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354800.9576From flagellar assembly to DNA replication: CJSe's role in mitigating microbial antibiotic resistance genes. The emergence of Antibiotic Resistance Genes (ARGs) in Campylobacter jejuni (CJ) poses a severe threat to food safety and human health. However, the specific impact of CJ and its variants on ARGs and other related factors remains to be further elucidated. Herein, integrated metagenomic sequencing and co-occurrence network analysis approach were employed to investigate the impact of CJ and CJ incorporated with biogenic selenium (CJSe) on ARGs, flagellar assembly pathways, microbial communities, and DNA replication pathways in chicken manure. Compared to the Control (CON) and CJ groups, the CJSe group exhibited 2.4-fold increase selenium levels (P < 0.01) in chicken manure. Notable differences were also observed between the CJ and CJSe groups, with sequence results showing a CJ > CJSe > CON trend in total ARG copy numbers. Furthermore, the CJSe group showed 31.6 % fewer flagellar assembly genes compared to the CJ group. Additionally, compared to the CJ group, CJSe inhibited pathways such as basal body/hook (e.g., FliH, FliO, FliQ reduced by 25-52 %) and stator (MotB downregulated by 42.3 %), suppressing flagellar assembly. We also found that both CJ and CJSe influenced bacterial DNA replication pathways, with the former increasing ARG-carrying bacteria and the latter, under selenium-induced selective pressure, reducing ARG-carrying bacteria. Moreover, compared to the CJ group, the CJSe group showed a significantly lower 9.72 % copy number of total archaeal DNA replication genes. Furthermore, through intricate co-occurrence network analysis, we discovered the complex interplay between changes in ARGs and bacterial and archaeal DNA replication dynamics within the microbial community. These findings indicate that CJSe mitigates the threat posed by CJ and reduces ARG prevalence, while its dual functionality enables applications in biofortified crop production and soil remediation in selenium-deficient regions, thereby advancing circular economy systems. While the current study demonstrates CJSe's dual functionality under controlled conditions, future work will implement a dedicated ecological risk assessment framework encompassing Se speciation/leaching tests and non-target organism assays to confirm environmental safety under field-relevant scenarios. This approach aligns with sustainable strategies for food security and public health safeguarding.202541108960
616210.9538The resistance of BALB/cJ mice to Yersinia pestis maps to the major histocompatibility complex of chromosome 17. Yersinia pestis, the causative agent of plague, has been well studied at the molecular and genetic levels, but little is known about the role that host genes play in combating this highly lethal pathogen. We challenged several inbred strains of mice with Y. pestis and found that BALB/cJ mice are highly resistant compared to susceptible strains such as C57BL/6J. This resistance was observed only in BALB/cJ mice and not in other BALB/c substrains. Compared to C57BL/6J mice, the BALB/cJ strain exhibited reduced bacterial burden in the spleen and liver early after infection as well as lower levels of serum interleukin-6. These differences were evident 24 h postinfection and became more pronounced with time. Although a significant influx of neutrophils in the spleen and liver was exhibited in both strains, occlusive fibrinous thrombi resulting in necrosis of the surrounding tissue was observed only in C57BL/6J mice. In an effort to identify the gene(s) responsible for resistance, we measured total splenic bacteria in 95 F(2) mice 48 h postinfection and performed quantitative trait locus mapping using 58 microsatellite markers spaced throughout the genome. This analysis revealed a single nonrecessive plague resistance locus, designated prl1 (plague resistance locus 1), which coincides with the major histocompatibility complex of chromosome 17. A second screen of 95 backcrossed mice verified that this locus confers resistance to Y. pestis early in infection. Finally, eighth generation backcrossed mice harboring prl1 were found to maintain resistance in the susceptible C57BL/6J background. These results identify a novel genetic locus in BALB/cJ mice that confers resistance to Y. pestis.200818573896
306920.9531The hospital sink drain biofilm resistome is independent of the corresponding microbiota, the environment and disinfection measures. In hospitals, the transmission of antibiotic-resistant bacteria (ARB) may occur via biofilms present in sink drains, which can lead to infections. Despite the potential role of sink drains in the transmission of ARB in nosocomial infections, routine surveillance of these drains is lacking in most hospitals. As a result, there is currently no comprehensive understanding of the transmission of ARB and the dissemination of antimicrobial resistance genes (ARGs) and associated mobile genetic elements (MGEs) via sink drains. This study employed a multifaceted approach to monitor the total aerobic bacteria as well as the presence of carbapenemase-producing Enterobacterales (CPEs), the microbiota and the resistome of sink drain biofilms (SDBs) and hospital wastewater (WW) of two separate intensive care units (ICUs) in the same healthcare facility in France. Samples of