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477300.9972Draft genome analysis for Enterobacter kobei, a promising lead bioremediation bacterium. Lead pollution of the environment poses a major global threat to the ecosystem. Bacterial bioremediation offers a promising alternative to traditional methods for removing these pollutants, that are often hindered by various limitations. Our research focused on isolating lead-resistant bacteria from industrial wastewater generated by heavily lead-containing industries. Eight lead-resistant strains were successfully isolated, and subsequently identified through molecular analysis. Among these, Enterobacter kobei FACU6 emerged as a particularly promising candidate, demonstrating an efficient lead removal rate of 83.4% and a remarkable lead absorption capacity of 571.9 mg/g dry weight. Furthermore, E. kobei FACU6 displayed a remarkable a maximum tolerance concentration (MTC) for lead reaching 3,000 mg/L. To further investigate the morphological changes in E. kobei FACU6 in response to lead exposure, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed. These analyses revealed significant lead adsorption and intracellular accumulation in treated bacteria in contrast to the control bacterium. Whole-genome sequencing was performed to gain deeper insights into E. kobei's lead resistance mechanisms. Structural annotation revealed a genome size of 4,856,454 bp, with a G + C content of 55.06%. The genome encodes 4,655 coding sequences (CDS), 75 tRNA genes, and 4 rRNA genes. Notably, genes associated with heavy metal resistance and their corresponding regulatory elements were identified within the genome. Furthermore, the expression levels of four specific heavy metal resistance genes were evaluated. Our findings revealed a statistically significant upregulation in gene expression under specific environmental conditions, including pH 7, temperature of 30°C, and high concentrations of heavy metals. The outstanding potential of E. kobei FACU6 as a source of diverse genes related to heavy metal resistance and plant growth promotion makes it a valuable candidate for developing safe and effective strategies for heavy metal disposal.202338260751
471110.9969Multi-omics analysis reveals interactions between host and microbes in Bama miniature pigs during weaning. INTRODUCTION: There are complex interactions between host and gut microbes during weaning, many of the mechanisms are not yet fully understood. Previous research mainly focuses on commercial pigs, whereas limited information has been known about the host and gut microbe interactions in miniature pigs. METHODS: To address the issue in Bama miniature piglets that were weaned 30 days after birth, we collected samples on days 25 and 36 for metabolomics, transcriptomics, and microgenomics analysis. RESULTS AND DISCUSSION: The average daily weight gain of piglets during weaning was only 58.1% and 40.6% of that during 0-25 days and 36-60 days. Metabolomic results identified 61 significantly different metabolites (SDMs), of which, the most significantly increased and decreased SDMs after weaning were ectoine and taurocholate, respectively, indicating the occurrence of inflammation. Metagenomic analysis identified 30 significantly different microbes before and after weaning. Bacteria related to decreasing intestinal inflammation, such as Megasphaera, Alistipes and Bifidobacterium, were enriched before weaning. While bacteria related to infection such as Chlamydia, Clostridium, Clostridioides, and Blautia were enriched after weaning. The carbohydrate enzymes CBM91, CBM13, GH51_1, and GH94 increase after weaning, which may contribute to the digestion of complex plant fibers. Furthermore, we found the composition of antibiotic resistance genes (ARGs) changed during weaning. Transcriptomic analysis identified 147 significantly differentially expressed genes (DEGs). The upregulated genes after weaning were enriched in immune response categories, whereas downregulated genes were enriched in protein degradation. Combining multi-omics data, we identified significant positive correlations between gene MZB1, genera Alistipes and metabolite stachydrine, which involve anti-inflammatory functions. The reduced abundance of bacteria Dialister after weaning had strong correlations with the decreased 2-AGPE metabolite and the downregulated expression of RHBDF1 gene. Altogether, the multi-omics study reflects dietary changes and gut inflammation during weaning, highlighting complex interactions between gut microbes, host genes and metabolites."202439723142
