Comparative genomics of plant growth promoting phosphobacteria isolated from acidic soils.

Despite being one of the most abundant elements in soil, phosphorus (P) often becomes a limiting macronutrient for plants due to its low bioavailability, primarily locked away in insoluble organic and inorganic forms. Phosphate solubilizing and mineralizing bacteria, also called phosphobacteria, isolated from P-deficient soils have emerged as a promising biofertilizer alternative, capable of converting these recalcitrant P forms into plant-available phosphates. Three such phosphobacteria strains-Serratia sp. RJAL6, Klebsiella sp. RCJ4, and Enterobacter sp. 198-previously demonstrated their particular strength as plant growth promoters for wheat, ryegrass, or avocado under abiotic stresses and P deficiency. Comparative genomic analysis of their draft genomes revealed several genes encoding key functionalities, including alkaline phosphatases, isonitrile secondary metabolites, enterobactin biosynthesis and genes associated to the production of indole-3-acetic acid (IAA) and gluconic acid. Moreover, overall genome relatedness indexes (OGRIs) revealed substantial divergence between Serratia sp. RJAL6 and its closest phylogenetic neighbours, Serratia nematodiphila and Serratia bockelmanii. This compelling evidence suggests that RJAL6 merits classification as a novel species. This in silico genomic analysis provides vital insights into the plant growth-promoting capabilities and provenance of these promising PSRB strains. Notably, it paves the way for further characterization and potential application of the newly identified Serratia species as a powerful bioinoculant in future agricultural settings.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Genotypic analyses of IncHI2 plasmids from enteric bacteria.

Incompatibility (Inc) HI2 plasmids are large (typically > 200 kb), transmissible plasmids that encode antimicrobial resistance (AMR), heavy metal resistance (HMR) and disinfectants/biocide resistance (DBR). To better understand the distribution and diversity of resistance-encoding genes among IncHI2 plasmids, computational approaches were used to evaluate resistance and transfer-associated genes among the plasmids. Complete IncHI2 plasmid (N = 667) sequences were extracted from GenBank and analyzed using AMRFinderPlus, IntegronFinder and Plasmid Transfer Factor database. The most common IncHI2-carrying genera included Enterobacter (N = 209), Escherichia (N = 208), and Salmonella (N = 204). Resistance genes distribution was diverse, with plasmids from Escherichia and Salmonella showing general similarity in comparison to Enterobacter and other taxa, which grouped together. Plasmids from Enterobacter and other taxa had a higher prevalence of multiple mercury resistance genes and arsenic resistance gene, arsC, compared to Escherichia and Salmonella. For sulfonamide resistance, sul1 was more common among Enterobacter and other taxa, compared to sul2 and sul3 for Escherichia and Salmonella. Similar gene diversity trends were also observed for tetracyclines, quinolones, ß-lactams, and colistin. Over 99% of plasmids carried at least 25 IncHI2-associated conjugal transfer genes. These findings highlight the diversity and dissemination potential for resistance across different enteric bacteria and value of computational-based approaches for the resistance-gene assessment.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Regional dissemination of NDM-1 producing Enterobacter hormaechei ST1740, with a subset of strains co-producing VIM-4 or IMP-13, France, 2019 to 2022.

BackgroundFrom 2019 to 2022, the French National Reference Centre for Antibiotic Resistance (NRC) received a total of 25 isolates of Enterobacter hormaechei subsp. hoffmannii sequence type (ST)1740. All produced metallo-ß-lactamase(s) and were from the Lyon area.AimTo understand these strains' spread and evolution, more extended microbiological and molecular analyses were conducted.MethodsPatients' demographics and specimen type related to isolates were retrieved. All strains underwent short-read whole genome sequencing, and for 15, long-read sequencing to understand carbapenemase-gene acquisition. Clonal relationships were inferred from core-genome single nt polymorphisms (SNPs). Plasmids and the close genetic environment of each carbapenemase-encoding gene were analysed.ResultsPatients (10 female/15 male) were on average 56.6 years old. Seven isolates were recovered from infections and 18 through screening. With ≤ 27 SNPs difference between each other's genome sequences, the 25 strains represented a clone dissemination. All possessed a chromosome-encoded bla NDM-1 gene inside a composite transposon flanked by two IS3000. While spreading, the clone independently acquired a bla VIM-4-carrying plasmid of IncHI2 type (n = 12 isolates), or a bla IMP-13-carrying plasmid of IncP-1 type (n = 1 isolate). Of the 12 isolates co-producing NDM-1 and VIM-4, seven harboured the colistin resistance gene mcr9.2; the remaining five likely lost this gene through excision.ConclusionThis long-term outbreak was caused by a chromosome-encoded NDM-1-producing ST1740 E. hormaechei subsp. hoffmannii clone, which, during its dissemination, acquired plasmids encoding VIM-4 or IMP-13 metallo-ß-lactamases. To our knowledge, IMP-13 has not prior been reported in Enterobacterales in France. Epidemiological and environmental investigations should be considered alongside microbiological and molecular ones.