SDB and WW were collected on a monthly basis, from January to April 2023, in the neonatal (NICU) and the adult (AICU) ICUs of Grenoble Alpes University Hospital. In the NICU, sink drain disinfection with surfactants was performed routinely. In the AICU, routine disinfection is not carried out. Culturable aerobic bacteria were quantified on non-selective media, and CPEs were screened using two selective agars. Isolates were identified by MALDI-TOF MS, and antibiotic susceptibility testing (AST) was performed on Enterobacterales and P. aeruginosa. The resistome was analyzed by high-throughput qPCR targeting >80 ARGs and MGEs. The overall bacterial microbiota was assessed via full-length 16S rRNA sequencing. No CPEs were isolated from SDBs in either ICU by bacterial culture. Culture-independent approaches revealed an overall distinct microbiota composition of the SDBs in the two ICUs. The AICU SDBs were dominated by pathogens containing Gram-negative bacterial genera including Pseudomonas, Stenotrophomona, Klebsiella, and Gram-positive Staphylococcus, while the NICU SDBs were dominated by the Gram-negative genera Achromobacter, Serratia, and Acidovorax, as well as the Gram-positive genera Weisella and Lactiplantibacillus. In contrast, the resistome of the SDBs exhibited no significant differences between the two ICUs, indicating that the abundance of ARGs and MGEs is independent of microbiota composition and disinfection practices. The AICU WW exhibited more distinct aerobic bacteria than the NICU WW. In addition, the AICU WW yielded 15 CPEs, whereas the NICU WW yielded a single CPE. All the CPEs were characterized at the species level. The microbiota of the NICU and AICU WW samples differed from their respective SDBs and exhibited distinct variations over the four-month period:the AICU WW contained a greater number of genes conferring resistance to quinolones and integron integrase genes, whereas the NICU WW exhibited a higher abundance of streptogramin resistance genes. Our study demonstrated that the resistome of the hospital SDBs in the two ICUs of the investigated healthcare institute is independent of the microbiota, the environment, and the local disinfection measures. However, the prevalence of CPEs in the WW pipes collecting the waste from the investigated drains differed. These findings offer valuable insights into the resilience of resistance genes in SDBs in ICUs, underscoring the necessity for innovative strategies to combat antimicrobial resistance in clinical environments.202540483807
516330.9513Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep. BACKGROUND: The integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control. METHODS: Eggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content. RESULTS: The heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep. CONCLUSIONS: The present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.202438429820
999740.9512RNAi screen of DAF-16/FOXO target genes in C. elegans links pathogenesis and dauer formation. The DAF-16/FOXO transcription factor is the major downstream output of the insulin/IGF1R signaling pathway controlling C. elegans dauer larva development and aging. To identify novel downstream genes affecting dauer formation, we used RNAi to screen candidate genes previously identified to be regulated by DAF-16. We used a sensitized genetic background [eri-1(mg366); sdf-9(m708)], which enhances both RNAi efficiency and constitutive dauer formation (Daf-c). Among 513 RNAi clones screened, 21 displayed a synthetic Daf-c (SynDaf) phenotype with sdf-9. One of these genes, srh-100, was previously identified to be SynDaf, but twenty have not previously been associated with dauer formation. Two of the latter genes, lys-1 and cpr-1, are known to participate in innate immunity and six more are predicted to do so, suggesting that the immune response may contribute to the dauer decision. Indeed, we show that two of these genes, lys-1 and clc-1, are required for normal resistance to Staphylococcus aureus. clc-1 is predicted to function in epithelial cohesion. Dauer formation exhibited by daf-8(m85), sdf-9(m708), and the wild-type N2 (at 27°C) were all enhanced by exposure to pathogenic bacteria, while not enhanced in a daf-22(m130) background. We conclude that knockdown of the genes required for proper pathogen resistance increases pathogenic infection, leading to increased dauer formation in our screen. We propose that dauer larva formation is a behavioral response to pathogens mediated by increased dauer pheromone production.201021209831
140050.9512Comparative genomic analysis of Escherichia coli strains obtained from continuous imipenem stress evolution. The carbapenem-resistant Escherichia coli has aroused increasing attention worldwide, especially in terms of imipenem (IMP) resistance. The molecular mechanism of IMP resistance remains unclear. This study aimed to explore the resistance mechanisms of IMP in E. coli. Susceptible Sx181-0-1 strain was induced into resistance strains by adaptive laboratory evolution. The drug resistance spectrum was measured using the disk diffusion and microbroth dilution methods. Whole-genome sequencing and resequencing were used to analyze the nonsynonymous single-nucleotide polymorphisms (nsSNPs) between the primary susceptible strain and resistant strains. The expression levels of these genes with nsSNPs were identified by