Multiomics analysis reveals the presence of a microbiome in the gut of fetal lambs. - Related Documents




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516001.0000Multiomics analysis reveals the presence of a microbiome in the gut of fetal lambs. OBJECTIVE: Microbial exposure is critical to neonatal and infant development, growth and immunity. However, whether a microbiome is present in the fetal gut prior to birth remains debated. In this study, lambs delivered by aseptic hysterectomy at full term were used as an animal model to investigate the presence of a microbiome in the prenatal gut using a multiomics approach. DESIGN: Lambs were euthanised immediately after aseptic caesarean section and their cecal content and umbilical cord blood samples were aseptically acquired. Cecal content samples were assessed using metagenomic and metatranscriptomic sequencing to characterise any existing microbiome. Both sample types were analysed using metabolomics in order to detect microbial metabolites. RESULTS: We detected a low-diversity and low-biomass microbiome in the prenatal fetal gut, which was mainly composed of bacteria belonging to the phyla Proteobacteria, Actinobacteria and Firmicutes. Escherichia coli was the most abundant species in the prenatal fetal gut. We also detected multiple microbial metabolites including short chain fatty acids, deoxynojirimycin, mitomycin and tobramycin, further indicating the presence of metabolically active microbiota. Additionally, bacteriophage phiX174 and Orf virus, as well as antibiotic resistance genes, were detected in the fetal gut, suggesting that bacteriophage, viruses and bacteria carrying antibiotic resistance genes can be transmitted from the mother to the fetus during the gestation period. CONCLUSIONS: This study provides strong evidence that the prenatal gut harbours a microbiome and that microbial colonisation of the fetal gut commences in utero.202133589511
254310.9993Capturing the antibiotic resistome of preterm infants reveals new benefits of probiotic supplementation. BACKGROUND: Probiotic use in preterm infants can mitigate the impact of antibiotic exposure and reduce rates of certain illnesses; however, the benefit on the gut resistome, the collection of antibiotic resistance genes, requires further investigation. We hypothesized that probiotic supplementation of early preterm infants (born < 32-week gestation) while in hospital reduces the prevalence of antibiotic resistance genes associated with pathogenic bacteria in the gut. We used a targeted capture approach to compare the resistome from stool samples collected at the term corrected age of 40 weeks for two groups of preterm infants (those that routinely received a multi-strain probiotic during hospitalization and those that did not) with samples from full-term infants at 10 days of age to identify if preterm birth or probiotic supplementation impacted the resistome. We also compared the two groups of preterm infants up to 5 months of age to identify persistent antibiotic resistance genes. RESULTS: At the term corrected age, or 10 days of age for the full-term infants, we found over 80 antibiotic resistance genes in the preterm infants that did not receive probiotics that were not identified in either the full-term or probiotic-supplemented preterm infants. More genes associated with antibiotic inactivation mechanisms were identified in preterm infants unexposed to probiotics at this collection time-point compared to the other infants. We further linked these genes to mobile genetic elements and Enterobacteriaceae, which were also abundant in their gut microbiomes. Various genes associated with aminoglycoside and beta-lactam resistance, commonly found in pathogenic bacteria, were retained for up to 5 months in the preterm infants that did not receive probiotics. CONCLUSIONS: This pilot survey of preterm infants shows that probiotics administered after preterm birth during hospitalization reduced the diversity and prevented persistence of antibiotic resistance genes in the gut microbiome. The benefits of probiotic use on the microbiome and the resistome should be further explored in larger groups of infants. Due to its high sensitivity and lower sequencing cost, our targeted capture approach can facilitate these surveys to further address the implications of resistance genes persisting into infancy without the need for large-scale metagenomic sequencing. Video Abstract.202236008821
