Identification and Characterization of a Highly Active Hyaluronan Lyase from Enterobacter asburiae.

Hyaluronic acid (HA) is a well-known functional marine polysaccharide. The utilization and derivative development of HA are of great interest. Hyaluronan lyase has wide application prospects in the production of HA oligosaccharides and lower molecular weight HA. In this study, a strain of Enterobacter asburiae CGJ001 with high hyaluronan lyase activity was screened from industrial wastewater. This strain exhibited an impressive enzyme activity of 40,576 U/mL after being incubated for 14 h. Whole genome sequencing analysis revealed that E. asburiae CGJ001 contained a cluster of genes involved in HA degradation, transport, and metabolism. A newly identified enzyme responsible for glycosaminoglycan degradation was designated as HylEP0006. A strain of E. coli BL21(DE3)/pET-22b(+)-hylEP0006 was successfully constructed. HylEP0006 exhibited optimal degradation at 40 °C and pH 7.0, showing a high activity of 950,168.3 U/mg. HylEP0006 showed specific activity against HA. The minimum degradation fragment of HylEP0006 was hyaluronan tetrasaccharides, and HylEP0006 could efficiently degrade HA into unsaturated disaccharides (HA2), with HA2 as the final product. These characteristics indicate that HylEP0006 has a potential application prospect for the extraction and utilization of hyaluronic acid.

Emergence and transmission of the high-risk ST78 clone of OXA-48-producing Enterobacter hormaechei in a single hospital in Taiwan.

Carbapenem-resistant Enterobacter cloacae complex is a significant global healthcare threat, particularly carbapenemase-producing Enterobacter hormaechei (CPEH). From January 2017 to January 2021, twenty-two CPEH isolates from a regional teaching hospital in central Taiwan were identified with the carriage of carbapenemase genes blaKPC-2, blaIMP-8, and predominantly blaOXA-48. Over 80% of these CPEH strains clustered into the high-risk ST78 lineage, carrying a blaOXA-48 IncL plasmid (pOXA48-CREH), nearly identical to the endemic plasmid pOXA48-KP in ST11 Klebsiella pneumoniae. This OXA-48-producing ST78 lineage disseminated clonally from 2018 to 2021 and transferred pOXA48-CREH to ST66 and ST90 E. hormaechei. An IMP-8-producing ST78 strain harbouring a blaIMP-8-carrying pIncHI2 plasmid appeared in 2018, and by late 2020, a KPC-2-producing ST78 strain was identified after acquiring a novel blaKPC-2-carrying IncFII plasmid. These findings suggest that the high-risk ST78 lineage of E. hormaechei has emerged as the primary driver behind the transmission of CPEH. ST78 has not only acquired various carbapenemase-gene-carrying plasmids but has also facilitated the transfer of pOXA48-CREH to other lineages. Continuous genomic surveillance and targeted interventions are urgently needed to control the spread of emerging CPEH clones in hospital settings.

Gut symbiont-derived sphingosine modulates vector competence in Aedes mosquitoes.

The main vectors of Zika virus (ZIKV) and dengue virus (DENV) are Aedes aegypti and Ae. albopictus, with Ae. aegypti being more competent. However, the underlying mechanisms remain unclear. Here, we find Ae. albopictus shows comparable vector competence to ZIKV/DENV with Ae. aegypti by blood-feeding after antibiotic treatment or intrathoracic injection. This suggests that midgut microbiota can influence vector competence. Enterobacter hormaechei_B17 (Eh_B17) is isolated from field-collected Ae. albopictus and conferred resistance to ZIKV/DENV infection in Ae. aegypti after gut-transplantation. Sphingosine, a metabolite secreted by Eh_B17, effectively suppresses ZIKV infection in both Ae. aegypti and cell cultures by blocking viral entry during the fusion step, with an IC50 of approximately 10 µM. A field survey reveals that Eh_B17 preferentially colonizes Ae. albopictus compared to Ae. aegypti. And field Ae. albopictus positive for Eh_B17 are more resistant to ZIKV infection. These findings underscore the potential of gut symbiotic bacteria, such as Eh_B17, to modulate the arbovirus vector competence of Aedes mosquitoes. As a natural antiviral agent, Eh_B17 holds promise as a potential candidate for blocking ZIKV/DENV transmission.