real-time quantitative PCR (RT-qPCR). Resistance phenotype appeared in the induced 15th generation (induction time = 183 h). Sx181-32 and Sx181-256, which had the minimum inhibitory concentrations of IMP of 8 and 64 µg ml-1, were isolated during continuous subculture exposed to increasing concentrations of IMP, respectively. A total of 19 nsSNPs were observed both in Sx181-32 and Sx181-256, distributed in rpsU, sdaC, zwf, ttuC, araJ, dacC, mrdA, secF, dacD, lpxD, mrcB, ftsI, envZ, and two unknown function genes (orf01892 and orf01933). Among these 15 genes, five genes (dacC, mrdA, lpxD, mrcB, and ftsI) were mainly involved in cell wall synthesis. The mrdA (V338A, L378P, and M574I) and mrcB (P784L, A736V, and T708A) had three amino acid substitutions, respectively. The expression levels of rpsU, ttuC, and orf01933 were elevated in both Sx181-32 and Sx181-256 compared to Sx181-0-1. The expression levels of these genes were elevated in Sx181-256, except for araJ. Bacteria developed resistance to antimicrobials by regulating various biological processes, among which the most involved is the cell wall synthesis (dacC, mrdA, lpxD, mrcB, and ftsI). The combination mutations of mrdA, envZ, and ftsI genes may increase the resistance to IMP. Our study could improve the understanding of the molecular mechanism of IMP resistance in E. coli.202235147175
354760.9512Occurrence of 40 sanitary indicators in French digestates derived from different anaerobic digestion processes and raw organic wastes from agricultural and urban origin. This study investigated the sanitary quality of digestates resulting from the mesophilic anaerobic digestion (AD) of urban and agricultural organic wastes (OWs). 40 sanitary indicators, including pathogenic bacteria, antimicrobial resistance genes, virulence factor genes, and mobile genetic elements were evaluated using real-time PCR and/or droplet digital PCR. 13 polycyclic aromatic hydrocarbons (PAHs) and 13 pharmaceutical products (PHPs) were also measured. We assessed agricultural OWs from three treatment plants to study the effect of different AD processes (feeding mode, number of stages, pH), and used three laboratory-scale reactors to study the effect of different feed-supplies (inputs). The lab-scale reactors included: Lab1 fed with 97% activated sludge (urban waste) and 3% cow manure; Lab2 fed with 85% sludge-manure mixture supplemented with 15% wheat straw (WS); and Lab3 fed with 81% sludge-manure mixture, 15% WS, and 4% zeolite powder. Activated sludge favored the survival of the food-borne pathogens Clostridium perfringens and Bacillus cereus, carrying the toxin-encoding genes cpe and ces, respectively. Globally, the reactors fed with fecal matter supplemented with straw (Lab2) or with straw and zeolite (Lab3) had a higher hygienization efficiency than the reactor fed uniquely with fecal matter (Lab1). Three pathogenic bacteria (Enterococcus faecalis, Enterococcus faecium, and Mycobacterium tuberculosis complex), a beta-lactam resistance gene (bla (TEM)), and three mobile genetic elements (intI1, intI2, and IS26) were significantly decreased in Lab2 and Lab3. Moreover, the concentrations of 11 PAHs and 11 PHPs were significantly lower in Lab2 and Lab3 samples than in Lab1 samples. The high concentrations of micropollutants, such as triclosan, found in Lab1, could explain the lower hygienization efficiency of this reactor. Furthermore, the batch-fed reactor had a more efficient hygienization effect than the semi-continuous reactors, with complete removal of the ybtA gene, which is involved in the production of the siderophore yersiniabactin, and significant reduction of intI2 and tetO. These data suggest that it is essential to control the level of chemical pollutants in raw OWs to optimize the sanitary quality of digestates, and that adding co-substrate, such as WS, may overcome the harmful effect of pollutants.202439165575
616170.9505Unraveling radiation resistance strategies in two bacterial strains from the high background radiation area of Chavara-Neendakara: A comprehensive whole genome analysis. This paper reports the results of gamma irradiation experiments and whole genome sequencing (WGS) performed on vegetative cells of two radiation resistant bacterial strains, Metabacillus halosaccharovorans (VITHBRA001) and Bacillus paralicheniformis (VITHBRA024) (D10 values 2.32 kGy and 1.42 kGy, respectively), inhabiting the top-ranking high background radiation area (HBRA) of Chavara-Neendakara placer deposit (Kerala, India). The present investigation has been carried out in the context that information on strategies of bacteria having mid-range resistance for gamma radiation is inadequate. WGS, annotation, COG and KEGG analyses and manual curation of genes helped us address the possible pathways involved in the major domains of radiation resistance, involving recombination repair, base excision repair, nucleotide excision repair and mismatch repair, and the antioxidant genes, which the candidate could activate to survive under ionizing radiation. Additionally, with the help of these data, we could compare the candidate strains with that of the extremely radiation resistant model bacterium Deinococccus radiodurans, so as to find the commonalities existing in their strategies of resistance on the one hand, and also the rationale behind the difference in D10, on the other. Genomic analysis of VITHBRA001 and VITHBRA024 has further helped us ascertain the difference in capability of radiation resistance between the two strains. Significantly, the genes such as uvsE (NER), frnE (protein protection), ppk1 and ppx (non-enzymatic metabolite production) and those for carotenoid biosynthesis, are endogenous to VITHBRA001, but absent in VITHBRA024, which could explain the former's better radiation resistance. Further, this is the first-time study performed on any bacterial population inhabiting an HBRA. This study also brings forward the two species whose radiation resistance has not been reported thus far, and add to the knowledge on radiation resistant capabilities of the phylum Firmicutes which are abundantly observed in extreme environment.202438857267