315520.9969In silico mapping of microbial communities and stress responses in a porcine slaughterhouse and pork products through its production chain, and the efficacy of HLE disinfectant. The use of shotgun metagenomic sequencing to understand ecological-level spread of microbes and their genes has provided new insights for the prevention, surveillance and control of microbial contaminants in the slaughterhouse environment. Here, microbial samples were collected from products and surrounding areas though a porcine slaughter process; shotgun metagenomic DNA-sequencing of these samples revealed a high community diversity within the porcine slaughterhouse and pork products, in zones originating from animal arrival through to the sale zones. Bacteria were more prevalent in the first zones, such as arrival- and anesthesia-zones, and DNA viruses were prevalent in the scorching-and-whip zone, animal products and sale zone. Data revealed the dominance of Firmicutes and Proteobacteria phyla followed by Actinobacteria, with a clear shift in the relative abundance of lactic acid bacteria (mainly Lactobacillus sp.) from early slaughtering steps to Proteobacteria and then to viruses suggesting site-specific community compositions occur in the slaughterhouse. Porcine-type-C oncovirus was the main virus found in slaughterhouse, which causes malignant diseases in animals and humans. As such, to guarantee food safety in a slaughterhouse, a better decipher of ecology and adaptation strategies of microbes becomes crucial. Analysis of functional genes further revealed high abundance of diverse genes associated with stress, especially in early zones (animal and environmental surfaces of arrival zone with 57,710 and 40,806 genes, respectively); SOS responsive genes represented the most prevalent, possibly associated with genomic changes responsible of biofilm formation, stringent response, heat shock, antimicrobial production and antibiotic response. The presence of several antibiotic resistance genes suggests horizontal gene transfer, thus increasing the likelihood for resistance selection in human pathogens. These findings are of great concern, with the suggestion to focus control measures and establish good disinfection strategies to avoid gene spread and microbial contaminants (bacteria and viruses) from the animal surface into the food chain and environment, which was achieved by applying HLE disinfectant after washing with detergent.202032846568
456530.9969Nodules-associated Klebsiella oxytoca complex: genomic insights into plant growth promotion and health risk assessment. The swift emergence of antibiotic resistance genes (ARGs) across interconnected One Health compartments poses a significant global threat. Although plant growth-promoting (PGP) bacteria possess numerous attributes beneficial to host plants, many of these bacteria also harbor ARGs, necessitating a focused assessment of their negative implications. In this context, here we performed whole genome sequencing of 14 PGP endophytic strains isolated from root nodules of faba beans, belonging to three Klebsiella oxytoca species complex (KoSC): K. grimontii (n = 5), K. michiganensis (n = 5), and K. pasteurii (n = 4). We performed comparative genomics, molecular typing, and pangenome analyses on these strains. We identified significant diversity within the KoSC population, classifying the strains into five sequence types (STs), three of which are novel to this study (ST-542, ST-569, and ST-629). Phylogenomic analysis revealed that the bacterial strains clustered more closely by ST than by their source of isolation. Annotation of gene clusters indicated that all assembled genomes are enriched with genes involved in PGP activities, alongside a robust array of genes conferring tolerance to abiotic stresses. Importantly, our findings disclosed that the 14 assembled genomes harbored multiple ARGs, conferring resistance to various antibiotic classes, with 71% of the population classified as multidrug-resistant based on the in vitro antibiotic susceptibility assay. Furthermore, all genomes contained an array of virulence factors critical for survival, pathogenesis, biofilm formation, and root colonization. In conclusion, this study substantiates the hypothesis that certain PGP bacteria may serve as potential reservoirs of multidrug resistance, posing significant public health risks. Thus, the future advancement of bacteria-based biofertilizers should integrate environmental considerations and monitor their impact on antibiotic resistance dissemination in soil ecosystems.202540375127
471440.9969Screening and genome analysis of heat-resistant and antioxidant lactic acid bacteria from Holstein cow milk. BACKGROUND: Heat stress significantly impacts dairy cows, primarily through oxidative stress, which undermines their health. The problem is exacerbated by the ongoing global warming trend. Lactic acid bacteria (LAB) are safe, economical, and readily accessible options for enhancing the host's antioxidant defenses and preventing oxidative damage. They have been proven effective in alleviating heat stress-related damage, making them an excellent choice for protecting dairy cows from the adverse effects of heat stress. METHOD: In this study, five strains of LAB from Holstein cow milk (Lactobacillus plantarum L5, L14, L17, L19, L20) were evaluated for their heat resistance and antioxidant capacity by evaluating the growth characteristics and tolerance of the strains under high-temperature conditions, as well as their H(2)O(2) tolerance, free radical scavenging ability (DPPH, OH(-), ABTS), reducing ability, and EPS production ability. Furthermore, we employed Caco-2 cells to assess the adhesion rate of the strain, thereby confirming its ability to successfully colonize the host's intestinal tract and ensuring the effective execution of its probiotic functions. The strain with excellent heat resistance and antioxidant capacity was then subjected to genomic analysis to gain insight into the molecular mechanisms behind their heat resistance, antioxidant capacity, and safety. RESULTS: Among the two strains, Lactobacillus plantarum L19 emerges as a highly promising candidate. The