VIM-1-producing Enterobacter asburiae with mobile colistin resistance genes from wastewaters.

BACKGROUND: Wastewaters are considered as important players in the spread of antimicrobial resistance, thus affecting the health of humans and animals. Here, we focused on wastewaters as a possible source of carbapenemase-producing Enterobacterales for the environment. METHODS: A total of 180 presumptive coliforms from hospital and municipal wastewaters, and a river in the Czech Republic were obtained by selective cultivation on meropenem-supplemented media and tested for presence of carbapenemase-encoding genes by PCR. Strains carrying genes of interest were characterized by testing antimicrobial susceptibility, carbapenemase production and combination of short- and long- read whole-genome sequencing. The phylogenetic tree including publicly available genomes of Enterobacter asburiae was conducted using Prokka, Roary and RAxML. RESULTS: Three VIM-producing Enterobacter asburiae isolates, members of the Enterobacter cloacae complex, were detected from hospital and municipal wastewaters, and the river. The blaVIM-1 gene was located within a class 1 integron that was carried by different F-type plasmids and one non-typeable plasmid. Furthermore, one of the isolates carried plasmid-borne colistin-resistance gene mcr-10, while in another isolate chromosomally located mcr-9 without colistin resistance phenotype was detected. In addition, the analysis of 685 publicly available E. asburiae genomes showed they frequently carry carbapenemase genes, highlighting the importance of this species in the emergence of resistance to last-line antibiotics. CONCLUSION: Our findings pointed out the important contribution of hospital and community wastewaters in transmission of multi-drug resistant pathogens.

pQEB1: a hospital outbreak plasmid lineage carrying bla KPC-2.

While conducting genomic surveillance of carbapenemase-producing Enterobacteriaceae (CPE) from patient colonisation and clinical infections at Birmingham's Queen Elizabeth Hospital (QE), we identified an N-type plasmid lineage, pQEB1, carrying several antibiotic resistance genes, including the carbapenemase gene bla KPC-2. The pQEB1 lineage is concerning due to its conferral of multidrug resistance, its host range and apparent transmissibility, and its potential for acquiring further resistance genes. Representatives of pQEB1 were found in three sequence types (STs) of Citrobacter freundii, two STs of Enterobacter cloacae, and three species of Klebsiella. Hosts of pQEB1 were isolated from 11 different patients who stayed in various wards throughout the hospital complex over a 13 month period from January 2023 to February 2024. At present, the only representatives of the pQEB1 lineage in GenBank were carried by an Enterobacter hormaechei isolated from a blood sample at the QE in 2016 and a Klebsiella pneumoniae isolated from a urine sample at University Hospitals Coventry and Warwickshire (UHCW) in May 2023. The UHCW patient had been treated at the QE. Long-read whole-genome sequencing was performed on Oxford Nanopore R10.4.1 flow cells, facilitating comparison of complete plasmid sequences. We identified structural variants of pQEB1 and defined the molecular events responsible for them. These have included IS26-mediated inversions and acquisitions of multiple insertion sequences and transposons, including carriers of mercury or arsenic resistance genes. We found that a particular inversion variant of pQEB1 was strongly associated with the QE Liver speciality after appearing in November 2023, but was found in different specialities and wards in January/February 2024. That variant has so far been seen in five different bacterial hosts from six patients, consistent with recent and ongoing inter-host and inter-patient transmission of pQEB1 in this hospital setting.

The effect of combinations of a glyphosate-based herbicide with various clinically used antibiotics on phenotypic traits of Gram-negative species from the ESKAPEE group.