608080.9505Metagenomic Insights into the Taxonomic and Functional Features of Traditional Fermented Milk Products from Russia. Fermented milk products (FMPs) contain probiotics that are live bacteria considered to be beneficial to human health due to the production of various bioactive molecules. In this study, nine artisanal FMPs (kefir, ayran, khurunga, shubat, two cottage cheeses, bryndza, khuruud and suluguni-like cheese) from different regions of Russia were characterized using metagenomics. A metagenomic sequencing of ayran, khurunga, shubat, khuruud and suluguni-like cheese was performed for the first time. The taxonomic profiling of metagenomic reads revealed that Lactococcus species, such as Lc. lactis and Lc. cremoris prevailed in khuruud, bryndza, one sample of cottage cheese and khurunga. The latter one together with suluguni-like cheese microbiome was dominated by bacteria, affiliated to Lactobacillus helveticus (32-35%). In addition, a high proportion of sequences belonging to the genera Lactobacillus, Lactococcus and Streptococcus but not classified at the species level were found in the suluguni-like cheese. Lactobacillus delbrueckii, as well as Streptococcus thermophilus constituted the majority in another cottage cheese, kefir and ayran metagenomes. The microbiome of shubat, produced from camel's milk, was significantly distinctive, and Lentilactobacillus kefiri, Lactobacillus kefiranofaciens and Bifidobacterium mongoliense represented the dominant components (42, 7.4 and 5.6%, respectively). In total, 78 metagenome-assembled genomes with a completeness ≥ 50.2% and a contamination ≤ 8.5% were recovered: 61 genomes were assigned to the Enterococcaceae, Lactobacillaceae and Streptococcaceae families (the Lactobacillales order within Firmicutes), 4 to Bifidobacteriaceae (the Actinobacteriota phylum) and 2 to Acetobacteraceae (the Proteobacteria phylum). A metagenomic analysis revealed numerous genes, from 161 to 1301 in different products, encoding glycoside hydrolases and glycosyltransferases predicted to participate in lactose, alpha-glucans and peptidoglycan hydrolysis as well as exopolysaccharides synthesis. A large number of secondary metabolite biosynthetic gene clusters, such as lanthipeptides, unclassified bacteriocins, nonribosomal peptides and polyketide synthases were also detected. Finally, the genes involved in the synthesis of bioactive compounds like β-lactones, terpenes and furans, nontypical for fermented milk products, were also found. The metagenomes of kefir, ayran and shubat was shown to contain either no or a very low count of antibiotic resistance genes. Altogether, our results show that traditional indigenous fermented products are a promising source of novel probiotic bacteria with beneficial properties for medical and food industries.202338276185
713290.9505Impact of blending for direct potable reuse on premise plumbing microbial ecology and regrowth of opportunistic pathogens and antibiotic resistant bacteria. Little is known about how introducing recycled water intended for direct potable reuse (DPR) into distribution systems and premise plumbing will affect water quality at the point of use, particularly with respect to effects on microbial communities and regrowth. The examination of potential growth of opportunistic pathogens (OPs) and spread of antibiotic resistance genes (ARGs), each representing serious and growing public health concerns, by introducing DPR water has not previously been evaluated. In this study, the impact of blending purified DPR water with traditional drinking water sources was investigated with respect to treatment techniques, blending location, and blending ratio. Water from four U.S. utility partners was treated in bench- and pilot-scale treatment trains to simulate DPR with blending. Water was incubated in simulated premise plumbing rigs made of PVC pipe containing brass coupons to measure regrowth of total bacteria (16S rRNA genes, heterotrophic plate count), OPs (Legionella spp., Mycobacterium spp., Pseudomonas aeruginosa), ARGs (qnrA, vanA), and an indicator of horizontal gene transfer and multi-drug resistance (intI1). The microbial community composition was profiled and the resistome (i.e., all ARGs present) was characterized in select samples using next generation sequencing. While regrowth of total bacteria (16S rRNA genes) from the start of the incubation through week eight consistently occurred across tested scenarios (Wilcoxon, p ≤ 0.0001), total bacteria were not more abundant in the water or biofilm of any DPR scenario than in the corresponding conventional potable condition (p ≥ 0.0748). Regrowth of OP marker genes, qnrA, vanA, and intI1 were not significantly greater in water or biofilm for any DPR blends treated with advanced oxidation compared to corresponding potable water (p ≥ 0.1047). This study of initial bacteria colonizing pipes after introduction of blended DPR water revealed little evidence (i.e., one target in one water type) of exacerbated regrowth of total bacteria, OPs, or ARGs in premise plumbing.201930594092
