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β-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent β-lactam resistance in Enterobacter cloacae. However, NagZ’s role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ’s function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

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Raising prices of organic fertilizers, environmental relation of their usage and need for global foodstuff, placed a topic of universal interest in cultivation of legume plants for human nutrients and soil fertility supplementation. Three rates of N fertilizer were studied in the field: 0, 20 and 40 kg N/ha. Three bacterial strains were identified and inoculated in the experiment: Enterobacter asburiae (E. asburiae), Klebsiella quasipneumoniae (K. quasipneumoniae) and Enterobacter cloacae (E. cloacae). The soil samples were taken before and after experiment for chemical attributes analysis. The effect of N2 fertilizer and 3 bacterial strains was on soil chemical properties, yield components and yield of peanut plant. The results of present study showed that application of different N2 fertilizer rates was a little influence on the N2-fixing ability of rhizosphere bacteria, but high effect was significant at the peanut nodule number. In the 3 N fertilizer rates and 3 bacteria, which only had N2 fertilizer rate of 40 kg N/ha and E. asburiae significantly raised the maximum yield component and yield of peanut, while there was the lowest yield component and yield of peanut was observed in N fertilizer rate application of 0 kg N/ha and E. cloacae inoculation. The differences in rhizosphere bacterium strains between different N fertilizer rates was better than that caused by the nitrogen fertilizer amount. This could be due to the substantial improvement in soil chemical attributes at different soils, especially in, pH, OM and total N, as these significantly impacted the soil structure. [ABSTRACT FROM AUTHOR]

ABSTRACT Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae. Single deletions of the genes encoding the HU subunits (hupA and hupB) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans-complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria.IMPORTANCET6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae, affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis.

β-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent β-lactam resistance in Enterobacter cloacae . However, NagZ's role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ's function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