387420.9992Culture-enriched human gut microbiomes reveal core and accessory resistance genes. BACKGROUND: Low-abundance microorganisms of the gut microbiome are often referred to as a reservoir for antibiotic resistance genes. Unfortunately, these less-abundant bacteria can be overlooked by deep shotgun sequencing. In addition, it is a challenge to associate the presence of resistance genes with their risk of acquisition by pathogens. In this study, we used liquid culture enrichment of stools to assemble the genome of lower-abundance bacteria from fecal samples. We then investigated the gene content recovered from these culture-enriched and culture-independent metagenomes in relation with their taxonomic origin, specifically antibiotic resistance genes. We finally used a pangenome approach to associate resistance genes with the core or accessory genome of Enterobacteriaceae and inferred their propensity to horizontal gene transfer. RESULTS: Using culture-enrichment approaches with stools allowed assembly of 187 bacterial species with an assembly size greater than 1 million nucleotides. Of these, 67 were found only in culture-enriched conditions, and 22 only in culture-independent microbiomes. These assembled metagenomes allowed the evaluation of the gene content of specific subcommunities of the gut microbiome. We observed that differentially distributed metabolic enzymes were associated with specific culture conditions and, for the most part, with specific taxa. Gene content differences between microbiomes, for example, antibiotic resistance, were for the most part not associated with metabolic enzymes, but with other functions. We used a pangenome approach to determine if the resistance genes found in Enterobacteriaceae, specifically E. cloacae or E. coli, were part of the core genome or of the accessory genome of this species. In our healthy volunteer cohort, we found that E. cloacae contigs harbored resistance genes that were part of the core genome of the species, while E. coli had a large accessory resistome proximal to mobile elements. CONCLUSION: Liquid culture of stools contributed to an improved functional and comparative genomics study of less-abundant gut bacteria, specifically those associated with antibiotic resistance. Defining whether a gene is part of the core genome of a species helped in interpreting the genomes recovered from culture-independent or culture-enriched microbiomes.201930953542
771630.9992Metagenomic analysis fecal microbiota of dysentery-like diarrhoea in a pig farm using next-generation sequencing. Porcine enteric diseases including swine dysentery involves a wide range of possible aetiologies and seriously damages the intestine of pigs of all ages. Metagenomic next-generation sequencing is commonly used in research for detecting and analyzing pathogens. In this study, the feces of pigs from a commercial swine farm with dysentery-like diarrhea was collected and used for microbiota analysis by next-generation sequencing. While Brachyspira spp. was not detected in diarrheal pig fecal samples, indicating that the disease was not swine dysentery. The quantity of microbial population was extremely lowered, and the bacterial composition was altered with a reduction in the relative abundance of the probiotics organisms, Firmicutes and Bacteroidetes, with an increase in pathogens like Fusobacterium and Proteobacteria, in which the specific bacteria were identified at species-level. Viral pathogens, porcine circovirus type 2, porcine lymphotropic herpesviruses 1, and porcine mastadenovirus A were also detected at pretty low levels. Carbohydrate-active enzymes (CAZy) analysis indicated that the constitute of Firmicutes and Bacteroidete were also changed. Further, the Kyoto Encyclopedia of Genes and Genomes (KEGG) alignment analysis indicated that the microbiota of diarrheal pigs had a lower ability in utilizing energy sources but were enriched in multi-drug resistance pathways. Comprehensive Antibiotic Resistance Database (CARD) and Virulence Factors of Pathogenic Bacteria (VFDB) analysis indicated that genes for elfamycin and sulfonamide resistance and the iron uptake system were enriched in diarrheal pigs. This revealed potential bacterial infection and can guide antibiotic selection for treating dysentery. Overall, our data suggested that alterations in both the population and functional attributes of microbiota in diarrheal pigs with decreased probiotic and increased pathogenic microorganisms. These results will help elucidate the mechanism of dysentery-like diarrhea and the development of approaches to control the disease.202337915946
254440.9992Antibiotic resistance potential of the healthy preterm infant gut microbiome. BACKGROUND: Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. RESULTS: Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. CONCLUSIONS: We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.201728149696