7726100.9504Distribution and comparison of bacterial communities in HVAC systems of two university buildings: Implications for indoor air quality and public health. The installation of HVAC systems in building is meant to enhance indoor air quality as well as increase comfort to occupants. However, HVAC systems have also become a vehicle of contamination of indoor air with potentially pathogenic microorganisms. DNA was extracted from ten HVAC filter dust samples collected from two buildings and subjected to high throughput sequencing analysis to determine the bacterial community structure. Further, the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software was used to predict the potential functional capabilities of the bacterial communities. Sequencing analysis led to the identification of five major bacterial phyla, including Proteobacteria, Cyanobacteria, Actinobacteria, Firmicutes and Bacteroidetes. At genus level, Mycobacterium, Bacillus, Cupriavidus, Hyphomicrobium and Mesorhizobium were the most dominant. With the exception of the later two bacterial genera, the first three are potential pathogens whose presence in HVAC systems poses a significant public health risk, especially among immunocompromised individuals. Nine pathways associated with antibiotics resistance and bacterial pathogenicity were identified, including polymyxin resistance and peptidoglycan biosynthesis pathways. Further, investigation of the relationship between the detected bacterial meta-communities and predicted potential virulence factors (antibiotic resistance and pathogenic genes) led to the detection of 350 positive associations among 43 core bacteria, 2 pathogenic genes (sitA and uidA) and 14 resistance genes. Overall, the heterogeneous nature of microorganisms found in HVAC systems observed in this study shows that HVAC systems are the origin of airborne infections in indoor environments, and must be periodically cleaned and disinfected to avoid the build-up of pathogens, and the subsequent exposure of human occupants of these pathogens.202133415530
7131110.9503Longitudinal study of the short- and long-term effects of hospitalisation and oral trimethoprim-sulfadiazine administration on the equine faecal microbiome and resistome. BACKGROUND: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids. METHODS: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0-D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14-D21), during 5 days of oral TMS treatment (D22-D26) and after discharge from the hospital up to 6 months later (D27-D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples. RESULTS: Hospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3")-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased 6 months later. CONCLUSIONS: Oral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimise long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment. Video Abstract.202336850017
5170120.9501Synergistic effect of imp/ostA and msbA in hydrophobic drug resistance of Helicobacter pylori. BACKGROUND: Contamination of endoscopy equipment by Helicobacter pylori (H. pylori) frequently occurs after endoscopic examination of H. pylori-infected patients. In the hospital, manual pre-cleaning and soaking in glutaraldehyde is an important process to disinfect endoscopes. However, this might not be sufficient to remove H. pylori completely, and some glutaraldehyde-resistant bacteria might survive and be passed to the next patient undergoing endoscopic examination through unidentified mechanisms. We identified an Imp/OstA protein associated with glutaraldehyde resistance in a clinical strain, NTUH-C1, from our previous study. To better understand and manage the problem of glutaraldehyde resistance, we further investigated its mechanism. RESULTS: The minimal inhibitory concentrations (MICs) of glutaraldehyde andexpression of imp/ostA RNA in 11 clinical isolates from the National Taiwan University Hospital were determined. After glutaraldehyde treatment, RNA expression in the strains with the MICs of 4-10 microg/ml was higher than that in strains with the MICs of 1-3 microg/ml. We examined the full-genome expression of strain NTUH-S1 after glutaraldehyde treatment using a microarray and found that 40 genes were upregulated and 31 genes were downregulated. Among the upregulated genes, imp/ostA and msbA, two putative lipopolysaccharide biogenesis genes, were selected for further characterization. The sensitivity to glutaraldehyde or hydrophobic drugs increased in both of imp/ostA and msbA single mutants. The imp/ostA and msbA double mutant was also hypersensitive to these chemicals. The lipopolysaccharide contents decreased in individual imp/ostA and msbA mutants and dramatically reduced in the imp/ostA and msbA double mutant. Outer membrane permeability assay demonstrated that the imp/ostA and msbA double mutation resulted in the increase of outer membrane permeability. Ethidium bromide accumulation assay demonstrated that MsbA was involved in efflux of hydrophobic drugs. CONCLUSION: The expression levels of imp/ostA and msbA were correlated with glutaraldehyde resistance in clinical isolates after glutaraldehyde treatment. Imp/OstA and MsbA play a synergistic role in hydrophobic drugs resistance and lipopolysaccharide biogenesis in H. pylori.200919594901