The emission of glyphosate and antibiotic residues from human activities threatens the diversity and functioning of the microbial community. This study examines the impact of a glyphosate-based herbicide (GBH) and common antibiotics on Gram-negative bacteria within the ESKAPEE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli). Ten strains, including type and multidrug-resistant strains for each species were analysed and eight antibiotics (cefotaxime, meropenem, aztreonam, ciprofloxacin, gentamicin, tigecycline, sulfamethoxazole-trimethoprim, and colistin) were combined with the GBH. While most combinations yielded additive or indifferent effects in 70 associations, antagonistic effects were observed with ciprofloxacin and gentamicin in five strains. GBH notably decreased the minimum inhibitory concentration of colistin in eight strains and displayed synergistic activity with meropenem against metallo-ß-lactamase (MBL)-producing strains. Investigation into the effect of GBH properties on outer membrane permeability involved exposing strains to a combination of this GBH and vancomycin. Results indicated that GBH rendered strains sensitive to vancomycin, which is typically ineffective against Gram-negative bacteria. Furthermore, we examined the impact of GBH in combination with three carbapenem agents on 14 strains exhibiting varying carbapenem-resistance mechanisms to assess its effect on carbapenemase activity. The GBH efficiently inhibited MBL activity, demonstrating similar effects to EDTA (ethylenediaminetetraacetic acid). Chelating effect of GBH may have multifaceted impacts on bacterial cells, potentially by increasing outer membrane permeability and inactivating metalloenzyme activity.

VacSol-ML(ESKAPE): Machine learning empowering vaccine antigen prediction for ESKAPE pathogens.

The ESKAPE family, comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., poses a significant global threat due to their heightened virulence and extensive antibiotic resistance. These pathogens contribute largely to the prevalence of nosocomial or hospital-acquired infections, resulting in high morbidity and mortality rates. To tackle this healthcare problem urgent measures are needed, including development of innovative vaccines and therapeutic strategies. Designing vaccines involves a complex and resource-intensive process of identifying protective antigens and potential vaccine candidates (PVCs) from pathogens. Reverse vaccinology (RV), an approach based on genomics, made this process more efficient by leveraging bioinformatics tools to identify potential vaccine candidates. In recent years, artificial intelligence and machine learning (ML) techniques has shown promise in enhancing the accuracy and efficiency of reverse vaccinology. This study introduces a supervised ML classification framework, to predict potential vaccine candidates specifically against ESKAPE pathogens. The model's training utilized biological and physicochemical properties from a dataset containing protective antigens and non-protective proteins of ESKAPE pathogens. Conventional autoencoders based strategy was employed for feature encoding and selection. During the training process, seven machine learning algorithms were trained and subjected to Stratified 5-fold Cross Validation. Random Forest and Logistic Regression exhibited best performance in various metrics including accuracy, precision, recall, WF1 score, and Area under the curve. An ensemble model was developed, to take collective strengths of both the algorithms. To assess efficacy of our final ensemble model, a high-quality benchmark dataset was employed. VacSol-ML(ESKAPE) demonstrated outstanding discrimination between protective vaccine candidates (PVCs) and non-protective antigens. VacSol-ML(ESKAPE), proves to be an invaluable tool in expediting vaccine development for these pathogens. Accessible to the public through both a web server and standalone version, it encourages collaborative research. The web-based and standalone tools are available at http://vacsolml.mgbio.tech/.

Integrative transcriptomic and metabolomic analysis to elucidate the effect of gossypol on Enterobacter sp. GD5.

Gossypol, a yellow polyphenolic compound found in the Gossypium genus, is toxic to animals that ingest cotton-derived feed materials. However, ruminants display a notable tolerance to gossypol, attributed to the pivotal role of ruminal microorganisms in its degradation. The mechanisms of how rumen microorganisms degrade and tolerate gossypol remain unclear. Therefore, in this study, Enterobacter sp. GD5 was isolated from rumen fluid, and the effects of gossypol on its metabolism and gene expression were investigated using liquid chromatography-mass spectrometry (LC-MS) and RNA analyses. The LC-MS results revealed that gossypol significantly altered the metabolic profiles of 15 metabolites (eight upregulated and seven downregulated). The Kyoto Encyclopedia of Genes and Genomes analysis results showed that significantly different metabolites were associated with glutathione metabolism in both positive and negative ion modes, where gossypol significantly affected the biosynthesis of amino acids in the negative ion mode. Transcriptomic analysis indicated that gossypol significantly affected 132 genes (104 upregulated and 28 downregulated), with significant changes observed in the expression of catalase peroxidase, glutaredoxin-1, glutathione reductase, thioredoxin 2, thioredoxin reductase, and alkyl hydroperoxide reductase subunit F, which are related to antioxidative stress. Furthermore, Gene Ontology analysis revealed significant changes in homeostatic processes following gossypol supplementation. Overall, these results indicate that gossypol induces oxidative stress, resulting in the increased expression of antioxidative stress-related genes in Enterobacter sp. GD5, which may partially explain its tolerance to gossypol.