322150.9991Age influences the temporal dynamics of microbiome and antimicrobial resistance genes among fecal bacteria in a cohort of production pigs. BACKGROUND: The pig gastrointestinal tract hosts a diverse microbiome, which can serve to select and maintain a reservoir of antimicrobial resistance genes (ARG). Studies suggest that the types and quantities of antimicrobial resistance (AMR) in fecal bacteria change as the animal host ages, yet the temporal dynamics of AMR within communities of bacteria in pigs during a full production cycle remains largely unstudied. RESULTS: A longitudinal study was performed to evaluate the dynamics of fecal microbiome and AMR in a cohort of pigs during a production cycle; from birth to market age. Our data showed that piglet fecal microbial communities assemble rapidly after birth and become more diverse with age. Individual piglet fecal microbiomes progressed along similar trajectories with age-specific community types/enterotypes and showed a clear shift from E. coli/Shigella-, Fusobacteria-, Bacteroides-dominant enterotypes to Prevotella-, Megaspheara-, and Lactobacillus-dominated enterotypes with aging. Even when the fecal microbiome was the least diverse, the richness of ARGs, quantities of AMR gene copies, and counts of AMR fecal bacteria were highest in piglets at 2 days of age; subsequently, these declined over time, likely due to age-related competitive changes in the underlying microbiome. ARGs conferring resistance to metals and multi-compound/biocides were detected predominately at the earliest sampled ages. CONCLUSIONS: The fecal microbiome and resistome-along with evaluated descriptors of phenotypic antimicrobial susceptibility of fecal bacteria-among a cohort of pigs, demonstrated opposing trajectories in diversity primarily driven by the aging of pigs.202336624546
468360.9991Characterization of Bacteroides fragilis from the vagina of a giant panda (Ailuropoda melanoleuca) with vaginitis. BACKGROUND: Bacteroides fragilis is a prevalent anaerobic bacterium typically resides in the human vagina. It is known to potentially induce infections under specific conditions. Interestingly, there have been no previous reports of B. fragilis being isolated from the vagina of giant pandas. CASE PRESENTATION: A novel strain of anaerobic bacteria was isolated from the vaginal tract of a giant panda exhibiting symptoms of vaginitis. This strain, designated as GPBF01, was identified as Bacteroides fragilis, a species commonly found in the vaginal microbiome of humans and other animals. After purifying of the single colony, a series of evaluations were conducted including morphological examination, physiological and biochemical identification, antibiotic resistance analysis, resistance genes detection, 16S rRNA sequence, and phylogenetic tree sequence analysis to investigate its biological characteristics. The findings indicated the presence of a predominant anaerobic bacterium, which was identified as B. fragilis and temporarily named GPBF01 with unique biological traits not previously. CONCLUSIONS: This study is the first to report B. fragilis in the vaginal tract of giant pandas. The analysis of antibiotic resistance patterns among anaerobic bacteria, as conducted in this research, is critical for informing the selection of appropriate antimicrobial agents in the clinical treatment of vaginitis in this species. The findings of this report substantially enhance the scientific basis needed to understand the etiology and refine therapeutic approaches for vaginitis in giant pandas.202439605068
454470.9991Identification of aminoglycoside and β-lactam resistance genes from within an infant gut functional metagenomic library. The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2-512x), increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth.201425247417
771980.9991Different artificial feeding strategies shape the diverse gut microbial communities and functions with the potential risk of pathogen transmission to captive Asian small-clawed otters (Aonyx cinereus). Captive otters raised in zoos are fed different artificial diets, which may shape gut microbiota. The objective is to evaluate the impacts of two different artificial diets on microbial communities and function capabilities and short-chain fatty acid (SCFA) profiles in healthy otters' feces. A total of 16 Asian small-clawed otters in two groups (n = 8) were selected. Group A otters were fed raw loaches supplemented with commercial cat food (LSCF) diet, and group B otters were fed raw crucian diet. The communities and functional capabilities of microbiota in feces were assessed with metagenomic sequencing. Captive otters fed two kinds of diets possessed different gut microbial communities and functional capabilities. Various pathogenic bacteria, like Escherichia coli and Clostridium perfringens, were enriched in the samples from the two groups, respectively. Most of the differential pathways of nutrient metabolism were significantly enriched in group A, and the distributions of carbohydrate enzymes in the two groups significantly differed from each other. Multiple resistance genes markedly accumulated in fecal samples of the group A otters with LSCF diet. Higher concentrations of SCFAs were also observed in group A otters. Two feeding strategies were both likely to