6076130.9501Isolation and identification of mucin-degrading bacteria originated from human faeces and their potential probiotic efficacy according to host-microbiome enterotype. AIM: Mucin-degrading bacteria are known to be beneficial for gut health. We aimed to isolate human-derived mucin-degrading bacteria and identify potential probiotic characteristics and their effects on the bacterial community and short-chain fatty acid (SCFA) production according to three different enterotypes of the host. METHODS AND RESULTS: Bacteria with mucin decomposition ability from human faeces were isolated and identified by 16S rRNA sequencing and MALDI-TOF. Heat resistance, acid resistance, antibiotic resistance, and antibacterial activity were analysed in the selected bacteria. Their adhesion capability to the Caco-2 cell was determined by scanning electron microscopy. Their ability to alter the bacterial community and SCFA production of the isolated bacteria was investigated in three enterotypes. The three isolated strains were Bifidobacterium(Bif.) animalis SPM01 (CP001606.1, 99%), Bif. longum SPM02 (NR_043437.1, 99%), and Limosilactobacillus(L.) reuteri SPM03 (CP000705.1, 99%) deposited in Korean Collection for Type Culture (KCTC-18958P). Among them, Bif. animalis exhibited the highest mucin degrading ability. They exhibited strong resistance to acidic conditions, moderate resistance to heat, and the ability to adhere tightly to Caco-2 cells. Three isolated mucin-degrading bacteria incubation increased Lactobacillus in the faecal bacteria from Bacteroides and Prevotella enterotypes. However, only L. reuteri elevated Lactobacillus in the faecal bacteria from the Ruminococcus enterotype. B. longum and B. animalis increased the α-diversity in the Ruminococcus enterotype, while their incubation with other intestinal types decreased the α-diversity. Bifidobacterium animalis and L. reuteri increased the butyric acid level in faecal bacteria from the Prevotella enterotype, and L. reuteri elevated the acetic acid level in those from the Ruminococcus enterotype. However, the overall SCFA changes were minimal. CONCLUSIONS: The isolated mucin-degrading bacteria act as probiotics and modulate gut microbiota and SCFA production differently according to the host's enterotypes. SIGNIFICANCE AND IMPACT OF STUDY: Probiotics need to be personalized according to the enterotypes in clinical application.202235365862
6391140.9499Monitoring antibiotic resistomes and bacterial microbiomes in the aerosols from fine, hazy, and dusty weather in Tianjin, China using a developed high-volume tandem liquid impinging sampler. Accurate quantification of the airborne antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) is critically important to assess their health risks. However, the currently widely used high-volume filter sampler (HVFS) often causes the desiccation of the sample, interfering with subsequent bacterial culture. To overcome this limitation, a high-volume tandem liquid impinging sampler (HVTLIS) was developed and optimized to investigate the airborne bacterial microbiomes and antibiotic resistomes under different weathers in Tianjin, China. Results revealed that HVTLIS can capture significantly more diverse culturable bacteria, ARB, and ARGs than HVFS. Compared with fine and hazy weathers, dusty weather had significantly more diverse and abundant airborne bacteria, ARGs, and human opportunistic pathogens with the resistance to last-resort antibiotics of carbapenems and polymyxin B, implicating a potential human health threat of dusty bioaerosols. Intriguingly, we represented the first report of Saccharibacteria predominance in the bioaerosol, demonstrating that the potential advantage of HVTLIS in collecting airborne microbes.202032438084
9082150.9499GeneMates: an R package for detecting horizontal gene co-transfer between bacteria using gene-gene associations controlled for population structure. BACKGROUND: Horizontal gene transfer contributes to bacterial evolution through mobilising genes across various taxonomical boundaries. It is frequently mediated by mobile genetic elements (MGEs), which may capture, maintain, and rearrange mobile genes and co-mobilise them between bacteria, causing horizontal gene co-transfer (HGcoT). This physical linkage between mobile genes poses a great threat to public health as it facilitates dissemination and co-selection of clinically important genes amongst bacteria. Although rapid accumulation of bacterial whole-genome sequencing data since the 2000s enables study of HGcoT at the population level, results based on genetic co-occurrence counts and simple association tests are usually confounded by bacterial population structure when sampled bacteria belong to the same species, leading to spurious conclusions. RESULTS: We have developed a network approach to explore WGS data for evidence of intraspecies HGcoT and have implemented it in R package GeneMates ( github.com/wanyuac/GeneMates ). The package takes as input an allelic presence-absence matrix of interested genes and a matrix of