Influence of the two-component system PhoBR associated with phosphate-sensing capacity on the virulence mechanism of Vibrio alginolyticus.

Background: Multidrug efflux pumps significantly contribute to bacterial resistance to antimicrobial drugs. The oqxAB efflux pump gene in Klebsiella pneumoniae has been well studied due to its significant contribution to antibiotic resistance. Objectives: This study investigated how Ciprofloxacin at a sub-Minimum Inhibitory Concentration (sub-MIC) affected the expression of the oqxAB efflux pump gene in an isolate of Klebsiella pneumoniae that was MDR (Multidrug-Resistant) with strong biofilm-forming ability. Methodology: Clinical samples were collected from blood, wounds, respiratory infections, and urine for isolation of K. pneumoniae. The isolates were tested for antibiotic susceptibility. Then, the MDR with strong biofilm-forming ability K. pneumoniae isolate were selected and incubated with sub-MIC of ciprofloxacin at early, mid, and late stages of the logarithmic growth phase. The levels of expression of the oqxAB efflux pump genes were then quantified by RT-qPCR. Results: Thirty K. pneumoniae isolates were identified from clinical specimens and tested for antibiotic susceptibility. Exposure of multidrug-resistant, strong biofilm-forming isolate to a sub-MIC of Ciprofloxacin resulted in marked downregulation of efflux pump genes. OqxA expression decreased by 0.3-fold, 0.09-fold, and 0.0001-fold at the early, mid, and late logarithmic phases, respectively, while OqxB expression decreased by 0.08-fold, 0.05-fold, and 0.004-fold at the corresponding stages. Conclusion: The results highlight the significant impact of ciprofloxacin stress in suppressing the expression of the oqxAB efflux pump genes.

Revealing the role of quorum sensing molecules in the treatment of kitchen waste with composite bacterial agents: Performance, mechanisms, and scale-up verification.

Comparative transcriptomic analysis of pathogenic Vibrio parahaemolyticus YDE17 under iron-limited conditions.

Adhesin gene overexpression stimulates biofilm formation and catalytic performance in recombinant Escherichia coli.

Fecal Sludge (FS) is a well-known reservoir for multiple pathogenic organisms and antimicrobial-resistant E. coli. Contaminated drinking water (DW) is the primary route through which fecal-oral diseases are spread. This study examined the prevalence of Extended-spectrum beta-lactamase (ESBL)-producing E. coli in FS and adjacent DW samples collected within the Rohingya Camps. The DW and treated FS samples were screened for thermotolerant E. coli and the isolates were characterized for their ESBL-production ability, presence of major antibiotic resistance (AMR) genes, pathogenic genes, biofilm formation ability and phylogenetic clustering. Among the samples, 88.1% FS and 40.2% DW contained E. coli. From the selected isolates, 32.3% of FS and 12.5% of DW were ESBL-positive. Subsequently, 113 ESBL-FS and 56 ESBL-DW isolates were characterized based on their concurrent presence. Upon screening, blaCTX-M (81.1%) was the most prevalent AMR gene, followed by blaTEM (23.7%) and blaNDM-1 (14.8%). Enteroaggregative E. coli (EAEC) (17.2%) and enterotoxigenic E. coli (ETEC) (12.4%) were the most prevalent diarrheagenic pathotypes and 4.7% were extra-intestinal pathogenic E. coli (ExPEC) strains. Antimicrobial susceptibility testing (AST) revealed that 98.2% of isolates were multidrug-resistant. At 25°C, 34.3% of isolates formed strong biofilm. The ERIC (Enterobacterial Repetitive Intergenic Consensus sequences) phylogenetic clustering generated 16 clusters at a 70% similarity index. The detection of pathogenic isolates highlights the risk of severe infections within susceptible populations and a high percentage of multidrug resistance limits the treatment options.

Virulence and Antimicrobial Resistance Gene Profiling of Salmonella Isolated from Dead-in-Shell Eggs and Hatchery Environments with Emphasis on Class 1 Integron Gene Cassette Sequencing in XDR Strains.