facilitate the colonization and expansion of various pathogenic bacteria and the accumulation of resistance genes in the intestines of captive otters, suggesting that risk of pathogen transmission existed in the current feeding process. Commercial cat food could supplement various nutrients and provide a substrate for the production of SCFAs, which might be beneficial for the otters' intestinal fermentation and metabolism. IMPORTANCE: Captive otters fed with different diets possessed distinct gut microbial communities and functions, with the enrichment of several pathogens and multiple resistance genes in their gut microbiota. The current artificial feeding strategies had the possibility to accelerate the colonization and proliferation of various pathogenic bacteria in the intestines of otters and the spread of resistance genes, increasing the risk of diseases. In addition, supplementation with commercial cat food had benefits for otters' intestinal fermentation and the metabolism of gut microbiota.202439601555
315290.9991Daily fluctuation of Lactobacillus species and their antibiotic resistome in the colon of growing pigs. There are various types of bacteria inhabiting the intestine that help maintain the balance of the intestinal microbiota. Lactobacillus is one of the important beneficial bacteria and is widely used as a food starter and probiotic. In this study, we investigated the daily fluctuation of the colonic Lactobacillus species and their distribution of antibiotic resistance genes (ARGs) as well as antibiotic susceptibility in pigs. Metagenomic analysis revealed that genus Lactobacillus was one of the most dominant genera in the colon of growing pigs. Rhythmicity analysis revealed that 84 out of 285 Lactobacillus species exhibited rhythmic patterns. Lactobacillus johnsonii and Lactobacillus reuteri were the two most abundant lactobacilli with circadian oscillation, which increased during the day and decreased at night. The profile of the antibiotic resistome was modified over time within 24-h period. Elfamycin resistance genes were the most enriched class found in Lactobacillus. Furthermore, the seven strains of Lactobacillus isolated from the pig intestine mainly exhibited resistance to gentamicin, erythromycin, and lincomycin. The whole genome annotation of four Lactobacillus strains indicated the presence of multiple ARGs, including elfamycin resistance genes, however, the most abundant ARG was optrA in genome of four strains. These results indicate the presence of various Lactobacillus species harboring a large number of ARGs in the swine intestine. This implies that when using animal-derived lactobacilli, it is essential to assess antibiotic resistance to prevent further transmission between animals and the environment.202438336077
4654100.9991Early Bacterial Colonization and Antibiotic Resistance Gene Acquisition in Newborns. Several studies have recently identified the main factors contributing to the bacterial colonization of newborns and the dynamics of the infant microbiome development. However, most of these studies address large time periods of weeks or months after birth, thereby missing on important aspects of the early microbiome maturation, such as the acquisition of antibiotic resistance determinants during postpartum hospitalization. The pioneer bacterial colonization and the extent of its associated antibiotic resistance gene (ARG) dissemination during this early phase of life are largely unknown. Studies addressing resistant bacteria or ARGs in neonates often focus only on the presence of particular bacteria or genes from a specific group of antibiotics. In the present study, we investigated the gut-, the oral-, and the skin-microbiota of neonates within the first 72 h after birth using 16S rDNA sequencing approaches. In addition, we screened the neonates and their mothers for the presence of 20 different ARGs by directed TaqMan qPCR assays. The taxonomic analysis of the newborn samples revealed an important shift of the microbiota during the first 72 h after birth, showing a clear site-specific colonization pattern in this very early time frame. Moreover, we report a substantial acquisition of ARGs during postpartum hospitalization, with a very high incidence of macrolide resistance determinants and mecA detection across different body sites of the newborns. This study highlights the importance of antibiotic resistance determinant dissemination in neonates during hospitalization, and the need to investigate the implication of the mothers and the hospital environment as potential sources of ARGs.202032754449