Dramatic increase in antimicrobial resistance in ESKAPE clinical isolates over the 2010-2020 decade in India.

RATIONALE AND OBJECTIVES: ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) constitute a threat to humans worldwide. India is now the most populous country. The goal was to investigate the evolution of the rates of antimicrobial resistance in ESKAPE pathogens across India over the 2010-20 decade. METHODS: The data (89 studies) were retrieved from the Medline PubMed repository using specific keywords. RESULTS: The study of 20 177 ESKAPE isolates showed that A. baumannii isolates were the most represented (35.9%, n = 7238), followed by P. aeruginosa (25.3%, n = 5113), K. pneumoniae (19.5%, n = 3934), S. aureus (16.3%, n = 3286), E. faecium (2.6%, n = 517) and Enterobacter spp. (0.4%, n = 89). A notable increase in the resistance rates to antimicrobial agents occurred over the 2010-20 decade. The most important levels of resistance were observed in 2016-20 for A. baumannii (90% of resistance to the amoxicillin-clavulanate combination) and K. pneumoniae (81.6% of resistance to gentamycin). The rise in ß-lactamase activities was correlated with an increase in the positivity of Gram-negative isolates for ß-lactamase genes. CONCLUSIONS: This review highlighted that, in contrast to developed countries that kept resistance levels under control, a considerable increase in resistance to various classes of antibiotics occurred in ESKAPE pathogens in India over the 2010-2020 decade.

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani.

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.

High ceftazidime-avibactam resistance among carbapenem-resistant Enterobacter species: Data from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2014-2021.

OBJECTIVES: Trends in the susceptibility to ceftazidime-avibactam (CZA) and tigecycline (TGC) among Enterobacter species from different geographic areas are unknown.This study aimed to analyse the trends in CZA and TGC susceptibility changes across different continents from 2014 to 2021 utilizing Antimicrobial Testing Leadership and Surveillance (ATLAS) data. METHODS: A total of 23 669 isolates of Enterobacter species were collected over an 8-y period. RESULTS: The overall non-susceptibility rate of Enterobacter isolates to both CZA and TGC was 3.2%. India (16.5%), Guatemala (15.4%), and the Philippines (13.1%) exhibited the highest resistance to CZA. The increase in CZA resistance rates was particularly evident in Asia, with an increase from 4.0% to 8.3%, and in Latin America, from 1.5% to 5%. The non-susceptibility rate for TGC mildly increased in Africa/Middle East but decreased in other continents during the study period. The overall rate of carbapenem resistance increased from 2.9% in 2014-2017 to 4.3% in 2018-2021. Among carbapenem-resistant Enterobacter isolates, the CZA resistance rate was highest in Asia (87.4%), followed by Europe (69.2%) and Africa/Middle East (60.8%). Among the 380 Enterobacter isolates resistant to CZA and carbapenem, the most common genotype of carbapenemase genes was blaNDM (59.2%), followed by blaVIM (24.2%), blaOXA (4.2%), blaIMP (1.1%), and blaKPC (1.1%). The susceptibility of carbapenem-resistant Enterobacter to TGC remained high, with an overall susceptibility rate of 90%. CONCLUSIONS: The heterogeneous distribution of CZA resistance rates among different geographical regions highlights the divergent therapeutic options for drug-resistant Enterobacter species.