Enterobacter adelaidei sp. nov. Isolation of an extensively drug resistant strain from hospital wastewater in Australia and the global distribution of the species.

BACKGROUND: Enterobacter species are included among the normal human gut microflora and persist in a diverse range of other environmental niches. They have become important opportunistic nosocomial pathogens known to harbour plasmid-mediated multi-class antimicrobial resistance (AMR) determinants. Global AMR surveillance of Enterobacterales isolates shows the genus is second to Klebsiella in terms of frequency of carbapenem resistance. Enterobacter taxonomy is confusing and standard species identification methods are largely inaccurate or insufficient. There are currently 27 named species and a total of 46 taxa in the genus distinguishable via average nucleotide identity (ANI) calculation between pairs of genomic sequences. Here we describe an Enterobacter strain, ECC3473, isolated from the wastewater of an Australian hospital whose species could not be determined by standard methods nor by ribosomal RNA gene multi-locus typing. AIM: To characterise ECC3473 in terms of phenotypic and genotypic antimicrobial resistance, biochemical characteristics and taxonomy as well as to determine the global distribution of the novel species to which it belongs. METHODS: Standard broth dilution and disk diffusion were used to determine phenotypic AMR. The strain's complete genome, including plasmids, was obtained following long- and short read sequencing and a novel long/short read hybrid assembly and polishing, and the genomic basis of AMR was determined. Phylogenomic analysis and quantitative measures of relatedness (ANI, digital DNA-DNA hybridisation, and difference in G+C content) were used to study the taxonomic relationship between ECC3473 and Enterobacter type-strains. NCBI and PubMLST databases and the literature were searched for additional members of the novel species to determine its global distribution. RESULTS: ECC3473 is one of 21 strains isolated globally belonging to a novel Enterobacter species for which the name, Enterobacter adelaidei sp. nov. is proposed. The novel species was found to be resilient in its capacity to persist in contaminated water and adaptable in its ability to accumulate multiple transmissible AMR determinants. CONCLUSION: E. adelaidei sp. nov. may become increasingly important to the dissemination of AMR.

Strengthening bioremediation potential: Enterobacter ludwigii ES2 for combined nicosulfuron and Cd contamination through whole genome and microbial diversity community analysis.

Nicosulfuron and Cd are common pollutants that pose significant threats to the environment and human health, particularly under combined stress. This study is the first to remediate environmental nicosulfuron and Cd under combined stress using microbiological techniques. Enterobacter ludwigii ES2 was isolated, characterized, and demonstrated to degrade 93.80 % of nicosulfuron and remove 59.64 % of Cd within 4 d. Potential functional genes, including nicosulfuron degradation genes gstA, gstB, glnQ, glnP, mreB, and sixA, and Cd tolerance/removal-related genes mntA, mntB, mntH, dnaK, znuA, and zupt, were predicted by sequencing the whole genome of strain ES2, and their expression was verified by qRT-PCR. Strain ES2 managed oxidative stress induced by Cd through superoxide dismutase, glutathione, catalase, peroxidase, and malondialdehyde. Furthermore, to repair compound stress, up to 90.48 % of nicosulfuron and 67.74 % of Cd were removed. The community structure analysis indicated that Enterobacteriaceae, Sphingomonadaceae, and Gemmatimonadaceae were dominant populations, with ES2 stably colonizing and becoming the dominant bacterium. In summary, ES2 demonstrated significant potential in remediating nicosulfuron and Cd pollution from various perspectives, providing a solid theoretical foundation.

The microbiota comparative analysis of the characteristics between colorectal adenomatous polyps and normal mucosal intestinal.