strain exhibits robust growth even at high temperatures at 40°C and maintains a survival rate of 16.42% at the extreme temperature of 65°C. Furthermore, it demonstrates superior tolerance to hydrogen peroxide (27.3%), and possesses a notably higher free radical scavenging capacity with a high adhesion rate to Caco-2 cell (22.19%) compared to the other four strains tested. Genomic analysis revealed its' genome has 17 genes related to antioxidants and three genes related to heat resistance. Importantly, L19 lacks any resistance genes, ensuring its safety as a probiotic. CONCLUSION: The results imply that Lactobacillus plantarum L19 has the potential to serve as an effective food additive in mitigating damages associated with heat stress. This research offers a valuable reference for the prevention and management of heat stress in dairy cows, while also expanding the scope of applications for LAB derived from cow milk.202439611093
471350.9969Characterization and Preliminary Safety Evaluation of Akkermansia muciniphila PROBIO. In addition to providing certain health advantages to the host, a bacterial strain must possess a clearly defined safety profile to be regarded as a probiotic. In this study, we present a thorough and methodical assessment of the safety of a novel strain of bacteria, Akkermansia muciniphila PROBIO, which was isolated from human feces. Firstly, we examined the strain's overall features, such as its gastrointestinal tolerance and its physiological and biochemical traits. Next, we verified its genotoxic properties through bacterial reverse mutation and in vitro mammalian cell micronucleus assays. The drug sensitivity of A. muciniphila PROBIO was subsequently examined through an analysis of its antibiotic resistance genes. Additionally, the toxicological impact was verified through acute and sub-chronic toxicity studies. A genome-based safety assessment was conducted to gain further insights into gene function, including potential virulence factors and pathogenic properties. Finally, we assessed whether moxifloxacin resistance in A. muciniphila PROBIO is transferred using in vitro conjugation experiments. A. muciniphila PROBIO exhibited superior gastrointestinal tolerance, with no observed hematological or histopathological abnormalities. Moreover, the outcomes pertaining to mutagenic, clastogenic, or toxic impacts were found to be negative, even at exceedingly high dosages. Moreover, no adverse effects associated with the test substance were observed during the examination of acute and sub-chronic toxicity. Consequently, it was plausible to estimate the no-observed-adverse-effect level (NOAEL) to be 6.4 × 10(11) viable bacteria for an average individual weighing 70 kg. Additionally, only three potential drug resistance genes and one virulence factor gene were annotated. A. muciniphila PROBIO is naturally resistant to moxifloxacin, and resistance does not transfer. Collectively, the data presented herein substantiate the presumed safety of A. muciniphila PROBIO for its application in food.202438338577
471560.9969Genomic and stress resistance characterization of Lactiplantibacillus plantarum GX17, a potential probiotic for animal feed applications. Lactobacilli, recognized as beneficial bacteria within the human body, are celebrated for their multifaceted probiotic functions, including the regulation of intestinal flora, enhancement of body immunity, and promotion of nutrient absorption. This study comprehensively analyzed the genotypic and phenotypic characteristics of Lactiplantibacillus plantarum (L. plantarum) strains isolated from the intestines of healthy chicks and assessed their potential as probiotics. The assembled genome consists of 29,521,986 bp, and a total of 1,771 coding sequences (CDSs) were predicted. Based on the entire genome sequence analysis, 50 stress resistance genes and seven virulence factors were identified. The results of the phenotypic experiments showed that the strain had good resistance to high temperature, low temperature, acid, alkali, salt, artificial gastrointestinal fluid, and strong antioxidant capacity. Additionally, transcriptomic analysis confirmed that under stress conditions, the expression levels of key genes were significantly upregulated. Therefore, the phenotypic characteristics of L. plantarum GX17 align well with its genotypic features, demonstrating promising probiotic properties. This strain holds great potential as a probiotic candidate, and further investigation into its beneficial effects on human health is warranted. IMPORTANCE: In humans, Lactiplantibacillus plantarum may synergize with host microbiota to ameliorate dysbiosis-related pathologies, enhance immunomodulation, and facilitate micronutrient bioavailability. For livestock, its application could improve feed conversion ratios, suppress enteric pathogens through competitive exclusion, and mitigate antibiotic overuse, "a critical strategy in One Health frameworks." Further investigations into strain-specific mechanisms (e.g., postbiotic metabolites, quorum sensing regulation) are warranted to translate these genomic-phenotypic advantages into sustainable health solutions across species.202540919934