The emergence of drug-resistant Pseudomonas aeruginosa is an increasing global concern affecting human and animal health, food production systems, and environmental safety. This study investigated its occurrence, antimicrobial resistance, and biofilm-forming ability using phenotypic assays, and identify the biofilm-associated genetic markers in 120 cheese samples including 30 samples each of (Kariesh, Tallaga, Processed, and Romy) collected from various retail sources in Cairo and Giza, Egypt. Pseudomonas aeruginosa isolates were identified using both biochemical tests and molecular confirmation using 16 S rRNA gene. Antimicrobial susceptibility was evaluated using the disk diffusion method. Biofilm formation was assessed phenotypically through the microtiter plate and tube assays, while the biofilm-associated genes pelA and pslA were detected using PCR. Pseudomonas aeruginosa was detected in 18 samples (15%), and identification was confirmed through biochemical and molecular methods. Antimicrobial susceptibility testing revealed that 55% of isolates were extensively drug-resistant, while 45% exhibited multidrug resistance, with MAR indices ≥ 0.2 and an average MAR index of 0.68, indicating exposure to high-risk environments with frequent antibiotic use. Phenotypic assays showed strong biofilm-forming capabilities among isolates, with 100% positive by microtiter plate and 95% by tube method. Molecular screening further confirmed the prevalence of biofilm-associated genes, detecting pelA in 72% and pslA in 61% of isolates. Overall, Pseudomonas aeruginosa isolated from cheese samples exhibited substantial antimicrobial resistance and robust biofilm-forming ability, posing significant concerns to cheese quality and consumer health. These findings highlight the urgent need for continuous surveillance, improved dairy hygiene, effective sanitation, responsible antimicrobial practices, and alternative control strategies within the dairy sector to reduce potential public health hazards.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-026-57335-2.

Hospital-acquired infections are a major concern in healthcare settings, often involving Gram-negative bacilli that affect both patients and healthcare personnel. This study aimed to assess the antimicrobial effects of chlorhexidine and EDTA, alone and in combination, on Gram-negative bacilli isolated from carriers, along with their antibiotic resistance profiles and biofilm-forming abilities. This cross-sectional study collected 390 samples from hospital personnel, patients and environmental surfaces to investigate Gram-negative bacilli. Antibiotic susceptibility, biofilm formation, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of chlorhexidine, with and without EDTA, were also determined. PCR also was used to detect biocide resistance genes (qacE, qacEΔ1, CepA). A total of 120 Gram-negative bacilli were isolated and subsequently confirmed through biochemical tests. Klebsiella pneumoniae was the predominant bacterial species, accounting for 70% (n=84). Based on the present findings, 29 (24.1%) isolates were multidrug-resistant (MDR). ESBL and AmpC production were detected in 12.5% and 21% of isolates, respectively. Strong biofilm formation was observed in 58.3% of the samples. Chlorhexidine alone inhibited bacterial growth at a MIC₉₀ of 0.009%, with a bactericidal effect at 0.019%. When combined with EDTA, a synergistic effect was observed, reducing the MIC to 0.002% and the MBC to 0.004%, indicating enhanced antimicrobial activity (P ≤ 0.05). These findings indicate a considerable prevalence of antibiotic-resistant and biofilm-producing Gram-negative bacilli among carriers. Furthermore, the increased activity observed with the combination of chlorhexidine and EDTA suggests that this approach may represent a potentially useful option for decontamination and infection control in healthcare settings.