Unravelling the transcriptome response of Enterobacter sp. S-33 under varying temperature.

Enterobacter genus includes the bacteria occupying every aspect of environment, however, their adaptability at varying temperature is not clear. In the present study, we analyzed the transcriptome response of Enterobacter sp. S-33 and their functional genes under various temperatures (30-45 ℃) that were expressed and accumulated in cells under temperature-stress. During a temperature shift from 37 to 45 ℃, 165 genes showed differential expression including 112 up-regulated and 53 down-regulated. In particular, heat-shock genes such as CspA, 16 kDa heat shock protein A/B and transcriptional regulators such as LysR, TetR, and LuxR were differentially expressed, indicating the role of complex molecular mechanism of Enterobacter adapting to temperature stress. Similarly, genes associated to signal transduction, ABC transporters, iron homeostasis, and quorum sensing were also induced. The Gene ontology enrichment analysis of differentially expressed genes (DEGs) were categorized into "transmembrane transport", "tRNA binding", "hydrogen sulfide biosynthetic process" and "sulfate assimilation" terms. In addition, Kyoto Encyclopedia of Genes and Genomes pathways showed that ABC transporter as well as quorum sensing pathways were significantly enriched. Overall, current study has contributed to explore the adaptive molecular mechanisms of Enterobacter spp. upon temperature change, which further opens new avenues for future in-depth functional studies.

High genetic diversity and different type VI secretion systems in Enterobacter species revealed by comparative genomics analysis.

The human-pathogenic Enterobacter species are widely distributed in diverse environmental conditions, however, the understanding of the virulence factors and genetic variations within the genus is very limited. In this study, we performed comparative genomics analysis of 49 strains originated from diverse niches and belonged to eight Enterobacter species, in order to further understand the mechanism of adaption to the environment in Enterobacter. The results showed that they had an open pan-genome and high genomic diversity which allowed adaptation to distinctive ecological niches. We found the number of secretion systems was the highest among various virulence factors in these Enterobacter strains. Three types of T6SS gene clusters including T6SS-A, T6SS-B and T6SS-C were detected in most Enterobacter strains. T6SS-A and T6SS-B shared 13 specific core genes, but they had different gene structures, suggesting they probably have different biological functions. Notably, T6SS-C was restricted to E. cancerogenus. We detected a T6SS gene cluster, highly similar to T6SS-C (91.2%), in the remote related Citrobacter rodenitum, suggesting that this unique gene cluster was probably acquired by horizontal gene transfer. The genomes of Enterobacter strains possess high genetic diversity, limited number of conserved core genes, and multiple copies of T6SS gene clusters with differentiated structures, suggesting that the origins of T6SS were not by duplication instead by independent acquisition. These findings provide valuable information for better understanding of the functional features of Enterobacter species and their evolutionary relationships.

Multidrug-resistant pathogens contaminate river water used in irrigation in disenfranchised communities.

OBJECTIVES: The contamination of fresh surface waters poses a significant burden on human health and prosperity, especially in marginalized communities with limited resources and inadequate infrastructure. Here, we performed in-depth genomic analyses of multidrug-resistant bacteria (MDR-B) isolated from Al-Oueik river water that is used for irrigation of agricultural fields in a disenfranchised area that also hosts a makeshift Syrian refugee camp. METHODS: A composite freshwater sample was filtered. Faecal coliforms were counted and extended spectrum cephalosporins and/or ertapenem resistant bacteria were screened. Isolates were identified using MALDI-TOF-MS and analysed using whole-genome sequencing (WGS) to identify the resistome, sequence types, plasmid types, and virulence genes. RESULTS: Approximately 106 CFU/100 mL of faecal coliforms were detected in the water. Four drug-resistant Gram-negative bacteria were identified, namely Escherichia coli, Klebsiella pneumoniae, Enterobacter hormaechei, and Pseudomonas otitidis. Notably, the E. coli isolate harboured blaNDM-5 and a YRIN-inserted PBP3, representing an emerging public health challenge. The K. pneumoniae isolate carried blaSHV-187 as well as mutations in the gene encoding the OmpK37 porin. Enterobacter hormaechei and P. otitidis harboured blaACT-16 and blaPOM-1, respectively. CONCLUSION: This report provides comprehensive genomic analyses of MDR-B in irrigation water in Lebanon. Our results further support that irrigation water contaminated with faecal material can be a reservoir of important MDR-B, which can spread to adjacent agricultural fields and other water bodies, posing both public health and food safety issues. Therefore, there is an urgent need to implement effective water quality monitoring and management programs to control the proliferation of antibiotic-resistant pathogens in irrigation water in Lebanon.