322470.9969Assessing phenotypic and genotypic antibiotic resistance in bacillus-related bacteria isolated from biogas digestates. Antibiotic resistance poses a significant public health challenge, with biogas digestate, a byproduct of anaerobic digestion (AD), presenting potential risks when applied as a biofertilizer. Understanding the actual resistance levels in digestate is crucial for its safe application. While many studies have investigated antibiotic resistance in AD processes using culture-independent molecular methods, these approaches are limited by their reliance on reference databases and inability to account for gene expression, leading to potential inaccuracies in resistance assessment. This study addresses these limitations by combining culture-independent whole-genome sequencing (WGS) with culture-dependent phenotypic testing to provide a more accurate understanding of antibiotic resistance in digestate. We investigated the phenotypic and genotypic resistance profiles of 18 antibiotic-resistant bacteria (ARB) isolated from digestates produced from food waste and animal manure. Resistance was assessed using WGS and Estrip testing across 12 antibiotics from multiple classes. This is the first study to directly compare phenotypic and genotypic resistance in bacteria isolated from digestate, revealing significant discrepancies between the two methods. Approximately 30 % of resistance levels were misinterpreted when relying solely on culture-independent methods, with both over- and underestimation observed. These findings highlight the necessity of integrating both methods for reliable resistance assessments. Additionally, our WGS analysis indicated low potential for transferability of detected ARGs among the isolated ARB, suggesting a limited risk of environmental dissemination. This study provides new insights into antibiotic resistance in digestate and underscores the importance of integrating methodological approaches to achieve accurate evaluations of resistance risks.202539947064
676680.9969Effect of non-antibiotic factors on conjugative transfer of antibiotic resistance genes in aquaculture water. Aquaculture water with antibiotic resistance genes (ARGs) is escalating due to the horizontal gene transfer. Non-antibiotic stressors specifically found, including those from fishery feed and disinfectants, are potential co-selectors. However, the mechanisms underlying this process remains unclear. Intragenus and intergenus conjugative transfer systems of the antibiotic-resistant plasmid RP4 were established to examine conjugative transfer frequency under exposure to five widely used non-antibiotic factors in aquaculture water: iodine, oxolinic acid, NO(2)-N, NO(3)-N and H(2)O(2) and four different recipient bacteria: E. coli HB101, Citrobacter portucalensis SG1, Vibrio harveyi and Vibrio alginolyticus. The study found that low concentrations of non-antibiotic factors significantly promoted conjugative transfer, whereas high concentrations inhibited it. Moreover, the conjugation transfer efficiencies were significantly different with different bacterial species within (E. coli HB101 ∼ 10(-3) %) or cross genera (C. portucalensis SG1 ∼10(-5) %, V. harveyi ∼1 %). Besides, excessive exposure concentrations inhibited the expression of related genes and the generation of reactive oxygen species (ROS). Regulation of multiple related genes and ROS-induced SOS responses are common primary mechanisms. However, the mechanisms of non-antibiotic factors differ from those of standard antibiotics, with direct changes in cell membrane permeability potentially playing a dominant role. Additionally, variations among non-antibiotic factors and the specific characteristics of bacterial species contribute to differences in conjugation mechanisms. Notably, this study found that non-antibiotic factors could increase the frequency of intergeneric conjugation beyond that of intrageneric conjugation. Furthermore, non-antibiotic factors influenced by multiple transport systems may raise the risk of unintended cross-resistance, significantly amplifying the potential for resistance gene spread. This study underscores the significance of non-antibiotic factors in the propagation of ARGs, highlighting their role in advancing aquaculture development and protecting human health.202539615392
657790.9969Evaluating Microbial and Chemical Hazards in Commercial Struvite Recovered from Wastewater. Controlled struvite (NH(4)MgPO(4)·6H(2)O) precipitation has become a well-known process for nutrient recovery from wastewater treatment systems to alleviate the pressures of diminishing, finite rock phosphate reservoirs. Nonetheless, coprecipitation of potential microbial and chemical hazards is poorly understood. On the other hand, antimicrobial resistance (AMR) is a major global public health concern and wastewater is thought to disseminate resistance genes within bacteria. Fecal indicator bacteria (FIB) are typically used as measures of treatment quality, and with multiresistant E. coli and Enterococcus spp. rising in concern, the quantification of FIB can be used as a preliminary method to assess the risk of AMR. Focusing on struvite produced from full-scale operations, culture and qPCR methods were utilized to identify FIB, antibiotic resistance genes, and human enteric viruses in the final product. Detection of these hazards occurred in both wet and dry struvite samples indicating that there is a potential risk that needs further consideration. Chemical and biological analyses support the idea that the presence of other wastewater