Soybean [Glycine max (L.) Merrill] is a major leguminous and oilseed crop known for high protein contents, fulfilling the nutritional demand, particularly in regions dominated by staple crops. In recent years, salt stress has heavily limited crop productivity. Salt-tolerant plant growth-promoting bacteria (PGPB) can better serve as biofertilizers for improved crop productivity under saline conditions. In this study, fourteen bacteria were isolated from root nodules of soybean and further tested for salt-tolerance and in vitro plant growth-promoting (PGP) properties under saline stress. Nine bacterial isolates showed salt-tolerance at 2% NaCl. The indole-3-acetic acid (IAA) production of these bacteria ranged from 3.8 to 13.2µg mL- 1, wherein HSS-5 showed maximum production. All isolates showed positive results on nitrogen-free minimal (NFM) media and were capable to solubilize phosphorus ranging from 1.08 to 2.44 index. Six isolates exhibited biofilm formation ability. In hydrolytic enzyme activities, five isolates showed potential in cellulase activity and five in pectinase activity. Based on the in vitro testing, five potential isolates were selected for taxonomic identification and further evaluated under controlled and field conditions. Phylogenetic analysis revealed that HSS-2 is closely related to Sinorhizobium psoraleae, HSS-6 to Bradyrhizobium japonicum, HSS-10 to Agrobacterium tumefaciens, HSS-12 to Agrobacterium pusense and HSS-13 to Priestia megaterium. In pot assay, HSS-13, HSS-2, and HSS-6 significantly promoted plant growth, increasing plant biomass by 69.88%, 43.37% and 40.96%, respectively, as compared to the control whereas HSS-6 showed maximum grain yield under field trial. This research highlights the first report of an unexplored diversity of salt-tolerant non-rhizobial endophytic bacterial community associated with soybean nodules. Notably, the identification of salt-tolerant traditional nodule endophyte, Bradyrhizobium japonicum HSS-6 and the unexplored emergence of non-traditional Priestia megaterium HSS-13 as an effective nodule endophyte with strong PGP traits represent a previously unreported finding that opens up new avenues for sustainable production under saline conditions.

Development and application of a fast and efficient CRISPR/Cas12f -based genetic toolkit in Bacillus cereus GW-01.

The emerging fungal pathogen Candida auris is a serious global public health threat due to its ability to persist in healthcare environments and on human skin. Here, we report a prevalent cluster of clinical C. auris strains with enhanced biofilm formation, a contributor to environmental and skin persistence. Genomic analyses show that the clinical isolates with enhanced biofilm formation ability carry a loss-of-function mutation in the WOR2 locus, which encodes a white-opaque switching regulator in Candida species. Analysis of 13,314 published genomes revealed that 3104 strains (23.3%) harbor nonsense or frameshift mutations in WOR2, indicating frequent clinical occurrence. Deletion of WOR2 in a clinical strain markedly increased biofilm formation, whereas reintroduction of an intact WOR2 significantly attenuated biofilm development. Further analyses show that Wor2 inactivation upregulates GFC1, ALS4, and multiple biofilm-associated genes. Together, these findings reveal a key regulatory mechanism underlying biofilm development, environmental persistence, and transmission of C. auris.

Antibiotics can significantly disrupt gut microbiota homeostasis, reducing microbial diversity and causing dysbiosis associated with health issues. Gut biofilms play a critical role in resilience and stress tolerance of the intestinal ecosystem. Mucosal microbial communities also help restore the gut microbiota after interventions like probiotics, antibiotics, or fecal transplants. Previously, we developed a core bacterial consortium with strong in vitro biofilm-forming and stress-resilient properties, but its colonization ability and in vivo function remained unclear. In this study, we first validated the in vivo biofilm formation of the microbial consortium using a germ-free (GF) mouse model, then introduced single-, dual-, and multi-strain combinations with varying biofilm-forming abilities into specific-pathogen-free mice to assess their potential for recovering antibiotic-disrupted gut microbiota. Our findings indicate that the robust, in vitro-selected consortium continued to form substantial biofilms in GF mouse intestines. 16S rRNA sequencing showed that, compared to single- or dual-strain treatments, administering the core consortium significantly increased microbial richness and diversity. The gut microbiota of consortium-treated mice more closely resembled healthy controls, suggesting the core consortium has superior potential to restore healthy gut microbiota. Overall, our research demonstrates the core consortium markedly improves gut microbiota resilience to antibiotic-induced disruptions in mice, accelerates restoration of health-associated taxa, and reestablishes gut homeostasis. This approach could transform probiotic interventions from passive supplementation to active ecological engineering, providing a theoretical and experimental basis for next generation of engineered probiotics and microbiome restoration therapies.