Insight into a natural novel histidine decarboxylase gene deletion in Enterobacter hormaechei RH3 from traditional Sichuan-style sausage.

Histamine (HIS) is primarily formed from decarboxylated histidine by certain bacteria with histidine decarboxylase (hdc) activity and is the most toxic biogenic amine. Hdc, which is encoded by the hdc gene, serves as a key enzyme that controls HIS production in bacteria. In this paper, we characterized the changes in microbial and biogenic amines content of traditional Sichuan-style sausage before and after storage and demonstrated that Enterobacteriaceae play an important role in the formation of HIS. To screen for Enterobacteriaceae with high levels of HIS production, we isolated strain RH3 which has a HIS production of 2.27 mg/mL from sausages stored at 37°C for 180 days, using selective media and high-performance liquid chromatography. The strain RH3 can produce a high level of HIS after 28 h of fermentation with a significant hysteresis. Analysis of the physicochemical factors revealed that RH3 still retained its ability to partially produce HIS in extreme environments with pH 3.5 and 10.0. In addition, RH3 exhibited excellent salt tolerance (6.0% NaCl and 1.0% NaNO2 ). Subsequently, RH3 was confirmed as Enterobacter hormaechei with hdc gene deletion by PCR, western blot, and whole-genome sequencing analysis. Furthermore, RH3 exhibited pathogenicity rate of 75.60% toward the organism, indicating that it was not a food-grade safe strain, and demonstrated a high level of conservation in intraspecific evolution. The results of this experiment provide a new reference for studying the mechanism of HIS formation in microorganisms. PRACTICAL APPLICATION: This study provides a new direction for investigating the mechanism of histamine (HIS) formation by microorganisms and provides new insights for further controlling HIS levels in meat products. Further research can control the key enzymes that form HIS to control HIS levels in food.

Genome sequence-based identification of Enterobacter strains and description of Enterobacter pasteurii sp. nov.

IMPORTANCE: Accurate taxonomy is essential for microbial biological resource centers, since the microbial resources are often used to support new discoveries and subsequent research. Here, we used genome sequence data, alongside matrix-assisted laser desorption/ionization time-of-flight mass spectrometer biotyper-based protein profiling, to accurately identify six Enterobacter cloacae complex strains. This approach effectively identified distinct species within the E. cloacae complex, including Enterobacter asburiae, "Enterobacter xiangfangensis," and Enterobacter quasihormaechei. Moreover, the study revealed the existence of a novel species within the Enterobacter genus, for which we proposed the name Enterobacter pasteurii sp. nov. In summary, this study demonstrates the significance of adopting a genome sequence-driven taxonomy approach for the precise identification of bacterial strains in a biological resource center and expands our understanding of the E. cloacae complex.

Endophytic Alkalotolerant Plant Growth-Promoting Bacteria Render Maize (Zea mays L.) Growth Under Alkaline Stress.

This study investigates the role of bacterial endophytes from extreme alkaline environments in alleviating alkaline stress and plant development. Stressful environmental factors, such as soil acidity and alkalinity/sodicity, frequently affect plant development. In the present study, alkaline-tolerant endophytic strains were isolated from three plant species Saccharum munja, Calotropis procera, and Chenopodium album, and 15 out of the total of 48 isolates were selected for further examination of their abiotic stress tolerance. Molecular analysis based on 16S rRNA gene sequencing revealed strains from Enterobacter, Acinetobacter, Stenotrophomonas, Bacillus, Lysinibacillus, and Mammaliicoccus genera. Out of 15 isolates based on their quantitative PGP traits and abiotic stress tolerance, 6 were finally selected for greenhouse experiments. Under alkaline conditions, results demonstrated that the strains from the genera Enterobacter, Bacillus, Stenotrophomonas, and Lysinibacillus had beneficial effects on maize growth. These findings suggest that using a combination of bacteria with multiple plant growth-promoting attributes could be a sustainable approach to enhance agricultural yield, even in a challenging alkaline environment. The study concludes that the application of bacterial endophytes from plants growing in extremely alkaline environments might provide other plants with similar stress-tolerance abilities. The outcome of the study provides a basis for future exploration of the mechanisms underlying endophyte-induced stress tolerance.