core-genome single-nucleotide polymorphisms, performs association tests with linear mixed models controlled for population structure, produces a network of significantly associated alleles, and identifies clusters within the network as plausible co-transferred alleles. GeneMates users may choose to score consistency of allelic physical distances measured in genome assemblies using a novel approach we have developed and overlay scores to the network for further evidence of HGcoT. Validation studies of GeneMates on known acquired antimicrobial resistance genes in Escherichia coli and Salmonella Typhimurium show advantages of our network approach over simple association analysis: (1) distinguishing between allelic co-occurrence driven by HGcoT and that driven by clonal reproduction, (2) evaluating effects of population structure on allelic co-occurrence, and (3) direct links between allele clusters in the network and MGEs when physical distances are incorporated. CONCLUSION: GeneMates offers an effective approach to detection of intraspecies HGcoT using WGS data.202032972363
3535160.9499Bacillus licheniformis-fermented products and enramycin differentially modulate microbiota and antibiotic resistome in the cecal digesta of broilers. Since antibiotic resistance is a global health issues, the use of antibiotics in animal feed for growth promotion has been restricted in many countries. Bacillus licheniformis probiotic is a potential alternative to antibiotics for increasing poultry performance. Through metagenomic sequencing, this study investigated the effects of B. licheniformis-fermented products (BLFPs) and enramycin on the microbial community composition and antibiotic resistance gene (ARG) distribution in the cecal digesta of broilers at the age of 35 d. In total, 144 one-day-old male broiler chicks (Ross 308) were randomly assigned to 4 dietary treatments as follows: basal diet (control [C] group), basal diet plus 10 mg/kg enramycin (E group), basal diet plus 1 g/kg BLFPs (L group), and basal diet plus 3 g/kg BLFPs (H group), with 6 replicate cages per treatment group and 6 birds per cage. The results indicated that the cecal alpha diversity (richness and evenness) of bacterial species was higher in the H group than in the C group. Principal coordinate analysis of microbiota and the ARG composition indicated clear differences among the cecal samples of the groups. In the cecal digesta, the abundance of active bacteria associated with probiotic properties, such as Lactobacillus crispatus and Akkermansia muciniphila, was higher in the H group than in the other groups. Enramycin treatment promoted the expression of peptide (bcrA), glycopeptide (vanRI), and lincosamide (lsaE) resistance genes but inhibited the expression of aminocoumarin (parY) and pleuromutilin (TaeA) resistance genes. BLFP (1 and 3 g/kg) treatment suppressed the expression of aminoglycoside (ANT(6)-Ib), streptogramin (vatB), and peptide (ugd) resistance genes but enhanced the expression of macrolide (efrA) and aminocoumarin (novA) resistance genes. The abundance of peptide resistance genes in Bacteroides spp. was lower in the H group than in the C group. The abundance of lincosamide resistance genes in Lactobacillus spp. was higher in the E group than in the other groups. These results demonstrated that differential changes in the structure of 3 g/kg BLFPs and enramycin-induced cecal microbial communities accompany changes in the abundance of bacterial hosts carrying specific ARGs in the cecal microbiota of broilers.202235841645
2472170.9499A 'Tuba Drain' incorporated in sink drains reduces counts of antibiotic-resistant bacterial species at the plughole: a blinded, randomized trial in 36 sinks in a hospital outpatient department with a low prevalence of sink colonization by antibiotic-resistant species. BACKGROUND: Multi-resistant Gram-negative bacteria (GNB) survive in hospital drains in traps that contain water and may ascend into the sink because of splashes, or biofilm growth. AIM: To investigate whether the 'Tuba Drain' (TD) a long, bent, continually descending copper tube between the sink outlet and the trap prevents the ascent of bacteria. METHODS: After initial laboratory tests confirmed that the TD prevented bacteria in the U-bend from splashing upwards into the sink outlet, TDs were assessed in a randomized, blinded trial in a hospital outpatient department built in 2019. Sinks were paired into those with a similar clinical exposure and each member of each pair was randomized to receive either new, standard plumbing up to and including the trap (18 sinks) or the same new standard plumbing but including the TD inserted between the sink outlet and trap. Bacterial counts in swabs from the sink outlets were determined blindly before and monthly after the plumbing change for a year. GNB that are associated with clinical infection and carriage of resistance genes, Pseudomonas aeruginosa, Acinetobacter baumanii, Stenotrophomonas maltophilia and all Enterobacterales were the organisms of primary interest and termed target bacteria. FINDINGS: The TDs fitted into the required spaces and functioned without problems. The geometric means (over months) of the counts of target bacteria in TD-plumbed sinks was lower than those in their paired controls, P=0.012 (sign test, two-tailed). Prevalence of target bacteria in sinks was low. CONCLUSION: TDs were effective for reducing target bacteria in sinks.202539515476