Non-fermenting Gram-negative bacilli (NFGNB) have emerged as important opportunistic pathogens associated with bloodstream infections, particularly in critical care settings where antimicrobial resistance poses a growing challenge. This study investigated the prevalence of NFGNB in bloodstream infections, with emphasis on Stenotrophomonas maltophilia, including its antimicrobial susceptibility profile, virulence-associated traits, and resistance determinants. In this prospective study (2023-2025) conducted at a tertiary care centre in Mangalore, Karnataka, India, NFGNB isolated from bloodstream infections were identified by MALDI-TOF MS. S. maltophilia isolates were further evaluated for antimicrobial susceptibility, biofilm formation, extracellular virulence enzyme production, cytotoxicity on Vero cells, and selected drug resistance genes. A total of 442 NFGNB were identified, among which Acinetobacter species were predominant (163; 36.69%), followed by Burkholderia species (79; 17.86%), S maltophilia (53; 11.99%), and Pseudomonas species (47; 10.65%). S. maltophilia demonstrated retained in vitro susceptibility to trimethoprim-sulfamethoxazole, levofloxacin, moxifloxacin, and minocycline, while resistance was observed against multiple β-lactam agents and aminoglycosides. Most isolates exhibited moderate to strong biofilm-forming ability, frequent production of protease, lipase, and lecithinase, and concentration-dependent cytotoxic effects, with strong biofilm producers showing greater cytotoxicity. The association observed between biofilm formation, enzyme production, and cytotoxicity suggests a coordinated virulence phenotype that may contribute to persistence and pathogenicity in bloodstream infections. Stenotrophomonas maltophilia emerged as a clinically significant bloodstream pathogen characterized by multidrug resistance and multiple virulence-associated traits. These findings highlight the importance of rapid species-level identification, appropriate antimicrobial selection guided by standardized susceptibility criteria, and recognition of virulence characteristics for improved management, surveillance, and infection control of S. maltophilia bloodstream infections.

Klebsiella pneumoniae is a prominent pathogen causing life-threatening bloodstream infections. Although biofilm formation and resistance to human serum are well-recognized virulence traits, their interrelatedness during K. pneumoniae bloodstream infections remains unclear. Here, we hypothesize that biofilm production is related to K. pneumoniae's ability to thrive in human serum and, therefore, may predict the strains' ability for serum survival. We analyzed 57 clinical, genetically diverse classical K. pneumoniae strains and characterized their survival and biofilm-producing ability in human serum. Serum survival patterns revealed three serum resistance categories-Low, Mid, and High. In addition, the biofilm biomass produced by the strains correlated with their serum resistance level (P < 0.001), and 3D biofilm visualization using confocal microscopy further confirmed that biofilm extracellular polysaccharide substances and biomass patterns were consistent with the serum resistance categories. Moreover, we revealed a direct correlation between the level of biofilm formation and the strain's serum survival level (R2 = 0.696), a prerequisite for systemic K. pneumoniae dissemination. As biofilm formation in serum reflects both survival and biofilm-forming ability, we assessed biofilm formation in defined modified basal medium (BM2), to rule out serum-mediated killing, and discovered a strong and significant association between the serum resistance category and BM2 biofilm biomass (P < 0.0001). By applying regression models, we discovered that biofilm formation serves as a significant predictor for bacterial survival in serum. Overall, our findings establish biofilm production in K. pneumoniae as a biomarker of serum survival and may open a new avenue for predicting bloodstream infection risk in clinical settings.IMPORTANCEBloodstream infections caused by Klebsiella pneumoniae are devastating life-threatening infections worldwide. Understanding the survival strategies of K. pneumoniae in the bloodstream is critical for elucidating key aspects of bacterial pathogenicity and developing new diagnostic and therapeutic modalities. Although serum survival is a recognized virulence trait necessary to thrive in the bloodstream, the relationship between serum resistance and biofilm formation, a multicellular organization that may protect bacteria from bloodstream stressors, remains poorly understood. In this article, we demonstrate biofilm production in human serum by clinical classical K. pneumoniae strains for the first time and discovered a direct correlation between the level of biofilm biomass formation and the degree of serum survival in human serum and in defined modified basal medium. These findings offer insights into the importance of biofilm production in K. pneumoniae serum resistance and may be used to develop future therapeutic strategies targeting bloodstream infections.

