The role of Ni- and Cd-resistant rhizobacteria in promoting the growth of rice seedlings and alleviating the combined phytotoxicity of Ni and Cd.

The problem of potentially toxic metal pollution is increasingly acute with the development of human society. In this study, we investigated the remediation of nickel (Ni) and cadmium (Cd) co-contamination through inoculating rice with three new-isolated Ni- and Cd-resistant plant growth-promoting rhizobacteria (PGPR) Y3, Y4, and Y5. These three strains possessed growth-promoting properties, including 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, the ability of indoleacetic acid (IAA) production, phosphate solubilization, siderophores production, and exopolysaccharide (EPS) development. According to 16S rDNA sequence homology, strains Y3, Y4, and Y5 were identified as Pseudomonas sp., Chryseobacterium sp., and Enterobacter sp., respectively. Based on the results of rice germination experiments conducted under combined toxicity, we set the contamination concentrations for Ni2+ at 20 µg mL-1 and Cd2+ at 40 µg mL-1. Then we conducted potting experiments at these concentration levels to study the effects of strains Y3, Y4, and Y5 on rice growth under synergistic Ni and Cd stress. The results indicated that the inoculated strains Y3, Y4, and Y5 were effective in promoting the growth of rice seedlings under the combined stress of Ni and Cd, and conferring tolerance to Ni and Cd by increasing the antioxidant enzyme activities of the seedlings. Among them, strain Y3 exhibited stronger ACC deaminase activity, IAA production capacity, and EPS production capacity, showing the most pronounced growth-promoting effect on rice. It was demonstrated that after inoculation with strain Y3, the germination rate of rice seeds increased by 43 %, the fresh weight of stems improved by 35 %, and the chlorophyll content enhanced by 70 % and other growth-promoting phenomena. Additionally, under Ni and Cd stress, strain Y5 performed better than strain Y4 in terms of IAA production capacity and its influence on rice root growth, suggesting that IAA production might play a specifically essential role in root growth under Ni and Cd stress.

Nosocomial cluster of patients infected with imipenemase-1-producing Enterobacter ludwigii.

Introduction. Imipenemase (IMI) enzymes are an uncommon class A carbapenemases that have been isolated from aquatic environments and, occasionally, from clinical isolates of Enterobacterales.Aim. We describe a cluster of three patients infected by IMI-1 carbapenemase-producing Enterobacter ludwigii (IMI-1-Elud) in a tertiary university hospital in Gran Canaria, Spain.Methodology. Antimicrobial susceptibility was determined using the Vitek2 AST-N355 card and antibiotic gradient strips. The modified carbapenem inactivation method (CIM) test was performed in cases where the ertapenem MIC value was higher than 0.125 mg l-1. The carbapenemase was identified by PCR and DNA microarray and later characterized by whole-genome next-generation sequencing (NGS) with Illumina.Results. Three patients presented thoracic or abdominal infections caused by IMI-1-Elud ST1677 from 14 June 2022 to 14 July 2022. All patients underwent at least one gastroscopy during their admission, and two of them were located in adjoining rooms. Isolates were resistant to carbapenems, colistin and fosfomycin but susceptible to ciprofloxacin. IMI/NMC-A carbapenemase was detected by PCR and hybridization test and confirmed by NGS as IMI-1. All patients underwent at least one gastroscopy, and two of them were in nearby rooms. Patients showed microbiological and clinical improvement following focus drainage and targeted antibiotic treatment with a fluoroquinolone.Conclusions. This study reports the first documented global outbreak of patients infected with IMI-1-Elud. The source appeared to be related to endoscopes. Contact transmission may also have played a role. A screening method such as the modified CIM test is crucial for detecting less common carbapenemases that might not be identified by rapid molecular or immunochromatographic tests, as these often do not include bla IMI genes, which could lead to the undetected dissemination of carbapenemase-producing Enterobacterales. Effective infection source control and targeted treatment are essential for achieving a favourable clinical outcome.

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.

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.

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.

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.

Semantic search using protein large language models detects class II microcins in bacterial genomes.