components can impact struvite formation through ion and microbial interference. While heavy metal concentrations met current fertilizer standards, the presence of K, Na, Ca, and Fe ions can impact struvite purity yet provide benefit for agricultural uses. Additionally, the quantified hazards detected varied among struvite samples produced from different methods and sources, thus indicating that production methods could be a large factor in the risk associated with wastewater-recovered struvite. In all, coprecipitation of metals, fecal indicator bacteria, antimicrobial resistance genes, and human enteric viruses with struvite was shown to be likely, and future engineered wastewater systems producing struvite may require additional step(s) to manage these newly identified public health risks.201930964655
4772100.9968Molecular identification of Proteus mirabilis, Vibrio species leading to CRISPR-Cas9 modification of tcpA and UreC genes causing cholera and UTI. Heavy metal accumulation increases rapidly in the environment due to anthropogenic activities and industrialization. The leather and surgical industry produces many contaminants containing heavy metals. Cadmium, a prominent contaminant, is linked to severe health risks, notably kidney and liver damage, especially among individuals exposed to contaminated wastewater. This study aims to leverage the natural cadmium resistance mechanisms in bacteria for bioaccumulation purposes. The industrial wastewater samples, characterized by an alarming cadmium concentration of 29.6 ppm, 52 ppm, and 76.4 ppm-far exceeding the recommended limit of 0.003 ppm-were subjected to screening for cadmium-resistant bacteria using cadmium-supplemented media with CdCl(2). 16S rRNA characterization identified Vibrio cholerae and Proteus mirabilis as cadmium-resistant bacteria in the collected samples. Subsequently, the cadmium resistance-associated cadA gene was successfully amplified in Vibrio species and Proteus mirabilis, revealing a product size of 623 bp. Further analysis of the identified bacteria included the examination of virulent genes, specifically the tcpA gene (472 bp) associated with cholera and the UreC gene (317 bp) linked to urinary tract infections. To enhance the bioaccumulation of cadmium, the study proposes the potential suppression of virulent gene expression through in-silico gene-editing tools such as CRISPR-Cas9. A total of 27 gRNAs were generated for UreC, with five selected for expression. Similarly, 42 gRNA sequences were generated for tcpA, with eight chosen for expression analysis. The selected gRNAs were integrated into the lentiCRISPR v2 expression vector. This strategic approach aims to facilitate precise gene editing of disease-causing genes (tcpA and UreC) within the bacterial genome. In conclusion, this study underscores the potential utility of Vibrio species and Proteus mirabilis as effective candidates for the removal of cadmium from industrial wastewater, offering insights for future environmental remediation strategies.202438609487
6574110.9968Exploiting microplastics and the plastisphere for the surveillance of human pathogenic bacteria discharged into surface waters in wastewater effluent. Discharge from wastewater treatment plants (WWTPs) is a well-characterised source of human pathogens and antimicrobial resistance genes entering the environment. However, determining whether pathogens released from effluent into surface waters are viable, and consequently pose a risk to human health, is hindered by the use of transient grab-sampling monitoring approaches. Here we present a novel surveillance system using low-cost microparticles (polyethylene, cork and rubber) deployed upstream and downstream of a WWTP effluent pipe, that exploits the ability of bacterial pathogens to form biofilms. Using quantitative culture-based and molecular methods, viable E. coli, Klebsiella spp., Citrobacter spp., and Enterococcus spp. were identified after only 24-hour of deployment. Moreover, these pathogens were continually present at each timepoint (2, 4, 6, 8, 10, 14 and 23 days) as biofilm communities matured, with all pathogens detected at higher concentrations downstream of the WWTP effluent pipe. Long-read whole genome sequencing revealed a suite of plasmids, virulence genes and antimicrobial resistance genes in bacterial pathogens isolated from biofilms formed downstream of the effluent pipe. Furthermore, recognising that pathogens are typically present at proportionally low concentrations within mixed biofilm communities, total biofilm pathogenicity was confirmed using a Galleria mellonella infection model. Full-length 16S rRNA gene sequencing revealed that human pathogens present in microplastic biofilms (the 'plastisphere') dominated the microbial community of infected G. mellonella larvae within 24 hr, suggesting these bacteria remained highly virulent. Overall, this study demonstrated the efficacy of an easy-to-deploy system for the surveillance and rapid detection of pathogenic bacteria being discharged from point-source pollution. We envisage that if used as part of an integrated environmental management approach, this approach could help to reduce the public and environmental health risks of human pathogens and antimicrobial resistance genes, by monitoring viable human pathogens entering surface waters.202540184703