Genomic and biotechnological potential of a novel oil-degrading strain Enterobacter kobei DH7 isolated from petroleum-contaminated soil.

In this study, a novel oil-degrading strain Enterobacter kobei DH7 was isolated from petroleum-contaminated soil samples from the industrial park in Taolin Town, Lianyungang, China. The whole genome of the strain was sequenced and analyzed to reveal its genomic potential. The oil degradation and growth conditions including nitrogen, and phosphorus sources, degradation cycle, biological dosing, pH, and oil concentration were optimized to exploit its commercial application. The genome of the DH7 strain contains 4,705,032 bp with GC content of 54.95% and 4653 genes. The genome analysis revealed that there are several metabolic pathways and enzyme-encoding genes related to oil degradation in the DH7 genome, such as the paa gene cluster which is involved in the phenylacetic acid degradation pathway, and complete degradation pathways for fatty acid and benzoate, genes related to chlorinated alkanes and olefins degradation pathway including adhP, frmA, and adhE, etc. The strain DH7 under the optimized conditions has demonstrated a maximum degradation efficiency of 84.6% after 14 days of treatment using synthetic oil, which comparatively displays a higher oil degradation efficiency than any Enterobacter species known to date. To the best of our knowledge, this study presents the first-ever genomic studies related to the oil degradation potential of any Enterobacter species. As Enterobacter kobei DH7 has demonstrated significant oil degradation potential, it is one of the good candidates for application in the bioremediation of oil-contaminated environments.

Whole genomic sequence of Enterobacter sichuanensis AJI 2411 - A plant growth promoting rhizobacteria.

Enterobacter sichuanensis AJI 2411 is a rhizobacteria displaying plant growth promoting potentials, which was isolated from the rhizosphere of soybeans in Ede, Osun State, Nigeria. The full genome of Enterobacter sichuanensis AJI 2411 was sequenced and reported in this study to shed light on the molecular mechanisms that aids the bacteria's plant growth-promoting abilities.

Genome-Based Epidemiologic Analysis of VIM/IMP Carbapenemase-Producing Enterobacter spp., Poland.

We sequenced all nonduplicate 934 VIM/IMP carbapenemase-producing Enterobacterales (CPE) reported in Poland during 2006-2019 and found ≈40% of the isolates (n = 375) were Enterobacter spp. During the study period, incidence of those bacteria gradually grew in nearly the entire country. The major factor affecting the increase was clonal spread of several E. hormaechei lineages responsible for multiregional and interregional outbreaks (≈64% of all isolates), representing mainly the pandemic sequence type (ST) 90 or the internationally rare ST89 and ST121 clones. Three main VIM-encoding integron types efficiently disseminated across the clone variants (subclones) with various molecular platforms. Those variants were predominantly Pseudomonas aeruginosa-derived In238-like elements, present with IncHI2+HI2A, IncFII+FIA, IncFIB, or IncN3 plasmids, or chromosomal genomic islands in 30 Enterobacter STs. Another prevalent type, found in 34 STs, were In916-like elements, spreading in Europe recently with a lineage of IncA-like plasmids.

Molecular characterization and in-depth genome analysis of Enterobacter sp. S-16.