Class II microcins are antimicrobial peptides that have shown some potential as novel antibiotics. However, to date, only 10 class II microcins have been described, and the discovery of novel microcins has been hampered by their short length and high sequence divergence. Here, we ask if we can use numerical embeddings generated by protein large language models to detect microcins in bacterial genome assemblies and whether this method can outperform sequence-based methods such as BLAST. We find that embeddings detect known class II microcins much more reliably than does BLAST and that any two microcins tend to have a small distance in embedding space even though they typically are highly diverged at the sequence level. In data sets of Escherichia coli, Klebsiella spp., and Enterobacter spp. genomes, we further find novel putative microcins that were previously missed by sequence-based search methods. IMPORTANCE: Antibiotic resistance is becoming an increasingly serious problem in modern medicine, but the development pipeline for conventional antibiotics is not promising. Therefore, alternative approaches to combat bacterial infections are urgently needed. One such approach may be to employ naturally occurring antibacterial peptides produced by bacteria to kill competing bacteria. A promising class of such peptides are class II microcins. However, only a small number of class II microcins have been discovered to date, and the discovery of further such microcins has been hampered by their high sequence divergence and short length, which can cause sequence-based search methods to fail. Here, we demonstrate that a more robust method for microcin discovery can be built on the basis of a protein large language model, and we use this method to identify several putative novel class II microcins.

Halotolerant Enterobacter asburiae A103 isolated from the halophyte Salix linearistipularis: Genomic analysis and growth-promoting effects on Medicago sativa under alkali stress.

Soil salinization negatively affects plant growth and threatens food security. Halotolerant plant growth-promoting bacteria (PGPB) can alleviate salt stress in plants via diverse mechanisms. In the present study, we isolated salt-tolerant bacteria with phosphate-solubilizing abilities from the rhizosphere of Salix linearistipularis, a halophyte distributed in saline-alkali soils. Strain A103 showed high phosphate solubilization activity and was identified as Enterobacter asburiae based on genome analysis. In addition, it can produce indole-3-acetic acid (IAA), siderophores, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Genome mining has also revealed the presence of several functional genes involved in the promotion of plant growth. Inoculation with A103 markedly improved alfalfa growth in the presence of 100 mM NaHCO3. Under alkali stress, the shoot and root dry weights after bacterial inoculation improved by 42.9 % and 21.9 %, respectively. Meanwhile, there was a 35.9-37.1 % increase in the shoot and root lengths after treatment with A103 compared to the NaHCO3-treated group. Soluble sugar content, peroxidase and catalase activities increased in A103-inoculated alfalfa under alkaline stress. A significant decrease in the malondialdehyde content was observed after treatment with strain A103. Metabolomic analysis indicated that strain A103 positively regulated alkali tolerance in alfalfa through the accumulation of metabolites, such as homocarnosine, panthenol, and sorbitol, which could reduce oxidative damage and act as osmolytes. These results suggest that halophytes are valuable resources for bioprospecting halotolerant beneficial bacteria and that the application of halotolerant growth-promoting bacteria is a natural and efficient strategy for developing sustainable agriculture.

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.

Honeybee gut bacterial strain improved survival and gut microbiota homeostasis in Apis mellifera exposed in vivo to clothianidin.

Pesticides are causing honeybee mortality worldwide. Research carried out on honeybees indicates that application of pesticides has a significant impact on the core gut community, which ultimately leads to an increase in the growth of harmful pathogens. Disturbances caused by pesticides also affect the way bacterial members interact, which results in gut microbial dysbiosis. Administration of beneficial microbes has been previously demonstrated to be effective in treating or preventing disease in honeybees. The objective of this study was to measure under in vivo conditions the ability of two bacterial strains (the Enterobacter sp. and Pantoea sp.) isolated from honeybee gut to improve survival and mitigate gut microbiota dysbiosis in honeybees exposed to a sublethal clothianidin dose (0.1 ppb). Both gut bacterial strains were selected for their ability to degrade clothianidin in vitro regardless of their host-microbe interaction characteristics (e.g., beneficial, neutral, or harmful). To this end, we conducted cage trials on 4- to 6-day-old newly emerging honeybees. During microbial administration, we jointly monitored the taxonomic distribution and activity level of bacterial symbionts quantifying 16S rRNA transcripts. First, curative administration of the Pantoea sp. strain significantly improved the survival of clothianidin-exposed honeybees compared to sugar control bees (i.e., supplemented with sugar [1:1]). Second, curative administration of the Enterobacter sp. strain significantly mitigated the clothianidin-induced dysbiosis observed in the midgut structural network, but without improving survival. IMPORTANCE: The present work suggests that administration of bacterial strains isolated from honeybee gut may promote recovery of gut microbiota homeostasis after prolonged clothianidin exposure, while improving survival. This study highlights that gut bacterial strains hold promise for developing efficient microbial formulations to mitigate environmental pesticide exposure in honeybee colonies.

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.

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.