Diabetic foot ulcers (DFUs) are a major complication of diabetes mellitus and a leading cause of morbidity, amputation, and mortality among affected patients. In this study, five Pseudomonas aeruginosa isolates recovered from DFUs of diabetic patients in Egypt were investigated. Antimicrobial susceptibility was evaluated using the disc diffusion method and broth microdilution assay, while biofilm-forming ability was phenotypically assessed using the crystal violet assay. All isolates exhibited multidrug-resistant (MDR) phenotypes, with two isolates classified as pan-drug resistant (PDR). Moreover, all isolates demonstrated a strong capacity for biofilm formation. Whole-genome sequencing (WGS) and subsequent bioinformatic analysis revealed three sequence types, ST369, ST664, and ST773, corresponding to serotypes O6, O2, and O11, respectively, with ST664 and ST773 representing high-risk clones. These high-risk clones carried horizontally transferable integrative conjugative elements (ICEs) encoding metal resistance and anti-phage defense systems. The isolates also harboured a diverse resistome comprising blaNDM-1, blaPER-1, rmtB, qnrVC1, aac(3), ant(4')-IIb, aph(3')-VIb, sul1, tet(G), and cmlA9 in addition to biocide resistance genes (qacE, triABC). Colistin resistance in three isolates was associated with L71R pmrA mutation. Virulence profiling revealed a minimum of 230 genes associated with adhesion, biofilm formation, toxin production, and secretion systems. Notably, high-risk clones (ST773 and ST664) carried an extensive array of anti-phage defense systems, up to 32 per genome, potentially reducing the efficacy of bacteriophage-based therapeutic alternatives. Collectively, these findings highlight the remarkable genomic plasticity and adaptive resilience of P. aeruginosa in chronic diabetic wounds, emphasizing the urgent need for improved infection control practices, genomic surveillance, and the development of novel therapeutic strategies for effective DFU management. To our knowledge, this is the first genomic characterization of MDR P. aeruginosa isolates from DFUs in Egypt.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-026-05145-x.

Role of 2-phenylethanol in regulating population density and biocontrol efficacy of Scheffersomyces spartinae W9 via quorum sensing.

Characterization of antimicrobial resistance and biofilm formation in Escherichia coli isolated from wild boars.

Biofilm formation is a critical virulence mechanism in pathogens such as Klebsiella pneumoniae, a Gram-negative, encapsulated bacterium that has emerged as a zoonotic threat capable of infecting both humans and animals. Its biofilm-forming ability is closely associated with catheter-related and urinary tract infections. Given its potential to cross species barriers and cause significant public health concern, elucidating the environmental cues and conserved molecular pathways driving biofilm formation is essential for developing cross-species prevention strategies. Here we found that K. pneumoniae exhibited significantly greater biofilm-forming efficiency in urine than in nutrient-rich medium under comparable biomass conditions. Transposon-insertion sequencing (Tn-seq) identified 19 fitness genes essential for optimal growth in urine, most involved in the de novo biosynthesis of amino acids, particularly arginine, methionine, and isoleucine. Urine represents an amino acid-starved (AAS) environment for K. pneumoniae, modulating c-di-GMP signaling to promote biofilm formation. Eight diguanylate cyclase (DGC, c-di-GMP synthesis) genes, four phosphodiesterase (PDE, c-di-GMP degradation) genes, and four DGC+PDE genes were significantly regulated in response to urine. Furthermore, transcriptomic analysis comparing K. pneumoniae grown in urine with that grown in M9 medium revealed significant activation of genes associated with exopolysaccharide (EPS) biosynthesis, including those encoding lipopolysaccharides (LPS), capsules, peptidoglycan, and enterobacterial common antigen (ECA). Notably, K. pneumoniae increases EPS biosynthesis under the iron-limited conditions in urine, further promoting biofilm development. In conclusion, AAS-mediated c-di-GMP signaling and iron limitation are key drivers of biofilm formation by K. pneumoniae in urine, providing mechanistic insights that may guide strategies to disrupt biofilm formation.

Unraveling the antibacterial mechanism of exogenous autoinducers against Shewanella putrefaciens revealed by metabolomics and its potential application on preserving large yellow croaker (Pseudosciaena crocea).