3875110.9991Ecological insights into the microbiology of food using metagenomics and its potential surveillance applications. A diverse array of micro-organisms can be found on food, including those that are pathogenic or resistant to antimicrobial drugs. Metagenomics involves extracting and sequencing the DNA of all micro-organisms on a sample, and here, we used a combination of culture and culture-independent approaches to investigate the microbial ecology of food to assess the potential application of metagenomics for the microbial surveillance of food. We cultured common foodborne pathogens and other organisms including Escherichia coli, Klebsiella/Raoultella spp., Salmonella spp. and Vibrio spp. from five different food commodities and compared their genomes to the microbial communities obtained by metagenomic sequencing following host (food) DNA depletion. The microbial populations of retail food were found to be predominated by psychrotrophic bacteria, driven by the cool temperatures in which the food products are stored. Pathogens accounted for a small percentage of the food metagenome compared to the psychrotrophic bacteria, and cultured pathogens were inconsistently identified in the metagenome data. The microbial composition of food varied amongst different commodities, and metagenomics was able to classify the taxonomic origin of 59% of antimicrobial resistance genes (ARGs) found on food to the genus level, but it was unclear what percentage of ARGs were associated with mobile genetic elements and thus transferable to other bacteria. Metagenomics may be used to survey the ARG burden, composition and carriage on foods to which consumers are exposed. However, food metagenomics, even after depleting host DNA, inconsistently identifies pathogens without enrichment or further bait capture.202539752189
4541120.9991Gut microbiome of endangered Tor putitora (Ham.) as a reservoir of antibiotic resistance genes and pathogens associated with fish health. BACKGROUND: Tor putitora, the largest freshwater fish of the Indian subcontinent, is an endangered species. Several factors have been attributed towards its continuous population decrease, but very little is known about the gut microbiome of this fish. Also, the fish gut microbiome serves as a reservoir of virulence factors and antibiotic resistance determinants. Therefore, the shotgun metagenomic approach was employed to investigate the taxonomic composition and functional potential of microbial communities present in the gut of Tor putitora, as well as the detection of virulence and antibiotic resistance genes in the microbiome. RESULTS: The analysis of bacterial diversity showed that Proteobacteria was predominant phylum, followed by Chloroflexi, Bacteroidetes, and Actinobacteria. Within Proteobacteria, Aeromonas and Caulobacter were chiefly present; also, Klebsiella, Escherichia, and plant symbionts were noticeably detected. Functional characterization of gut microbes endowed the virulence determinants, while surveillance of antibiotic resistance genes showed the dominance of β-lactamase variants. The antibiotic-resistant Klebsiella pneumoniae and Escherichia coli pathovars were also detected. Microbial genome reconstruction and comparative genomics confirmed the presence of Aeromonads, the predominant fish pathogens. CONCLUSIONS: Gut microbiome of endangered Tor putitora consisted of both commensals and opportunistic pathogens, implying that factors adversely affecting the non-pathogenic population would allow colonization and proliferation of pathogens causing diseased state in asymptomatic Tor putitora. The presence of virulence factors and antibiotic resistance genes suggested the potential risk of dissemination to other bacteria due to horizontal gene transfer, thereby posing a threat to fish and human health. The preservation of healthy gut microflora and limited use of antibiotics are some of the prerequisites for the conservation of this imperilled species.202032787773
3322130.9990Combined analysis of metagenomic data revealed consistent changes of gut microbiome structure and function in inflammatory bowel disease. AIMS: To reveal the consistency and discrepancy in the gut microbial structure and function in inflammatory bowel disease (IBD) patients from different regions. METHODS AND RESULTS: Gut microbes, antibiotic resistance genes (ARGs) and virulence factors genes (VFGs) were analysed using metagenome data from three cohorts. The abundance of Escherichia coli extensively increased in IBD patients, whereas Subdoligranulum unclassified decreased dramatically in IBD patients from three countries. Escherichia coli showed a positive correlation with multiple ARGs and VFGs in cohorts from China and the United States, including multidrug-related resistance genes and Capsule and LOS-related virulence factors genes. Escherichia coli biofilm synthesis pathways significantly enriched in IBD patients from three different regions. Notably, Subdoligranulum unclassified and Eubacterium hallii were negatively related to ARGs and VFGs. CONCLUSIONS: Consistent changes of microbiome structure and function were observed in IBD patients from three different regions. As pathogenic bacteria, E. coli may accelerate IBD progression through encapsulation in biofilms by upregulating antibiotic resistance in Crohn's disease patients. Subdoligranulum unclassified and E. hallii may be beneficial for IBD patients and could serve as potential probiotics for IBD treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work dispels worries about the regional differences in gut microbial changes in IBD patients and provides useful guidance for more rational microbiome-based therapies.202134008889