Transmission of global clones of NDM-producing Enterobacterales and interspecies spread of IncX3 plasmid harbouring blaNDM-5 in Tokyo.

OBJECTIVE: The aim of this study is to characterise the molecular characteristics of NDM-producing Enterobacterales, which have been on the increase in recent years in Japan, where IMP-producing bacteria are dominant among carbapenemase-producing Enterobacterales. METHODS: We collected 21 strains of NDM-producing Enterobacterales detected between 2015 and 2022 at five hospitals in Tokyo and performed illumina whole genome sequencing. For the seven selected strains, nanopore long-read sequencing was also performed to characterise the plasmids harbouring blaNDM. RESULTS: Fourteen strains were Escherichia coli and all carried blaNDM-5. Among these strains, eight and three were sequence type (ST) 410 and ST167, respectively, and both groups of strains were spread clonally in different hospitals. Two strains of Klebsiella pneumoniae ST147 carrying blaNDM-1 were detected in a hospital, and these strains had also spread clonally. The remainder included Enterobacter hormaechei, Klebsiella quasipneumoniae, Citrobacter amalonaticus, and Klebsiella michiganensis. Plasmid analysis revealed that an identical IncX3 plasmid harbouring blaNDM-5 was shared among four strains of different bacterial species (E. coli, C. amalonaticus, K. michiganensis, and E. hormaechei) detected at the same hospital. In addition, a Klebsiella quasipneumoniae strain detected at a different hospital also carried an IncX3 plasmid with a similar genetic structure. CONCLUSIONS: Nosocomial spread of multiple multidrug-resistant global clones and transmission of IncX3 plasmids harbouring blaNDM-5 among multiple species were detected as the major pathways of spread of NDM-producing Enterobacterales in Tokyo. Early detection of carriers and measures to prevent nosocomial spread are important to prevent further spread of NDM-producing organisms.

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.

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.

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.

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.

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.

The therapeutic potential of indole hybrids, dimers, and trimers against drug-resistant ESKAPE pathogens.

Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter (ESKAPE) species as causative agents are characterized by increased levels of resistance toward multiple classes of first-line as well as last-resort antibiotics and represent serious global health concerns, creating a critical need for the development of novel antibacterials with therapeutic potential against drug-resistant ESKAPE species. Indole derivatives with structural and mechanistic diversity demonstrated broad-spectrum antibacterial activity against various clinically important pathogens including drug-resistant ESKAPE. Moreover, several indole-based agents that are exemplified by creatmycin have already been used in clinics or under clinical trials for the treatment of bacterial infections, demonstrating that indole derivatives hold great promise for the development of novel antibacterials. This review is an endeavor to highlight the current scenario of indole hybrids, dimers, and trimers with therapeutic potential against drug-resistant ESKAPE pathogens, covering articles published from 2020 to the present, to open new avenues for the exploration of novel antidrug-resistant ESKAPE candidates.

Whole-Genome Analysis of Extensively Drug-Resistant Enterobacter hormaechei Isolated from a Patient with Non-Hodgkin's Lymphoma.

BACKGROUND: Currently, the Enterobacteriaceae species are responsible for a variety of serious infections and are already considered a global public health problem, especially in underdeveloped countries, where surveillance and monitoring programs are still scarce and limited. Analyses were performed on the complete genome of an extensively antibiotic-resistant strain of Enterobater hormaechei, which was isolated from a patient with non-Hodgkin's lymphoma, who had been admitted to a hospital in the city of Manaus, Brazil. METHODS: Phenotypical identification and susceptibility tests were performed in automated equipment. Total DNA extraction was performed using the PureLink genomic DNA mini-Kit. The genomic DNA library was prepared with Illumina Microbial Amplicon Prep and sequenced in the MiSeq Illumina Platform. The assembly of the whole-genome and individual analyses of specific resistance genes extracted were carried out using online tools and the Geneious Prime software. RESULTS: The analyses identified an extensively resistant ST90 clone of E. hormaechei carrying different genes, including blaCTX-M-15, blaGES-2, blaTEM-1A, blaACT-15, blaOXA-1 and blaNDM-1, [aac(3)-IIa, aac(6')-Ian, ant(2″)-Ia], [aac(6')-Ib-cr, (qnrB1)], dfrA25, sul1 and sul2, catB3, fosA, and qnrB, in addition to resistance to chlorhexidine, which is widely used in patient antisepsis. CONCLUSIONS: These findings highlight the need for actions to control and monitor these pathogens in the hospital environment.