Introduction: Carbapenemase-producing Gram-negative bacilli are a worldwide problem, which represent a health concern because most of their resistance mechanisms are encoded by plasmids and therefore easily transmissible in hospital settings. Many methods have been proposed to detect such resistance, but screening is still challenging. Recently, the modified carbapenem inactivation method has shown promising results; however, more studies need to be performed. Hence, this study aimed to evaluate the performance of the mCIM in carbapenem-resistant Enterobacteriaceae and non-fermenting Gram-negative bacilli isolates. Materials and methods: From a microbial collection with molecular characterization of carbapenemase genes previously conducted, 100 Gram-negative bacilli isolates were selected: fifty-two non-carbapenemase-producing and 48 carbapene- mase-producing isolates. The mCIM was performed according to the CLSI guidelines, and to assess the performance of the method, sensitivity and specifi- city were calculated. Results: The sensitivity of the mCIM observed in this study was 96% (46/48), and the specificity was 96,2% (50/52). Most of the Gram-negative bacilli carrying a carbapenemase gene were mCIM-positive. Moreover, in carbapenem-resistant isolates that do not produce a carbapenemase (En- terobacteriaceae and non-fermenters), the results of the mCIM were negative. Conclusion: Overall, the mCIM provides a good alternative for the screening of carbapenemase-producing Gram-negative bacilli. Our findings highlight that mCIM is a sensitive and specific method to assess carbapenemase production in non-fermenting Gram-negative bacilli and Enterobacteriaceae such as Enterobacter cloacae. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is a gastrointestinal disease linked with GIT microbial dysbiosis. The present study has targeted the comparative analysis of virulent factor FadA from gut-associated bacteria of CRC patients ( F. nucleatum ) and healthy individuals ( E. cloacae ). Methods: For this purpose, FadA protein sequences of fifteen strains of F. nucleatum and four strains of E. cloacae , were retrieved from the UniProt database. These sequences were analysed through VirulentPred, PSLpred, ProtParam, PFP-FunDSeqE, PROTEUS Structure Prediction Server, SWISS-MODEL, SAVES validation server, MEME suite 5.5.0, CAVER Web tool, Webserver VaxinPAD, HPEPDOCK and HDOCK servers. Results: FadA protein from F. nucleatum was found to exhibit significant differences as compared to E. nucleatum i.e. it exhibited helical configuration, cytoplasmic, periplasmic, outer-membrane and extracellular localisation, 2D structure comprising of 70-96% helix, 0% beta-sheet, 4-30% coils and 17-20 signal peptide residues, hydrophilicity, strongly acidic character and smaller number of antigenic epitopes. In contrast, FadA protein from E. nucleatum was found to have globular 3D configuration, cytoplasmic localisation, 2D structure (30-56% helix, 12-21% beta-sheet, 33-50% coils and 43 signal peptide residues), highly hydrophobic, slightly acidic and more number of antigenic epitopes. Docking analyses of virulent factors revealed their high binding affinities with previously reported inhibitory peptide and FAD-approved drug COX2. Conclusion: The wide range of differences not only provided us the reason for the role of FadA protein as a virulent factor in F. nucleatum but also might help us in designing virulent FadA protein inhibiting strategies including peptide-based vaccine adjuvants and drugs designing, modification of tunnels and catalytic pockets to reduce substrate binding and FAD approved drugs selection. Inhibition of this virulent factor in CRC patients' gut bacteria might result in oncogenesis regression and reduced death rate., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Enterobacter cloacae is an emerging pathogen isolated in healthcare-associated infections. A major virulence factor of this bacterium is the type VI secretion system (T6SS). The genome of E. cloacae harbors two T6SS gene clusters (T6SS-1 and T6SS-2), and the functional characterization of both systems showed that these two T6SSs are not expressed under the same conditions. Here, we report that the major histone-like protein HU positively regulates the expression of both T6SSs and, therefore, the function that each T6SS exerts in E. cloacae . Single deletions of the genes encoding the HU subunits ( hupA and hupB ) decreased mRNA levels of both T6SS. In contrast, the hupA hupB double mutant dramatically affected the T6SS expression, diminishing its transcription. The direct binding of HU to the promoter regions of T6SS-1 and T6SS-2 was confirmed by electrophoretic mobility shift assay. In addition, single and double mutations in the hup genes affected the ability of inter-bacterial killing, biofilm formation, adherence to epithelial cells, and intestinal colonization, but these phenotypes were restored when such mutants were trans -complemented. Our data broaden our understanding of the regulation of HU-mediated T6SS in these pathogenic bacteria., Importance: T6SS is a nanomachine that functions as a weapon of bacterial destruction crucial for successful colonization in a specific niche. Enterobacter cloacae expresses two T6SSs required for bacterial competition, adherence, biofilm formation, and intestinal colonization. Expression of T6SS genes in pathogenic bacteria is controlled by multiple regulatory systems, including two-component systems, global regulators, and nucleoid proteins. Here, we reported that the HU nucleoid protein directly activates both T6SSs in E. cloacae , affecting the T6SS-related phenotypes. Our data describe HU as a new regulator involved in the transcriptional regulation of T6SS and its impact on E. cloacae pathogenesis., Competing Interests: The authors declare no conflict of interest.

• Enterobacter cloacae clinical isolates were exposed to cefepime, meropenem and ceftazidime-avibactam • Increases in minimum inhibitory concentrations were greater for cefepime and meropenem than for ceftazidime-avibactam • Alterations in AmpC, OmpC and efflux proteins were observed in only 4/13 mutants • Isolates had alterations in genes not commonly associated with β-lactam resistance Five Enterobacter cloacae isolates were subjected to 10-day serial passage in broth microdilution with cefepime, meropenem, or ceftazidime-avibactam to evaluate increases in minimum inhibitory concentration (MIC) and resistance mechanisms after exposure. Post-exposure isolates displaying >2-fold changes from the parent isolate were analysed alongside the parent isolate. Increases in MIC were 4- to 256-fold (median: 16-fold) after cefepime exposure, 16- to 128-fold (64-fold) after meropenem, and 2- to 32-fold (8-fold) after ceftazidime-avibactam. Post-exposure isolates had diverse mechanisms, identified using a combination of short and long whole-genome sequencing. All agents selected for AmpC alterations in one isolate set. OmpC and TetA/AcrR regulator alterations were noted in meropenem and ceftazidime-avibactam post-exposure isolates of the same set. Other mutations in AmpC were noted when isolates were exposed to cefepime or ceftazidime-avibactam. A premature stop codon in the cell division inhibitor protein, MioC was observed when one parent isolate was exposed to any of the agents, indicating a cell persistence mechanism. Mutations in less common transporter systems and protein synthesis components were also noted. All agents showed cross-resistance to other β-lactams and resistance mechanisms were diverse, with some not usually associated with β-lactam resistance in Enterobacterales. This initial evaluation indicates that cefepime and meropenem select for isolates with higher MIC values compared to ceftazidime-avibactam. Further studies evaluating these findings should be performed for other species for which the primary β-lactam resistance mechanism is not gene acquisition. These studies should evaluate these observations in vivo to assess their translation into patient treatment policies. [ABSTRACT FROM AUTHOR]