8385140.9990Function and Phylogeny of Bacterial Butyryl Coenzyme A:Acetate Transferases and Their Diversity in the Proximal Colon of Swine. Studying the host-associated butyrate-producing bacterial community is important, because butyrate is essential for colonic homeostasis and gut health. Previous research has identified the butyryl coenzyme A (CoA):acetate-CoA transferase (EC 2.3.8.3) as a gene of primary importance for butyrate production in intestinal ecosystems; however, this gene family (but) remains poorly defined. We developed tools for the analysis of butyrate-producing bacteria based on 12 putative but genes identified in the genomes of nine butyrate-producing bacteria obtained from the swine intestinal tract. Functional analyses revealed that eight of these genes had strong But enzyme activity. When but paralogues were found within a genome, only one gene per genome encoded strong activity, with the exception of one strain in which no gene encoded strong But activity. Degenerate primers were designed to amplify the functional but genes and were tested by amplifying environmental but sequences from DNA and RNA extracted from swine colonic contents. The results show diverse but sequences from swine-associated butyrate-producing bacteria, most of which clustered near functionally confirmed sequences. Here, we describe tools and a framework that allow the bacterial butyrate-producing community to be profiled in the context of animal health and disease. IMPORTANCE: Butyrate is a compound produced by the microbiota in the intestinal tracts of animals. This compound is of critical importance for intestinal health, and yet studying its production by diverse intestinal bacteria is technically challenging. Here, we present an additional way to study the butyrate-producing community of bacteria using one degenerate primer set that selectively targets genes experimentally demonstrated to encode butyrate production. This work will enable researchers to more easily study this very important bacterial function that has implications for host health and resistance to disease.201627613689
3222150.9990Differences in gut metagenomes between dairy workers and community controls: a cross-sectional study. BACKGROUND: As a nexus of routine antibiotic use and zoonotic pathogen presence, the livestock farming environment is a potential hotspot for the emergence of zoonotic diseases and antibiotic resistant bacteria. Livestock can further facilitate disease transmission by serving as intermediary hosts for pathogens as they undergo evolution prior to a spillover event. In light of this, we are interested in characterizing the microbiome and resistome of dairy workers, whose exposure to the livestock farming environment places them at risk for facilitating community transmission of antibiotic resistant genes and emerging zoonotic diseases. RESULTS: Using shotgun sequencing, we investigated differences in the taxonomy, diversity and gene presence of the human gut microbiome of 10 dairy farm workers and 6 community controls, supplementing these samples with additional publicly available gut metagenomes. We observed greater abundance of tetracycline resistance genes and prevalence of cephamycin resistance genes in dairy workers' metagenomes, and lower average gene diversity. We also found evidence of commensal organism association with plasmid-mediated tetracycline resistance genes in both dairy workers and community controls (including Faecalibacterium prausnitzii, Ligilactobacillus animalis, and Simiaoa sunii). However, we did not find significant differences in the prevalence of resistance genes or virulence factors overall, nor differences in the taxonomic composition of dairy worker and community control metagenomes. CONCLUSIONS: This study presents the first metagenomics analysis of United States dairy workers, providing insights into potential risks of exposure to antibiotics and pathogens in animal farming environments. Previous metagenomic studies of livestock workers in China and Europe have reported increased abundance and carriage of antibiotic resistance genes in livestock workers. While our investigation found no strong evidence for differences in the abundance or carriage of antibiotic resistance genes and virulence factors between dairy worker and community control gut metagenomes, we did observe patterns in the abundance of tetracycline resistance genes and the prevalence of cephamycin resistance genes that is consistent with previous work.202337215025