6539120.9968An alternative material for an effective treatment technique proposal in the light of bibliometric profile of global scientific research on antibiotic resistance and Escherichia coli. Antibiotic resistance is considered by the countries to be a global health issue and a huge threat to public health. The reduction of resistant microorganisms from water/wastewater is of importance in environmental sciences since they are resistant in the aquatic environment. In this study, a bibliometric analysis of literature from the field of environmental science in water ecosystems from 2015 to 2019 was carried out using the keywords "Antibiotic Resistance (AR)" and "Escherichia coli". Furthermore, using the keywords of "Fresh Water," "Sea Water," and "Waste Water," 155, 52, and 57 studies were discovered, respectively. It is found that 217 studies of the total 2115 studies investigated on AR are mostly performed in the "Waste Water" by considering human health. Given the studies, an up-to-date solution should be proposed since the release of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) from wastewater treatment plants needs to be mitigated. For this reason, it is obvious that working on micro and macro ecosystems will increase the probability of solutions in antibiotic resistance. A discussion of removal techniques for coliform bacteria, particularly antibiotic resistant Escherichia coli, was presented. One of the unique values of this study is to offer an innovative solution that removing them by metal-organic frameworks (MOFs) are emerging crystalline hybrid materials. MOFs are used for environmental, biological, and food antimicrobial substances efficiently. Therefore, we can give inspiration to the future studies of antimicrobial resistance removal via adsorption using MOFs as adsorbents. Graphical Abstract.202033079229
9938130.9968Comparison of CRISPR-Cas9, CRISPR-Cas12f1, and CRISPR-Cas3 in eradicating resistance genes KPC-2 and IMP-4. Bacterial plasmid encoding antibiotic resistance could be eradicated by various CRISPR systems, such as CRISPR-Cas9, Cas12f1, and Cas3. However, the efficacy of these gene editing tools against bacterial resistance has not been systematically assessed and compared. This study eliminates carbapenem resistance genes KPC-2 and IMP-4 via CRISPR-Cas9, Cas12f1, and Cas3 systems, respectively. The eradication efficiency of the three CRISPR systems was evaluated. First, the target sites for the three CRISPR systems were designed within the regions 542-576 bp of the KPC-2 gene and 213-248 bp of the IMP-4 gene, respectively. The recombinant CRISPR plasmids were transformed into Escherichia coli carrying KPC-2 or IMP-4-encoding plasmid. Colony PCR of transformants showed that KPC-2 and IMP-4 were eradicated by the three different CRISPR systems, and the elimination efficacy was both 100.00%. The drug sensitivity test results showed that the resistant E. coli strain was resensitized to ampicillin. In addition, the three CRISPR plasmids could block the horizontal transfer of drug-resistant plasmids, with a blocking rate as high as 99%. Importantly, a qPCR assay was performed to analyze the copy number changes of drug-resistant plasmids in E. coli cells. The results indicated that CRISPR-Cas3 showed higher eradication efficiency than CRISPR-Cas9 and Cas12f1 systems. IMPORTANCE: With the continuous development and application of CRISPR-based resistance removal technologies, CRISPR-Cas9, Cas12f1, and Cas3 have gradually come into focus. However, it remains uncertain which system exhibits more potent efficacy in the removal of bacterial resistance. This study verifies that CRISPR-Cas9, Cas12f1, and Cas3 can eradicate the carbapenem-resistant genes KPC-2 and IMP-4 and restore the sensitivity of drug-resistant model bacteria to antibiotics. Among the three CRISPR systems, the CRISPR-Cas3 system showed the highest eradication efficiency. Although each system has its advantages and characteristics, our results provide guidance on the selection of the CRISPR system from the perspective of resistance gene removal efficiency, contributing to the further application of CRISPR-based bacterial resistance removal technologies.202540293254
6772140.9968Disinfectants facilitate the transformation of exogenous antibiotic resistance genes via multiple pathways. The prevalence and spread of multidrug-resistant (MDR) bacteria pose a global challenge to public health. Natural transformation is one of the essential ways for horizontal transfer of antibiotic resistance genes (ARGs). Although disinfectants are frequently used during COVID-19, little is known about whether these disinfectants are associated with the transformation of plasmid-borne ARGs. In our study, we assessed the effect of some disinfectants on bacterial transformation using resistance plasmids as extracellular DNA and E. coli DH5α as the recipient bacteria. The results showed that these disinfectants at environmentally relevant concentrations, including benzalkonium bromide (BB), benzalkonium chloride (BC) and polyhexamethylene guanidine hydrochloride (PHMG), significantly enhanced the transformation of plasmid-encoded ARGs. Furthermore, we investigated the mechanisms underlying the promotive effect of disinfectants on transformation. We revealed that the addition of disinfectants significantly increased the membrane permeability and promoted membrane-related genes expression. Moreover, disinfectants led to the boosted bacterial respiration, ATP production and flagellum motility, as well as increased expression of bacterial secretion system-related genes. Together, our findings shed insights into the spread of ARGs through bacterial transformation and indicate potential risks associated with the widespread use of disinfectants.202336857920