OBJECTIVE: The aim of this study is to systematically examine and compare the characteristics distinguishing colorectal adenomatous polyps from normal mucosal intestinal microbiota. METHODS: A total of 30 specimens were obtained from patients diagnosed with colorectal adenomatous polyps (adenoma group) who underwent endoscopic removal at Wenzhou People's Hospital between September 2021 and November 2021. Concurrently, 30 normal mucosal specimens were collected from patients without adenomatous polyps (control group). Subsequently, microbiome total DNA extraction was carried out, followed by PCR amplification targeting the V3-V4 region of the 16S rDNA. High-throughput sequencing was conducted using the Illumina MiSeq platform. Subsequent to sequencing, bioinformatics analysis was used to assess the diversity, composition, and functional aspects of the intestinal microbiota in both study groups. RESULTS: A notable dissimilarity in the microbiota structure was identified, specifically within the transverse colon, between these two groups ( P  < 0.05). Species composition analysis revealed that Escherichia , Fusobacterium , and Bacteroides were predominant bacteria in both groups, with Escherichia and Enterobacter displaying significant differences at the genera level between the control group and the adenoma group ( P  < 0.05). Correlation analysis and functional prediction demonstrated substantial disparities in interactions among dominant intestinal microbial genera within patients from both groups. Additionally, it was discovered that the intestinal microbiomes in patients in the adenoma group exhibited a significantly higher pathogenic potential. CONCLUSION: Upon conducting a comprehensive analysis, it was discerned that the microbiota present in the transverse colon of the control group exhibited distinctive characteristics that may contribute to the maintenance of intestinal health.

Enterobacter sp. HIT-SHJ4 isolated from wetland with carbon, nitrogen and sulfur co-metabolism and its implication for bioremediation.

Both autotrophic and heterotrophic denitrification are known as important bioprocesses of microbe-mediated nitrogen cycle in natural ecosystems. Actually, mixotrophic denitrification co-driven by organic matter and reduced sulfur substances are also common, especially in hypoxic environments such as estuarine sediments. However, carbon, nitrogen and sulfur co-metabolism during mixotrophic denitrification in natural water ecosystems has rarely been reported in detail. Therefore, this study investigated the co-metabolism of carbon, nitrogen and sulfur using samples collected from four distinct natural water ecosystems. Results demonstrated that samples from various sources all exhibited the ability for co-metabolism of carbon, nitrogen and sulfur. Microbial community analysis showed that Pseudomonas and Paracoccus were dominant bacteria ranging from 65.6% to 75.5% in mixotrophic environment. Enterobacter sp. HIT-SHJ4, a mixotrophic denitrifying strain which owned the capacity for co-metabolism of carbon, nitrogen and sulfur, was isolated and reported here for the first time. The strain preferred methanol as its carbon source and demonstrated remarkable efficiency for removing sulfide and nitrate with below 100 mg/L sulfide. Under weak acid conditions (pH 6.5-7.0), it exhibited enhanced capability in converting sulfide to elemental sulfur. Its bioactivity was evident within a temperature from 25 °C to 40 °C and C/N ratios from 0.75 to 3. This study confirmed the widespread presence of microbial-mediated synergistic carbon, nitrogen and sulfur metabolism in natural aquatic ecosystems. HIT-SHJ4 emerges as a novel strain, shedding light on carbon, nitrogen and sulfur co-metabolism in natural water bodies. Furthermore, it also serves as a promising candidate microorganism for in-situ ecological remediation, particularly in dealing with contamination posed by nitrate, sulfide, and organic matter.

Citrobacter spp. and Enterobacter spp. as reservoirs of carbapenemase blaNDM and blaKPC resistance genes in hospital wastewater.

Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gram-negative bacterial species. The presence of ß-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 ß-lactamase genes; blaTEM in 33.1%, blaCTX-M in 25.4%, blaKPC in 25.4%, blaNDM 8.8%, blaSHV in 5.3%, and blaOXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase blaKPC was found in six Citrobacter spp. and E. coli, while blaNDM was detected in two distinct Enterobacter spp. and E. coli. Notably, blaNDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase blaKPC and blaNDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains. IMPORTANCE: The significance of this study lies in its findings regarding the prevalence and diversity of antibiotic-resistant bacteria and genes identified in hospital wastewater in Mexico. The research underscores the urgent need for enhanced surveillance and prevention strategies to tackle the escalating challenge of antibiotic resistance, particularly evident through the elevated frequencies of carbapenemase genes such as blaKPC and blaNDM within the Enterobacteriaceae family. Moreover, the identification of these resistance genes on conjugative plasmids highlights the potential for widespread transmission via horizontal gene transfer. Understanding the mechanisms of antibiotic resistance in hospital wastewater is crucial for developing targeted interventions aimed at reducing transmission, thereby safeguarding public health and preserving the efficacy of antimicrobial therapies.