The overuse of N fertilizers has caused serious environmental problems (e.g., soil acidification, excessive N 2 O in the air, and groundwater contamination) and poses a serious threat to human health. Improving N fertilizer utilization efficiency and plant uptake is an alternative for N fertilizers overuses. Enterobacter cloacae is an opportunistic pathogen, also used as plant growth-promoting rhizobacteria (PGPR), has been widely presented in the fields of bioremediation and bioprotection. Here we developed a new N fixation-release model by combining biochar with E. cloacae. The efficiency of the model was evaluated using a greenhouse pot experiment with maize (Zea mays L.) as the test crop. The results showed that biochar combined with E. cloacae significantly increased the N content. The application of biochar combined with E. cloacae increased total N in soil by 33% compared with that of N fertilizers application. The N-uptake and utilization efficiency (NUE) in plant was increased 17.03% and 14.18%, respectively. The activities of urease, dehydrogenase and fluorescein diacetate hydrolase (FDA) was improved, the catalase (CAT) activity decreased. Analysis of the microbial community diversity revealed the abundance of Proteobacteria, Actinobacteria, Firmicutes, and Gemmatimonadetes were significantly improved. The mechanism under the model is that E. cloacae acted as N-fixation by capturing N 2 from air. Biochar served as carrier, supporting better living environment for E. cloacae , also as adsorbent adsorbing N from fertilizer and from fixed N by E. cloacae , the adsorption in turn slower the N release. Altogether, the model promotes N utilization by plants, improves the soil environment, and reduces N pollution. • N is fixed and slowly released to plants through a green sustainability model. • N fixation release model promotes N uptake by plants and improves N utilization. • Soil enzyme and microorganisms were positive effect via a N fixation-release model. • Controlling the slow release of N can reduce environmental pollution due to N loss. [ABSTRACT FROM AUTHOR]

It is well known that gut microbiota imbalance can promote the development of metabolic disease. Enterobacter cloacae (E. cloacae) is a kind of opportunistic pathogen in the intestine. Therefore, we hypothesized that E. cloacae accelerated the development of metabolic disease. To answer this question, we used E. cloacae to induce disease in guinea pigs. We used H&E staining to detect the pathological changes of liver and aorta and used Oil Red O staining to evaluate the lipid accumulation in the liver. And that we used a kit to detect AST content and used Western blot to detect protein levels in the liver. We found that E. cloacae could induce liver pathological changes and lipid accumulation as well as aortic wall pathological changes in guinea pigs. And E. cloacae increased the liver index to 5.94% and the serum AST level to 41.93 U/L. Importantly, E. cloacae activated liver high mobility group protein (HMGB1)/toll-like receptor 4 (TLR4)/myeloiddifferentiationfactor88 (MYD88)/nuclear factor-kappa B (NF-κB) signal and sterol regulatory element-binding protein 1c (SREBP-1c) and inhibited AMP-activated protein kinase (AMPK). We conclude that E. cloacae promote nonalcoholic fatty liver disease (NAFLD) by inducing inflammation and lipid accumulation, and E. cloacae also promote atherosclerosis. These findings are important for study on the pathogenesis and drug screening of NAFLD and atherosclerosis. [Display omitted] • E. cloacae aggravated NAFLD by activating HMGB1/TLR4/MyD88/NF-κB signal. • E. cloacae aggravated NAFLD by activating SREBP-1c and inhibiting AMPK. • E. cloacae accelerated the development of atherosclerosis. [ABSTRACT FROM AUTHOR]