8463150.9968Safety assessment of five candidate probiotic lactobacilli using comparative genome analysis. Micro-organisms belonging to the Lactobacillus genus complex are often used for oral consumption and are generally considered safe but can exhibit pathogenicity in rare and specific cases. Therefore, screening and understanding genetic factors that may contribute to pathogenicity can yield valuable insights regarding probiotic safety. Limosilactobacillus mucosae LM1, Lactiplantibacillus plantarum SK151, Lactiplantibacillus plantarum BS25, Limosilactobacillus fermentum SK152 and Lactobacillus johnsonii PF01 are current probiotics of interest; however, their safety profiles have not been explored. The genome sequences of LM1, SK151, SK152 and PF01 were downloaded from the NCBI GenBank, while that of L. plantarum BS25 was newly sequenced. These genomes were then annotated using the Rapid Annotation using Subsystem Technology tool kit pipeline. Subsequently, a command line blast was performed against the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) to identify potential virulence factors and antibiotic resistance (AR) genes. Furthermore, ResFinder was used to detect acquired AR genes. The query against the VFDB identified genes that have a role in bacterial survivability, platelet aggregation, surface adhesion, biofilm formation and immunoregulation; and no acquired AR genes were detected using CARD and ResFinder. The study shows that the query strains exhibit genes identical to those present in pathogenic bacteria with the genes matched primarily having roles related to survival and surface adherence. Our results contribute to the overall strategies that can be employed in pre-clinical safety assessments of potential probiotics. Gene mining using whole-genome data, coupled with experimental validation, can be implemented in future probiotic safety assessment strategies.202438361650
7608160.9968Evaluation of a constructed wetland for wastewater treatment: Addressing emerging organic contaminants and antibiotic resistant bacteria. The occurrence of emerging organic contaminants (EOCs) in wastewaters and the inability of the conventional wastewater treatments plants to deal with them have been pointed out several times over the last few years. As a result, remnants of those compounds released into the aquatic environment present a potential risk for public health. Constructed wetlands (CWs) have been proposed as environmentally friendly, low-cost alternative systems with satisfactory results for different types of contaminants. This study aimed to evaluate the efficiency of a CW system, planted with the halophyte Juncus acutus, to eliminate bisphenol A (BPA) and two antibiotics, namely ciprofloxacin (CIP) and sulfamethoxazole (SMX) under different operating conditions. The behavior of Escherichia coli and enterococcal populations in terms of changes in their resistance profile for the selected antibiotics and the abundance of two resistance genes (qnrA and sul1) were also examined. BPA and CIP were significantly removed by the CW, with an overall removal of 76.2% and 93.9% respectively and with the plants playing a vital role. In contrast, SMX was not significantly eliminated. Moreover, fluctuations in the antibiotic resistance profile of bacteria were observed. Treatment processes affected the response of the two selected bacterial indicators, depending on the conditions employed in each case. Furthermore, increased levels of resistance genes were monitored in the system effluent. This study indicates that CWs, as tertiary wastewater treatment systems, may demonstrate high removal rates for some but not all EOCs. This implies that each EOC identified in the feed stream should be tested assiduously by analyzing the final effluents before their reuse or discharge into water bodies.201931146037
5101170.9968Identification of Key Features Pivotal to the Characteristics and Functions of Gut Bacteria Taxa through Machine Learning Methods. BACKGROUND: Gut bacteria critically influence digestion, facilitate the breakdown of complex food substances, aid in essential nutrient synthesis, and contribute to immune system balance. However, current knowledge regarding intestinal bacteria remains insufficient. OBJECTIVE: This study aims to discover essential differences for different intestinal bacteria. METHODS: This study was conducted by investigating a total of 1478 gut bacterial samples comprising 235 Actinobacteria, 447 Bacteroidetes, and 796 Firmicutes, by utilizing sophisticated machine learning algorithms. By building on the dataset provided by Chen et al., we engaged sophisticated machine learning techniques to further investigate and analyze the gut bacterial samples. Each sample in the dataset was described by 993 unique features associated with gut bacteria, including 342 features annotated by the Antibiotic Resistance Genes Database, Comprehensive Antibiotic Research Database, Kyoto Encyclopedia of Genes and Genomes, and Virulence Factors of Pathogenic Bacteria. We employed incremental feature selection methods within a computational framework to identify the optimal features for classification. RESULTS: Eleven feature ranking algorithms selected several key features as pivotal to the characteristics and functions of gut bacteria. These features appear to facilitate the identification of specific gut bacterial species. Additionally, we established quantitative rules for identifying Actinobacteria, Bacteroidetes, and Firmicutes. CONCLUSION: This research underscores the significant potential of machine learning in studying gut microbes and enhances our understanding of the multifaceted roles of gut bacteria.202540671232