Emergence of a novel FRI-type carbapenemase; blaFRI-12 in Enterobacter asburiae located on an IncR plasmid.

Carbapenem-resistance in Enterobacter spp due to acquisition of mobile carbapenemases is of concern. An Enterobacter spp grew on ChromID CARBA medium and was positive for the mCIM carbapenemase detection assay. Susceptibility testing showed resistance to aztreonam and reduced susceptibility to imipenem. Conventional PCR using FRI primers detected a blaFRI gene. Whole genome sequencing reveled a new variant; blaFRI-12 was closest in sequence to blaFRI-5 differing by 13 amino acids and was found on a unique 110Kb IncR plasmid. Given the intrinsic nature of Enterobacter spp. to be carbapenem non-susceptible, blaFRI-types may be under reported globally.

Run-and-tumble kinematics of Enterobacter Sp. SM3.

The recent discovery of the peritrichous, swarm-competent bacterium Enterobacter sp. SM3 has offered a new opportunity to investigate the connection between bacterial swimming and swarming. Here, we report the run-and-tumble behavior of SM3 as planktonic swimming cells and as swarming cells diluted in liquid medium, drawing comparison between the two states. Swimming cells of SM3 run for an average of 0.77 s with a speed of approximately 30µm/s before tumbling. Tumbles last for a duration of 0.12 s on average and cause changes in direction averaging 69^{∘}. Swimming cells exposed to the common chemoattractant serine in bulk solution suppress the frequency of tumbles in the steady state, lengthening the average run duration and decreasing the average tumble angle. When exposed to aspartate, cells do not demonstrate a notable change in run-and-tumble parameters in the steady state. For swarming cells of SM3, the frequency of tumbles is reduced, with the average run duration being 50% longer on average than that of swimming cells in the same liquid medium. Additionally, the average tumble angle of swarming cells is smaller by 35%. These findings reveal that the newly identified species, SM3, performs run-and-tumble motility similar to other species of peritrichous bacteria such as E. coli, both in the swimming and swarming states. We present a simple mechanical model, which provides a physical understanding of the run-and-tumble behavior of peritrichous bacteria.

Some virulence genes are associated with antibiotic susceptibility in Enterobacter cloacae complex.

BACKGROUND: Enterobacter cloacae complex (ECC) including different species are isolated from different human clinical samples. ECC is armed by many different virulence genes (VGs) and they were also classified among ESKAPE group by WHO recently. The present study was designed to find probable association between VGs and antibiotic susceptibility in different ECC species. METHODS: Forty-five Enterobacter isolates that were harvested from different clinical samples were classified in four different species. Seven VGs were screened by PCR technique and antibiotic susceptibility assessment was performed by disk-diffusion assay. RESULT: Four Enterobacter species; Enterobacter cloacae (33.3%), Enterobacter hormaechei (55.6%), Enterobacter kobei (6.7%) and Enterobacter roggenkampii (4.4%) were detected. Minimum antibiotic resistance was against carbapenem agents and amikacin even in MDR isolates. 33.3% and 13.3% of isolates were MDR and XDR respectively. The rpoS (97.8%) and csgD (11.1%) showed maximum and minimum frequency respectively. Blood sample isolated were highly virulent but less resistant in comparison to the other sample isolates. The csgA, csgD and iutA genes were associated with cefepime sensitivity. CONCLUSION: The fepA showed a predictory role for differentiating of E. hormaechei from other species. More evolved iron acquisition system in E. hormaechei was hypothesized. The fepA gene introduced as a suitable target for designing novel anti-virulence/antibiotic agents against E. hormaechei. Complementary studies on other VGs and ARGs and with bigger study population is recommended.

Metal Nanoparticle-Based Biosensors for the Early Diagnosis of Infectious Diseases Caused by ESKAPE Pathogens in the Fight against the Antimicrobial-Resistance Crisis.