8382160.9990Transcriptional and Functional Analysis of Bifidobacterium animalis subsp. lactis Exposure to Tetracycline. Commercial probiotic bacteria must be tested for acquired antibiotic resistance elements to avoid potential transfer to pathogens. The European Food Safety Authority recommends testing resistance using microdilution culture techniques previously used to establish inhibitory thresholds for the Bifidobacterium genus. Many Bifidobacterium animalis subsp. lactis strains exhibit increased resistance to tetracycline, historically attributed to the ribosomal protection gene tet(W). However, some strains that harbor genetically identical tet(W) genes show various inhibition levels, suggesting that other genetic elements also contribute to observed differences. Here, we adapted several molecular assays to confirm the inhibition of B. animalis subsp. lactis strains Bl-04 and HN019 and employed RNA sequencing to assess the transcriptional differences related to genomic polymorphisms. We detected specific stress responses to the antibiotic by correlating ATP concentration to number of viable genome copies from droplet digital PCR and found that the bacteria were still metabolically active in high drug concentrations. Transcriptional analyses revealed that several polymorphic regions, particularly a novel multidrug efflux transporter, were differentially expressed between the strains in each experimental condition, likely having phenotypic effects. We also found that the tet(W) gene was upregulated only during subinhibitory tetracycline concentrations, while two novel tetracycline resistance genes were upregulated at high concentrations. Furthermore, many genes involved in amino acid metabolism and transporter function were upregulated, while genes for complex carbohydrate utilization, protein metabolism, and clustered regularly interspaced short palindromic repeat(s) (CRISPR)-Cas systems were downregulated. These results provide high-throughput means for assessing antibiotic resistances of two highly related probiotic strains and determine the genetic network that contributes to the global tetracycline response.IMPORTANCEBifidobacterium animalis subsp. lactis is widely used in human food and dietary supplements. Although well documented to be safe, B. animalis subsp. lactis strains must not contain transferable antibiotic resistance elements. Many B. animalis subsp. lactis strains have different resistance measurements despite being genetically similar, and the reasons for this are not well understood. In the current study, we sought to examine how genomic differences between two closely related industrial B. animalis subsp. lactis strains contribute to different resistance levels. This will lead to a better understanding of resistance, identify future targets for analysis of transferability, and expand our understanding of tetracycline resistance in bacteria.201830266728
4542170.9990Phylogenetic intermixing reveals stable fly-mediated circulation of mastitis-associated bacteria in dairy settings. Stomoxys flies are common blood-feeding pests on dairy farms and are suspected carriers of pathogenic bacteria due to their close association with manure and cattle hosts. While prior studies have used amplicon sequencing and culture-dependent methodologies to characterize the composition of the Stomoxys microbiota, little is known about strain-level acquisition of mastitis-causing bacteria from manure by Stomoxys or the functional diversity of Stomoxys-associated taxa. In this study, we address these key knowledge gaps by using whole genome sequencing to provide the first comparative genomic analysis of Stomoxys-derived Escherichia coli, Klebsiella pneumoniae, and Staphylococcaceae isolates. Our results show that fly and manure isolates collected from the same farm system are phylogenetically interspersed, with subsequent pairwise genome alignments revealing near-identical strains and plasmids shared between the two sources. We further identify a phylogenetic clade of Mammaliicoccus sciuri containing known mastitis agents associated with both flies and manure. Functional analysis reveals that this clade is highly enriched in xylose metabolism genes that are rare across other M. sciuri lineages, suggesting potential niche differentiation within the genus. Collectively, our results provide strong evidence for the acquisition of fecal-associated bacteria by adult Stomoxys flies, confirming the link between biting muscid flies and manure habitats. The intermixing of fly and manure isolates in clinically relevant taxonomic groups strongly suggests that flies serve as carriers of opportunistic mastitis-causing or other fecal-borne pathogens and may serve as important vehicles of pathogen dissemination across the dairy farm