3156180.9968Deciphering Resistome and Virulome Diversity in a Porcine Slaughterhouse and Pork Products Through Its Production Chain. We aimed to better understand resistome and virulome patterns on animal and process-area surfaces through a pig slaughterhouse to track possible contamination within the food production chain. Culture-dependent methods revealed high levels of microbial contamination, corresponding to mesophilic and pathogenic bacteria on both the animal and process-area surfaces mainly in the anesthesia (AA and AS) zone followed by "scorching and whip" (FA and FS) zone and also in the end products. To evaluate the potential risk of antibiotic resistance and virulence determinants, shotgun metagenomic DNA-sequencing of isolates from selected areas/products uncovered a high diversity and richness of antibiotic resistance genes (ARGs): 55-62 genes in the anesthesia area (AA and AS) and 35-40 in "animal-arrival zone" (MA and MS). The "scorching and whip" (FA and FS) area, however, exhibited lowered abundance of ARGs (1-6), indicating that the scalding and depilating process (an intermediate zone between "anesthesia" and "scorching and whip") significantly decreased bacterial load by 1-3 log(10) but also diminished the resistome. The high prevalence of antibiotic-inactivating enzyme genes in the "animal-arrival zone" (60-65%) and "anesthesia" area (56%) were mainly represented by those for aminoglycoside (46-51%) and lincosamide (14-19%) resistance, which did not reflect selective pressures by antibiotics most commonly used in pig therapy-tetracyclines and beta-lactams. Contrary to ARGs, greater number of virulence resistance genes were detected after evisceration in some products such as kidney, which reflected the poor hygienic practices. More than 19 general virulence features-mainly adherence, secretion system, chemotaxis and motility, invasion and motility were detected in some products. However, immune evasion determinants were detected in almost all samples analyzed from the beginning of the process, with highest amounts found from the anesthesia area. We conclude that there are two main sources of contamination in a pig slaughterhouse: the microorganisms carried on the animals' hide, and those from the evisceration step. As such, focussing control measures, e.g., enhanced disinfection procedures, on these contamination-source areas may reduce risks to food safety and consumer health, since the antibiotic and virulence determinants may spread to end products and the environment; further, ARG and virulence traits can exacerbate pathogen treatments.201830258416
8678190.9968Metagenomics-Guided Discovery of Potential Bacterial Metallothionein Genes from the Soil Microbiome That Confer Cu and/or Cd Resistance. Metallothionein (MT) genes are valuable genetic materials for developing metal bioremediation tools. Currently, a limited number of prokaryotic MTs have been experimentally identified, which necessitates the expansion of bacterial MT diversity. In this study, we conducted a metagenomics-guided analysis for the discovery of potential bacterial MT genes from the soil microbiome. More specifically, we combined resistance gene enrichment through diversity loss, metagenomic mining with a dedicated MT database, evolutionary trace analysis, DNA chemical synthesis, and functional genomic validation to identify novel MTs. Results showed that Cu stress induced a compositional change in the soil microbiome, with an enrichment of metal-resistant bacteria in soils with higher Cu concentrations. Shotgun metagenomic sequencing was performed to obtain the gene pool of environmental DNA (eDNA), which was subjected to a local BLAST search against an MT database for detecting putative MT genes. Evolutional trace analysis led to the identification of 27 potential MTs with conserved cysteine/histidine motifs different from those of known prokaryotic MTs. Following chemical synthesis of these 27 potential MT genes and heterologous expression in Escherichia coli, six of them were found to improve the hosts' growth substantially and enhanced the hosts' sorption of Cu, Cd, and Zn, among which MT5 led to a 13.7-fold increase in Cd accumulation. Furthermore, four of them restored Cu and/or Cd resistance in two metal-sensitive E. coli strains.IMPORTANCE The metagenomics-guided procedure developed here bypasses the difficulties encountered in classic PCR-based approaches and led to the discovery of novel MT genes, which may be useful in developing bioremediation tools. The procedure used here expands our knowledge on the diversity of bacterial MTs in the environment and may also be applicable to identify other functional genes from eDNA.202032111593