Enterobacter species are considered to be an opportunistic human pathogen owing to the existence of antibiotic-resistant strains and drug resides; however, the detailed analysis of the antibiotic resistance and virulence features in environmental isolates is poorly characterized. Here, in the study, we characterized the biochemical characteristics, and genome, pan-genome, and comparative genome analyses of an environmental isolate Enterobacter sp. S-16. The strain was identified as Enterobacter spp. by using 16S rRNA gene sequencing. To unravel genomic features, whole genome of Enterobacter sp. S-16 was sequenced using a hybrid assembly approach and genome assembly was performed using the Unicycler tool. The assembled genome contained the single conting size 5.3 Mbp, GC content 55.43%, and 4500 protein-coding genes. The genome analysis revealed the various gene clusters associated with virulence, antibiotic resistance, type VI secretion system (T6SS), and many stress tolerant genes, which may provide important insight for adapting to changing environment conditions. Moreover, different metabolic pathways were identified that potentially contribute to environmental survival. Various hydrolytic enzymes and motility functions equipped the strain S-16 as an active colonizer. The genome analysis confirms the presence of carbohydrate-active enzymes (CAZymes), and non-enzymatic carbohydrate-binding modules (CBMs) involved in the hydrolysis of complex carbohydrate polymers. Moreover, the pan-genome analysis provides detailed information about the core genes and shared genes with the closest related Enterobacter species. The present study is the first report showing the presence of YdhE/NorM in Enterobacter spp. Thus, the elucidation of genome sequencing will increase our understanding of the pathogenic nature of environmental isolate, supporting the One Health Concept.

Resistance to aztreonam-avibactam due to a mutation of SHV-12 in Enterobacter.

Aztreonam-avibactam is an important option against Enterobacterales producing metallo-ß-lactamases (MBLs). We obtained an aztreonam-avibactam-resistant mutant of an MBL-producing Enterobacter mori strain by induced mutagenesis. Genome sequencing revealed an Arg244Gly (Ambler position) substitution of SHV-12 ß-lactamase in the mutant. Cloning and susceptibility testing verified that the SHV-12 Arg244Gly substitution led to significantly reduced susceptibility to aztreonam-avibactam (MIC, from 0.5/4 to 4/4 mg/L) but with the loss of resistance to cephalosporins as tradeoff. Arg244 of SHV involves in the binding of avibactam by forming an arginine-mediated salt bridge and is a critical residue to interact with ß-lactams. Molecular modeling analysis demonstrated that the Arg244Gly substitution hindered the binding of avibactam to SHV with higher binding energy (from - 5.24 to -4.32 kcal/mol) and elevated inhibition constant Ki (from 143.96 to 677.37 µM) to indicate lower affinity. This substitution, however, resulted in loss of resistance to cephalosporins as tradeoff by impairing substrate binding. This represents a new aztreonam-avibactam resistance mechanism.

Multidrug-resistant Enterobacter spp. in wastewater and surface water: Molecular characterization of ß-lactam resistance and metal tolerance genes.

Among the ESKAPE group pathogens, Enterobacter spp. is an opportunistic Gram-negative bacillus, widely dispersed in the environment, that causes infections. In the present study, samples of hospital wastewater, raw and treated urban wastewater, as well as surface receiving water, were collected to assess the occurrence of multidrug-resistant (MDR) Enterobacter spp. A molecular characterization of ß-lactam antibiotic resistance and metal tolerance genes was performed. According to identification by MALDI-TOF MS, 14 isolates were obtained: 7 E. bugandensis, 5 E. kobei, and 2 E. cloacae. The isolates showed resistance mainly to ß-lactam antibiotics, including those used to treat infections caused by MDR bacteria. Multiple antibiotic resistance index was calculated for all isolates. It allowed verify whether sampling points showed a high risk due to antibiotic resistant Enterobacter spp., as well as to determine if the isolates have been in environments with a frequent antibiotic use. Twelve isolates showed ß-lactam antibiotic resistance gene, being the blaKPC widely detected. Regarding metal tolerance, 13 isolates showed at least two genes that encode metal tolerance mechanisms. Overall, metal tolerance mechanisms to silver, copper, mercury, arsenic and tellurium were found. New data on metal tolerance mechanisms dispersion and antibiotic-resistance characterization of the E. bugandensis and E. kobei species were here provided. The occurrence of MDR Enterobacter spp. in analyzed samples draws attention to an urgent need to put control measures into practice. It also evidences waterborne spread of clinically important antibiotic-resistant bacteria recognized as critical priority pathogens.