environment.IMPORTANCEBovine mastitis causes up to $32 billion dollars in losses annually in the global dairy industry. Opportunistic intramammary pathogens can be transmitted through incidental contact with bacteria in environmental reservoirs like manure. However, factors affecting the abundance, persistence, and spread of these bacteria are not well understood. Our research shows that mastitis pathogens are present in the guts of blood-feeding Stomoxys (stable) flies, which develop in cow feces and bite cows. Genomic analysis of isolates from flies, manure, and mastitis cases reveals that strains and antimicrobial resistance genes are shared between these sources. Further analysis of fly gut isolates shows virulence factors and possible niche specialization, identifying fly-associated clades with known mastitis agents from mastitic cows. This strongly suggests that Stomoxys flies play a role in the carriage and circulation of bovine mastitis pathogens from manure in dairy settings.202540748061
7403180.9990Effect of Enrofloxacin on the Microbiome, Metabolome, and Abundance of Antibiotic Resistance Genes in the Chicken Cecum. Enrofloxacin is an important antibiotic for the treatment of Salmonella infections in livestock and poultry. However, the effects of different concentrations of enrofloxacin on the bacterial and metabolite compositions of the chicken gut and changes in the abundance of resistance genes in cecum contents remain unclear. To investigate the effects of enrofloxacin on chickens, we orally administered different concentrations of enrofloxacin to 1-day-old chickens and performed 16S rRNA gene sequencing to assess changes in the gut microbiomes of chickens after treatment. The abundance of fluoroquinolone (FQ) resistance genes was measured using quantitative PCR. Metabolomics techniques were used to examine the cecal metabolite composition. We found that different concentrations of enrofloxacin had different effects on cecum microorganisms, with the greatest effect on cecum microbial diversity in the low-concentration enrofloxacin group at day 7. Enrofloxacin use reduced the abundance of beneficial bacteria such as Lactobacillaceae and Oscillospira. Furthermore, cecum microbial diversity was gradually restored as the chickens grew. In addition, enrofloxacin increased the abundance of resistance genes, and there were differences in the changes in abundance among different antibiotic resistance genes. Moreover, enrofloxacin significantly affected linoleic acid metabolism, amino acid metabolism, and signaling pathways. This study helps improve our understanding of how antibiotics affect host physiological activities and provides new insights into the rational use of drugs in poultry farming. The probiotics and metabolites that we identified could be used to modulate the negative effects of antibiotics on the host, which requires further study. IMPORTANCE In this study, we investigated changes in the cecum flora, metabolites, and abundances of fluoroquinolone antibiotic resistance genes in chickens following the use of different concentrations of enrofloxacin. These results were used to determine the effects of enrofloxacin on chick physiology and the important flora and metabolites that might contribute to these effects. In addition, these results could help in assessing the effect of enrofloxacin concentrations on host metabolism. Our findings could help guide the rational use of antibiotics and mitigate the negative effects of antibiotics on the host.202336840593
7731190.9990Biochemical Recurrence in Prostate Cancer Is Associated with the Composition of Lactobacillus: Microbiome Analysis of Prostatic Tissue. Many human pathologies, such as malignancy, are linked with specific bacteria and changes in the constituents of the microbiome. In order to examine the association between an imbalance of bacteria and prostate carcinoma, a comparison of the microbiomes present in patients with biochemical recurrence (BCR) or NO BCR (NBCR) was performed. Additionally, 16S rRNA-based next-generation sequencing was applied to identify the bacterial profiles within these tumors in terms of the bacteria and operational genes present. The percentage average taxonomic composition between the taxa indicated no difference between BCR and NBCR. In addition, alpha and beta diversity indices presented no distinction between the cohorts in any statistical method. However, taxonomic biomarker discovery indicated a relatively higher population of Lactobacillus in the NBCR group, and this finding was supported by PCR data. Along with that, differences in the operational activity of the bacterial genes were also determined. It is proposed that the biochemical recurrence was linked to the quantity of Lactobacillus present. The aim of this study was to investigate the microbiome involved in prostate carcinoma and the potential association between them.202337445601