8472180.9498Genetic architecture of resistance to plant secondary metabolites in Photorhabdus entomopathogenic bacteria. BACKGROUND: Entomopathogenic nematodes of the genus Heterorhabditis establish a symbiotic association with Photorhabdus bacteria. Together, they colonize and rapidly kill insects, making them important biological control agents against agricultural pests. Improving their biocontrol traits by engineering resistance to plant secondary metabolites (benzoxazinoids) in Photorhabdus symbiotic bacteria through experimental evolution has been shown to increase their lethality towards benzoxazinoid-defended larvae of the western corn rootworm, a serious crop pest of maize, and it is therefore a promising approach to develop more efficient biocontrol agents to manage this pest. To enhance our understanding of the genetic bases of benzoxazinoid resistance in Photorhabdus bacteria, we conducted an experimental evolution experiment with a phylogenetically diverse collection of Photorhabdus strains from different geographic origins. We cultured 27 different strains in medium containing 6-methoxy-2-benzoxazolinone (MBOA), a highly active benzoxazinoid breakdown product, for 35 24 h-cycles to select for benzoxazinoid-resistant strains. Then, we carried out genome-wide sequence comparisons to uncover the genetic alterations associated with benzoxazinoid resistance. Lastly, we evaluated the resistance of the newly isolated resistant Photorhabdus strains to eight additional bioactive compounds, including 2-benzoxazolinone (BOA), nicotine, caffeine, 6-chloroacetyl-2-benzoxazolinone (CABOA), digitoxin, fenitrothion, ampicillin, and kanamycin. RESULTS: We found that benzoxazinoid resistance evolves rapidly in Photorhabdus in a strain-specific manner. Across the different Photorhabdus strains, a total of nineteen nonsynonymous point mutations, two stop codon gains, and one frameshift were associated with higher benzoxazinoid resistance. The different genetic alterations were polygenic and occurred in genes coding for the EnvZ/OmpR two-component regulatory system, the different subunits of the DNA-directed RNA polymerase, and the AcrABZ-TolC multidrug efflux pump. Apart from increasing MBOA resistance, the different mutations were also associated with cross-resistance to 2-benzoxazolinone (BOA), nicotine, caffeine, and 6-chloroacetyl-2-benzoxazolinone (CABOA) and with collateral sensitivity to fenitrothion, ampicillin, and kanamycin. Targeted mutagenesis will provide a deeper mechanistic understanding, including the relative contribution of the different mutation types. CONCLUSIONS: Our study reveals several genomic features that are associated with resistance to xenobiotics in this important group of biological control agents and enhances the availability of molecular tools to develop better biological control agents, which is essential for more sustainable and ecologically friendly agricultural practices.202541168779
6834190.9497Landscape of antibiotic resistance genes and bacterial communities in groundwater on the Tibetan Plateau, and distinguishing their difference with low-altitude counterparts. Groundwater is a vital source of drinking water for Tibetans. Antibiotic resistance genes (ARGs) and bacterial communities in groundwater on the Tibetan Plateau remain unclear. Furthermore, the characterization of their differences between high-altitude and low-altitude groundwater is still unrevealed. Herein, 32 groundwater samples were collected on the plateau, and intra- and extracellular ARGs (iARGs and eARGs), and bacterial communities were characterised through qPCR assays to 19 ARGs and 16S rRNA sequencing. It showed top four abundant intra- and extracellular last-resort ARGs (LARGs) were bla(OXA-48), mcr-1, vanA, and vanB, whereas dominant common ARGs (CARGs) were tetA and ermB, respectively. CARGs had higher abundances than LARGs, and iARGs were more frequently detected than eARGs. Proteobacteria, an invasive resident phylum, and Firmicutes dominated eDNA release. Network analysis revealed all observed LARGs co-occurred with pathogenic and non-pathogenic bacteria. Community diversity was significantly associated with longitude and elevation, while nitrate correlated with ARGs. Comparative analysis demonstrated eARG frequencies and abundances were higher at high altitudes than at low altitudes. Additionally, Acinetobacter and Pseudomonas specifically dominated at high altitudes. This study reveals the widespread prevalence of ARGs, particularly LARGs, in groundwater on the less-disturbed Tibetan Plateau and underlines the potential risks associated with the LARG-carrying bacteria. ENVIRONMENTAL IMPLICATION: Antibiotic resistance genes (ARGs), which are defined as emerging environmental contaminants, are becoming a global concern due to their ability to confer antibiotic resistance to pathogens. Our findings highlight the prevalence of ARGs, particularly LARGs, in groundwater on the Tibetan Plateau, and the possibility that naturally-occurring pathogenic and non-pathogenic bacteria carry multiple LARGs. In addition, we further reveal differences in the distribution of ARGs and bacterial community between high-altitude and low-altitude groundwater. Collectively, our findings offer an important insight into the potential public risks related to groundwater on the Tibetan Plateau.202337595466