles were characterized using standard techniques. Ultraviolet–visible (UV‐Vis) spectra, X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FE‐SEM), transmission electron microscopy (TEM), and energy‐dispersive X‐ray spectroscopy (EDS) confirmed both nanoparticle formation, size, and morphology. Antimicrobial activity against Staphylococcus aureus (ATCC 6538), Morganella morganii, Kerstersia gyiorum, and Klebsiella pneumoniae was evaluated using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, showing a 62.5% greater efficacy of bimetallic NPs than ZnO alone. The 2,2‐diphenyl‐1‐picrylhydrazyl hydrate (DPPH) assay revealed that E‐CuO/ZnO NPs exhibited superior antioxidant activity with the lowest IC50 of 5.528 μg/mL, outperforming E‐ZnO NPs, which is attributed to the synergistic effect between ZnO and CuO NPs. The combination of E‐ZnO and E‐CuO/ZnO nanoparticles with ciprofloxacin (CIP) and ceftazidime (CAZ) was evaluated against MDR isolates. Synergistic interactions were observed particularly against K. pneumoniae. This study confirms effective E. hirae‐mediated synthesis and the enhanced antibacterial and antioxidant potential of CuO/ZnO NPs, supporting eco‐friendly strategies against MDR infections, with synergistic interactions observed with conventional antibiotics, particularly against K. pneumoniae, indicating that the nanoparticles can enhance antibiotic efficacy.

Background: This study was conducted to investigate the presence of antibiotic-resistant bacteria in avocados sold in Mersin, Turkey. The study is based on concerns about foodborne illnesses transmitted through fresh fruits and the increasing incidence of antimicrobial resistance (AMR), especially in developing countries. Methods: A total of 10 avocado samples were obtained from local markets and supermarkets. A total of 29 bacterial isolates were obtained from the samples, and identification was performed by the pour plate method. Antibiotic susceptibility tests were evaluated according to EUCAST (2024) criteria using the Kirby–Bauer disk diffusion method. Results: Total viable bacterial load was found to be (8.3 ± 1.2) × 10⁴ CFU/mL. Among the identified bacteria, Escherichia coli was detected at the highest rate (41.4%; n=12), while Klebsiella spp. was detected at the lowest rate (6.9%; n=2). Staphylococcus aureus, Enterobacter cloacae, and Bacillus cereus were also isolated. Two Escherichia coli strains were identified as multiply antibiotic resistant (MDR). 28.5% of E. cloacae isolates were resistant to ceftazidime (CAZ), while all S. aureus isolates (100%) were resistant to penicillin, ciprofloxacin, and levofloxacin. Conclusions: The findings reveal that the presence of fecal-derived antibiotic-resistant bacteria in fresh avocado samples poses a potential public health risk. This underscores the importance of controlling microbial contamination in fresh fruits and vegetables, strengthening food hygiene, and regularly monitoring antibiotic resistance.

BackgroundHemin is a major source of iron for pathogens in infectious niches. The FecIRA-like surface signaling cascade is a common regulatory system for iron acquisition by pathogens. This system consists of a FecA-like TonB-dependent transporter (TBDT), a FecR-like inner membrane anti-sigma factor, and a FecI-like extracytoplasmic function (ECF) sigma factor. Beyond iron acquisition, FecIRA-like systems have been reported to regulate additional physiological processes. The known hemin acquisition system in Stenotrophomonas maltophilia includes HemA, a TBDT; HemU, an inner membrane transporter; and the TonB1–ExbB1–ExbD1a–ExbD1b complex, a multi-subunit motor that energizes HemA. Fur and HemP are the primary regulators involved in hemin utilization. In this study, we identified a novel FecIRA-like regulatory system, i.e., HemI–HemR–HemAD.MethodsThe regulatory role of HemI was examined using promoter–xylE transcriptional fusion constructs and real-time PCR. Mutants associated with the hemI–hemR–hemAD operon were generated and evaluated for iron utilization, swimming motility, oxidative stress tolerance, and antibiotic susceptibility.ResultsThe hemI–hemR–hemAD operon was repressed by Fur–Fe2+ under iron-replete conditions. Its expression was partially derepressed under iron depletion and further derepressed in the presence of hemin; however, the operon showed no autoregulation. HemI was essential for hemin acquisition. The overexpression of hemI in the S. maltophilia KJ strain increased the susceptibility to levofloxacin (LVX) and trimethoprim–sulfamethoxazole (SXT). All S. maltophilia isolates examined displayed increased minimum inhibitory concentrations (MICs) for ceftazidime (CAZ) and minocycline (MIN) under the iron-depleted and hemin-available conditions; notably, the changes in the MICs of LVX and SXT were strain-dependent.ConclusionHemI, a novel ECF sigma factor, not only regulates hemin acquisition but also contributes to antibiotic susceptibility under iron-limited and hemin-available conditions.

Background/Objectives: Stenotrophomonas maltophilia (SM) is a significant cause of hospital-acquired infections in immunocompromised and critical care patients. This study investigates the impact of combining ceftazidime/avibactam (CZA) with conventional antibiotics on SM obtained from various sources in planktonic and biofilm cell cultures. Methods: Using broth microdilution, the MICs of different antibiotics, including CZA, were determined on 37 SM strains. CZA's bactericidal and synergistic effectiveness were examined through in vitro time-kill curve tests with tigecycline (TGC), chloramphenicol (CHL), levofloxacin (LVX), colistin (CS), and amikacin (AMK). In addition, synergistic activity was investigated against SM biofilm cell cultures, and antibiotic Mutant Prevention Concentrations (MPCs) were tested against SM isolates. Results: Compared to ceftazidime (CAZ), CZA was four times more efficient against 37 SM strains. Unlike TGC and CHL, CS, AMK, and CZA had 2-4 times higher MBCs than MICs. All studied antibiotics were bactericidal at 1× or 4× MIC doses against SM bacteria, except for CZA. The combinations of CZA with LVX and CZA with AMK or CS demonstrated synergistic effects in four out of seven (57%) strains and in three out of seven (43%) strains, respectively, when tested at doses equivalent to the MIC. Moreover, all antibiotic combinations with CZA showed a synergistic effect at dosages four times the MIC. Additionally, CZA and the tested drugs synergistically inhibited SM biofilm formation, and MPC values were 8-16 times the MIC. Conclusions: The results of this study indicate that combining CZA with LVX and CS was more effective against SM strains. These combinations might provide alternatives for treating SM pathogens in both planktonic and biofilm cell cultures.