The species included in the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and the genus Enterobacter) have a high capacity to develop antimicrobial resistance (AMR), a health problem that is already among the leading causes of death and could kill 10 million people a year by 2050. The generation of new potentially therapeutic molecules has been insufficient to combat the AMR "crisis", and the World Health Organization (WHO) has stated that it will seek to promote the development of rapid diagnostic strategies. The physicochemical properties of metallic nanoparticles (MNPs) have made it possible to design biosensors capable of identifying low concentrations of ESKAPE bacteria in the short term; other systems identify antimicrobial susceptibility, and some have been designed with dual activity in situ (bacterial detection and antimicrobial activity), which suggests that, in the near future, multifunctional biosensors could exist based on MNPs capable of quickly identifying bacterial pathogens in clinical niches might become commercially available. This review focuses on the use of MNP-based systems for the rapid and accurate identification of clinically important bacterial pathogens, exhibiting the necessity for exhaustive research to achieve these objectives. This review focuses on the use of metal nanoparticle-based systems for the rapid and accurate identification of clinically important bacterial pathogens.

Quantifying Membrane Alterations with Tailored Fluorescent Dyes: A Rapid Antibiotic Resistance Profiling Methodology.

Accurate detection of bacterial antibiotic sensitivity is crucial for theranostics and the containment of antibiotic-resistant infections. However, the intricate task of detecting and quantifying the antibiotic-induced changes in the bacterial cytoplasmic membrane, and their correlation with other metabolic pathways leading to antibiotic resistance, poses significant challenges. Using a novel class of 4-aminophthalimide (4AP)-based fluorescent dyes with precisely tailored alkyl chains, namely 4AP-C9 and 4AP-C13, we quantify stress-mediated alterations in E. coli membranes. Leveraging the unique depth-dependent positioning and environment-sensitive fluorescence properties of these dyes, we detect antibiotic-induced membrane damage through single-cell imaging and monitoring the fluorescence peak maxima difference ratio (PMDR) of the dyes within the bacterial membrane, complemented by other methods. The correlation between the ROS-induced cytoplasmic membrane damage and the PMDR of dyes quantifies sensitivity against bactericidal antibiotics, which correlates to antibiotic-induced lipid peroxidation. Significantly, our findings largely extend to clinical isolates of E. coli and other ESKAPE pathogens like K. pneumoniae and Enterobacter subspecies. Our data reveal that 4AP-Cn probes can potentially act as precise scales to detect antibiotic-induced membrane damage ("thinning") occurring at a subnanometer scale through the quantification of dyes' PMDR, making them promising membrane dyes for rapid detection of bacterial antibiotic resistance, distinguishing sensitive and resistant infections with high specificity in a clinical setup.

Inducible biosynthesis of bacterial cellulose in recombinant Enterobacter sp. FY-07.

Bacterial cellulose (BC) is an extracellular polysaccharide with myriad unique properties, such as high purity, water-holding capacity and biocompatibility, making it attractive in materials science. However, genetic engineering techniques for BC-producing microorganisms are rare. Herein, the electroporation-based gene transformation and the λ Red-mediated gene knockout method with a nearly 100 % recombination efficiency were established in the fast-growing and BC hyperproducer Enterobacter sp. FY-07. This genetic manipulation toolkit was validated by inactivating the protein subunit BcsA in the cellulose synthase complex. Subsequently, the inducible BC-producing strains from glycerol were constructed through inducible expression of the key gene fbp in the gluconeogenesis pathway, which recovered >80 % of the BC production. Finally, the BC properties analysis results indicated that the induced-synthesized BC pellicles were looser, more porous and reduced crystallinity, which could further broaden the application prospects of BC. To our best knowledge, this is the first attempt to construct the completely inducible BC-producing strains. Our work paves the way for increasing BC productivity by metabolic engineering and broadens the available fabrication methods for BC-based advanced functional materials.

Occurrence of multi-carbapenemase-producing Enterobacterales in a tertiary hospital in Madrid (Spain): A new epidemiologic scenario.

INTRODUCTION: Multi-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021-2022). METHODS: We collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected. RESULTS: Overall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.26-0.28 cases per 1000 admissions). OXA-48 + KPC-3 (7/23) and KPC-3 + VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam + aztreonam combination was synergistic in all metallo-ß-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1 + OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of a Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3 + VIM-1-ST29-K. pneumoniae, NDM-1 + OXA-48-ST147-K. pneumoniae and KPC-2 + VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1 + KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48 + KPC-3. CONCLUSION: M-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-ß-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures.