16S rDNA Libraries Research Articles

Study of the diversity and short-chain fatty acids production by the bacterial community in overweight and obese Mexican children.

Obesity and overweight are health problems of multifactorial etiology, which may include changes in the microbiome. In Mexico, more than 30% of the child population between 5 and 11years of age suffer from being overweight or are obese, which makes it a public health issue in progress. The purpose of this work was to measure the short-chain fatty acid concentration by high-performance liquid chromatography (HPLC), and to characterize the bacterial diversity by ion torrent semiconductor sequencing, of 16S rDNA libraries prepared from stools collected from a sample of well-characterized Mexican children for normal weight, overweight, and obese conditions by anthropometric and biochemical criteria. We found that triglyceride levels are increased in overweight and obese children, who presented altered propionic and butyric acid concentrations in feces. In addition, although the colon microbiota did not show a clear bacterial dysbiosis among the three conditions, the abundance of some particular bacteria was changed with respect to normal controls. We conclude from our results that the imbalance in the abundance of at least nine different bacteria as well as altered short-chain fatty acid concentration in feces is associated to the overweight and obese conditions of Mexican children.

Archaeal communities associated with roots of the common reed (Phragmites australis) in Beijing Cuihu Wetland.

The richness, phylogeny and composition of archaeal community associated with the roots of common reed (Phragmites australis) growing in the Beijing Cuihu Wetland, China was investigated using a 16S rDNA library. In total, 235 individual sequences were collected, and a phylogenetic analysis revealed that 69.4 and 11.5% of clones were affiliated with the Euryarchaeota and the Crenarchaeota, respectively. In Euryarchaeota, the archaeal community was dominated by species in following genera: Methanobacterium in the order Methanobacteriales (60.7%); Methanoregula and Methanospirillum in the order Methanomicrobiales (20.2%), and Methanomethylovorans, Methanosarcina and Methanosaeta in the order Methanosarcinales (17.2%). Of 27 sequences assigned to uncultured Crenarchaeota, 22 were grouped into Group 1.3, and five grouped into Group 1.1b. Hence, the archaeal communities associated with reed roots are largely involved in methane production, and, to a lesser extent, in ammonia oxidization. Quantification of the archaeal amoA gene indicated that ammonia oxidizing archaea were more numerous in the rhizosphere soil than in the root tissue or surrounding water. A total of 19.1% of the sequences were unclassified, suggesting that many unidentified archaea are probably involved in the reed wetland ecosystem.

Purifying the impure: sequencing metagenomes and metatranscriptomes from complex animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Increases in ‘Candidatus Liberibacter asiaticus’ viability and investigations of biofilm-like structures in citrus juice medium

Huanglongbing disease of citrus, associated with infection by the bacterium ‘Candidatus Liberibacter asiaticus’ (LAS), has spread rapidly in the US since 2005. Attempts to culture LAS in vitro have not yielded a consistently reproducible culture method; therefore, obtaining knowledge about the infection process is difficult. To determine conditions which sustain LAS viability, LAS inoculum obtained from seeds of fruit from infected pomelo trees (Citrus grandis ‘Mato Buntan’) was added to different media, and cell viability was monitored for several weeks using quantitative polymerase chain reaction (qPCR) in conjunction with ethidium monoazide (EMA). Among media tested, King’s B (K) did not support viability of LAS cells, while grapefruit juice (G) allowed LAS cells to survive in vitro for ~20 days. In media that sustained LAS viability, a reproducible biofilm-like substance was formed over time at the air-liquid interface of culture flasks and glass slides inserted in cultures. Fluorescence in situ hybridization (FISH) showed the biofilm contains aggregates of LAS cells, which was confirmed by qPCR. 16S rDNA libraries of the biofilm samples have been constructed and will be sequenced via Illumina next-generation sequencing to determine their bacterial composition. To elucidate why juice-based media prolongs LAS viability, the elemental nutrient compositions of the media and the biofilm were analyzed via inductively coupled plasma optical emission spectrometry (ICP-OES). Compositions were compared, and specific elements, such as potassium and calcium, were more abundant in media that sustain LAS cell viability. Results will contribute to future development of a culture medium for LAS.

Analysis of endophytic bacterial community composition by 16S rDNA clone library in Achnatherum inebrians

The endophytic bacterial diversity of drunken horse grass (DHG) (Achnatherum inebrians) growing in native habitat station in Xinjiang Province, China was analyzed by 16S rDNA cloning, amplified ribosomal DNA restriction analysis (ARDRA), and sequence homology comparison. To effectively exclude the interference of chloroplast DNA and mitochondrial DNA of DHG, a pair of bacterial PCR primers was selected to specifically amplify bacterial 16S rDNA sequences directly from DHG root tissues. Among 249 positive clones in the 16S rDNA library of endophytes, 57 OTUs were identified based on the similarity of the ARDRA banding profiles. Sequence analysis revealed diverse phyla of bacteria in the 16S rDNA library, which consisted of alpha, beta, gamma, delta, and epsilon subclasses of the Proteobacteria, Flavobacteria, Actinobacteria, Sphingobacteria, and uncultured bacteria. The dominant group was Proteobacteria, whie the most numerous genus was Bacillus. More than 8.00% of the total clones showed a high similarity to uncultured bacteria, suggesting that nonculturable bacteria make a significant part of DHG endophytic bacterial community. To our knowledge, this is the first report that endophytic bacteria associated with DHG are revealed by the culture-independent approach. The results suggest that the diversity of endophytic bacteria is abundant in DHG.

Identification of causative pathogens in mouse eyes with bacterial keratitis by sequence analysis of 16S rDNA libraries.

The clone library method using PCR amplification of the 16S ribosomal RNA (rRNA) gene was used to identify pathogens from corneal scrapings of C57BL/6-corneal opacity (B6-Co) mice with bacterial keratitis. All 10 samples from the eyes with bacterial keratitis showed positive PCR results. All 10 samples from the normal cornea showed negative PCR results. In all 10 PCR-positive samples, the predominant and second most predominant species accounted for 20.9 to 40.6% and 14.7 to 26.1%, respectively, of each clone library. The predominant species were Staphylococcus lentus, Pseudomonas aeruginosa, and Staphylococcus epidermidis. The microbiota analysis detected a diverse group of microbiota in the eyes of B6-Co mice with bacterial keratitis and showed that the causative pathogens could be determined based on percentages of bacterial species in the clone libraries. The bacterial species detected in this study were mostly in accordance with results of studies on clinical bacterial keratitis in human eyes. Based on the results of our previous studies and this study, the B6-Co mouse should be considered a favorable model for studying bacterial keratitis.

Effects of Elevated Carbon Dioxide on Soil Bacterial Community Structure

The soil bacteria diversity in different concentrations of CO2was investigated in the simulation test area, by using PCR (Polymerase Chain Reaction) -DGGE (Denaturing Gradient Gel Electrophoresis) and 16Sr-DNA library technology. The results showed that the bacteria diversity coefficient (P) got down to 0.8710, 0.8710 and 0.7742 from 0.9032 (from control area) when the concentration of CO2in soil reached 20000ppm, 40000ppm, 60000ppmrespectively. With the increasing concentration of CO2in soil, the abundance of the original low-density bacteria such asAsticcacaulis excentricus, etc. increased, while the abundance ofunclassified_Rhizobialesreduced significantly. Dominant bacteria such asBrucella melitensisetc. had the higher homology. It can be revealed that high concentration of CO2had a significant impact on the soil bacteria community diversity, while a weak influence on main bacterial species. Azotobacter was sensitive to the increasing of the CO2concentration. Great reducing of their abundance had an adverse effect on nitrogen-fixing capability of soil.

Rhizosphere heterogeneity shapes abundance and activity of sulfur-oxidizing bacteria in vegetated salt marsh sediments.

Salt marshes are highly productive ecosystems hosting an intense sulfur (S) cycle, yet little is known about S-oxidizing microorganisms in these ecosystems. Here, we studied the diversity and transcriptional activity of S-oxidizers in salt marsh sediments colonized by the plant Spartina alterniflora, and assessed variations with sediment depth and small-scale compartments within the rhizosphere. We combined next-generation amplicon sequencing of 16S rDNA and rRNA libraries with phylogenetic analyses of marker genes for two S-oxidation pathways (soxB and rdsrAB). Gene and transcript numbers of soxB and rdsrAB phylotypes were quantified simultaneously, using newly designed (RT)-qPCR assays. We identified a diverse assemblage of S-oxidizers, with Chromatiales and Thiotrichales being dominant. The detection of transcripts from S-oxidizers was mostly confined to the upper 5 cm sediments, following the expected distribution of root biomass. A common pool of species dominated by Gammaproteobacteria transcribed S-oxidation genes across roots, rhizosphere, and surrounding sediment compartments, with rdsrAB transcripts prevailing over soxB. However, the root environment fine-tuned the abundance and transcriptional activity of the S-oxidizing community. In particular, the global transcription of soxB was higher on the roots compared to mix and rhizosphere samples. Furthermore, the contribution of Epsilonproteobacteria-related S-oxidizers tended to increase on Spartina roots compared to surrounding sediments. These data shed light on the under-studied oxidative part of the sulfur cycle in salt marsh sediments and indicate small-scale heterogeneities are important factors shaping abundance and potential activity of S-oxidizers in the rhizosphere.

Diversity and Distribution of Sulfate-Reducing Bacteria in Jilin Oilfield

To better understand the roles of microorganisms in oil production, diversity and distribution of microbes in Jilin oilfield was studied. Firstly, chromosomal DNA wassuccessfully extracted directly from crude oil samples. Diversity and distribution of microbes was then analyzed based on 16S rDNA libraries. There were totally 21 OTUs were obtained through 16S rDNA sequencing. Of those OTUs, 13 are bacteria in whichProteobacteriais the major family, while 8 are archaea in whichMethanomicrobiais the dominant. Finally, SRB was found in all samples by amplifying theapsAgene using PCR. SRB found in Jilin oil samples belong toδ-Proteobacteria.

Effect of copper exposure on bacterial community structure and function in the sediments of Jiaozhou Bay, China

Microcosms were setup to investigate the possible impact of copper exposure on bacterial community structure and function in sediments of Jiaozhou Bay, China, by culture-independent microbial ecological techniques and community-level physiological profiling. Bacterial 16S rDNA libraries indicated that proportion of the bacteria in phyla Chloroflexi and Acidobacteria decreased, but that of Gammaproteobacteria and Planctomycetes slightly increased in copper-treated sediment. Denaturing gradient gel profiles showed that bacterial communities in control and copper exposed sediments developed into different directions, while the copper exposure did not change the pattern of ammonia oxidizing bacterial community. Microbial community-level physiological profiling revealed an obvious response to copper dosage. The copper pollution caused an acute decrease of carbon utilizing ability as well as bacterial functional diversity; the number of culturable heterotrophic bacteria was reduced by 90%. This study demonstrated that high copper input would obviously reduce culturable bacterial counts and seriously impact bacterial community function in marine sediments.

Diversity and novelty of actinobacteria in Arctic marine sediments

The actinobacterial diversity of Arctic marine sediments was investigated using culture-dependent and culture-independent approaches. A total of 152 strains were isolated from seven different media; 18 isolates were selected for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 18 isolates belonged to a potential novel genus and 10 known genera including Actinotalea, Arthrobacter, Brachybacterium, Brevibacterium, Kocuria, Kytococcus, Microbacterium, Micrococcus, Mycobacterium, and Pseudonocardia. Subsequently, 172 rDNA clones were selected by restriction fragment length polymorphism analysis from 692 positive clones within four actinobacteria-specific 16S rDNA libraries of Arctic marine sediments, and then these 172 clones were sequenced. In total, 67 phylotypes were clustered in 11 known genera of actinobacteria including Agrococcus, Cellulomonas, Demequina, Iamia, Ilumatobacter, Janibacter, Kocuria, Microbacterium, Phycicoccus, Propionibacterium, and Pseudonocardia, along with other, unidentified actinobacterial clones. Based on the detection of a substantial number of uncultured phylotypes showing low BLAST identities (<95 %), this study confirms that Arctic marine environments harbour highly diverse actinobacterial communities, many of which appear to be novel, uncultured species.

A primary assessment of the endophytic bacterial community in a xerophilous moss (Grimmia montana) using molecular method and cultivated isolates.

Investigating the endophytic bacterial community in special moss species is fundamental to understanding the microbial-plant interactions and discovering the bacteria with stresses tolerance. Thus, the community structure of endophytic bacteria in the xerophilous moss Grimmia montana were estimated using a 16S rDNA library and traditional cultivation methods. In total, 212 sequences derived from the 16S rDNA library were used to assess the bacterial diversity. Sequence alignment showed that the endophytes were assigned to 54 genera in 4 phyla (Proteobacteria, Firmicutes, Actinobacteria and Cytophaga/Flexibacter/Bacteroids). Of them, the dominant phyla were Proteobacteria (45.9%) and Firmicutes (27.6%), the most abundant genera included Acinetobacter, Aeromonas, Enterobacter, Leclercia, Microvirga, Pseudomonas, Rhizobium, Planococcus, Paenisporosarcina and Planomicrobium. In addition, a total of 14 species belonging to 8 genera in 3 phyla (Proteobacteria, Firmicutes, Actinobacteria) were isolated, Curtobacterium, Massilia, Pseudomonas and Sphingomonas were the dominant genera. Although some of the genera isolated were inconsistent with those detected by molecular method, both of two methods proved that many different endophytic bacteria coexist in G. montana. According to the potential functional analyses of these bacteria, some species are known to have possible beneficial effects on hosts, but whether this is the case in G. montana needs to be confirmed.

Diversity of endophytic bacteria in Caragana microphylla grown in the desert grassland of the Ningxia Hui autonomous region of China.

The diversity of endophytic bacteria in the sand-fixation plant Caragana microphylla was investigated by amplified rDNA restriction analysis and by sequence and phylogenetic comparisons of the 16S rRNA genes. A total of 24, 19, and 17 operational taxonomic units were identified from 16S rDNA libraries of the plant roots, stems and leaves, respectively. Homology analysis revealed a 92-100% identity of bacterial 16S rDNA sequences compared with those in the GenBank database. The bacteria identified by sequence homology fell into the following groups: α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, bacilli, and uncultured bacterium. Sequence analysis demonstrated that the roots were colonized predominantly by Bradyrhizobiaceae, while bacteria from Burkholderiaceae and Sphingomonadaceae were predominant in the stems and leaves, respectively. Additionally, the endophytic bacterial community in leaves was more diverse than those in the roots and stems. Overall, the most abundant bacteria in all three tissues analyzed were from the Sphingomonadaceae and Burkholderiaceae families, although many bacterial populations were found in only a single tissue. These results suggest that the bacterial population of C. microphylla is diverse.

Identification of key markers of normal and pathogenic microbiota determining health of periodontium by NGS-sequencing 16S-rDNA libraries of periodontal swabs

By using NGS-sequencing libraries of DNA from periodontal swabs with primers specific to V6 region of 16S rDNA prevalence of bacterial genera and species in periodontal microbiota of patients with aggressive periodontitis and healthy donors was analyzed. Six genera of putative periodontal protectors and eight periodontal pathogens were identified with respect to aggressive (but not chronic) periodontitis. Statistically relevant over-colonization by general Porphyromonas, Treponema, Synergistes, Tannerella, Filifactor, Ruminococcus, Parvimonas and Mycoplasma was found to be associated with the condition. From these, only three genera Porphyromonas, Treponema and Tannerella are traditionally considered as periodontal pathogens. Statistically confidential over-colonization by genus Veillonella was found in healthy patients. This genus should be considered as a relevant marker of a healthy periodontium. Genera Streptococcus, Bergeyella, Granulicatella, Kingella and Corynebacterium may be considered as putative periodontal protectors. Comparison of data of NGS-sequencing and real-time PCR demonstrated a good agreement if different PCR efficiency using independent primer pairs is taken into account.

Molecular Characterization and Analysis of Bacterial Diversity in &lt;I&gt;Aleurocanthus woglumi&lt;/I&gt; (Hemiptera: Aleyrodidae)

Aleurocanthus woglumi Ashby (Hemiptera: Aleyrodidae), commonly referred to as citrus blackfly, is a sap-sucking hemipteran insect. Although polyphagous, citrus is its most preferred host plant. Samples of this insect were collected from Murraya koenigii (L.). The cytochrome c oxidase subunit I gene (mtCO1)-based analysis by sequencing helped in molecular identification of the insect. Phylogenetic analysis of cytB-nd1-LrDNA showed the coevolution of A. woglumi with its primary bacterial symbiont Portiera. Sequencing a 16S rDNA library from insect DNA revealed three bacterial phylotypes, namely, Portiera, Wolbachia, and Erwinia chrysanthemi. Further, we used fluorescence in situ hybridization to visualize the endosymbionts in a whole mount of A. woglumi. Culturable bacteria were obtained on different media and were classified on the basis of 16S rDNA. In total, 30 bacterial phylotypes belonging to 14 different genera, namely, Bacillus, Kocuria, Micrococcus, Staphylococcus, Paenibacillus, Rhodococcus, Rummellibacillus, Arthrobacter, Curtobacterium, Psychrobacillus, Listeria, Brevibacillus, Bhargavae, and Pantoea, were isolated by culturable methods.

16S rDNA analysis of bacterial communities associated with the hyper accumulator Arabidopsis halleri grown on a Zn and Cd polluted soil

Having a glimpse of the relationships between the zinc and cadmium hyper accumulator Arabidopsis halleri and its rhizospheric bacteria may represent an interesting step towards the implementation of a rhizoremediation strategy based on this plant. Until now, these relationships remained unraveled. This work aimed to give some insights into the composition of the bacterial communities surrounding A. halleri when it grows on a soil harboring elevated amounts of zinc and cadmium, as well as into the influence that plant roots may exert on them. Industrial highly polluted soils containing different amounts of zinc and cadmium and surrounding A. halleri plants were sampled and their bacterial contents were harvested at various distances from A. halleri root system. Genetic diversity was assessed through DNA extraction, PCR amplification, 16S rDNA libraries construction, RFLP analysis and partial sequencing. Nine mini-libraries of hundred clones each were established and analyzed. Up to 481 independent clones were sequenced and identified at genus level. Twelve genera among 48 were found particularly well represented in the different sub-fractions analyzed. At class level, ten different classes were identified. Alpha-proteobacteria, Gamma-proteobacteria, Flavobacteria and Sphingobacteria were the main contributors to flora diversity. In the less polluted soil, roots proximity seems to affect negatively Alpha-proteobacteria and Flavobacteria and positively Gamma-proteobacteria. A rhizospheric selection limited to some specific genera was also observed when pollution raised.

Simultaneous biodegradation of phenanthrene and oxidation of arsenite by a dual-functional bacterial consortium

Bioremediation of co-contamination by polycyclic aromatic hydrocarbons (PAHs) and arsenite (As (III)) may be a cost-effective alternative to conventional chemical removal methods. In this study, a heterotrophic bacterial consortium capable of simultaneously degrading PAHs and oxidizing As (III) has been isolated. After 48 h of incubation, the bacterial consortium removed 71.4% of PHE and transformed 96.2% of As (III) to arsenate (As (V)) in liquid culture containing 200 mg l−1 of PHE and 60 mg l−1 of As (III) under aerobic conditions. A 16S rDNA library was constructed from the consortium to identify the dominant bacteria, which were Pseudomonas, Pusillimonas, Alcaligenes and Achromobacter spp. DGGE profiles showed considerable differences in community structure as the ratio of PHE and As (III) changed. When both PHE and As (III) were present, bacteria from the γ-Proteobacteria group (Pseudomonas) and β-Proteobacteria group (Achromobacter and Alcaligenes) were abundant. However, when only PHE was present, Pseudomonas and Achromobacter spp. were dominant, and when only As (III) was present, Pseudomonas and Alcaligenes spp. were the dominant species. The use of the dually functioning bacterial consortium provides a potential application for efficient biotransformation of PAHs and arsenite co-contamination systems.

Comparison of the diversity of root-associated bacteria in Phragmites australis and Typha angustifolia L. in artificial wetlands

Common reed (Phragmites australis) and narrow-leaved cattail (Typha angustifolia L.) are two plant species used widely in artificial wetlands constructed to treat wastewater. In this study, the community structure and diversity of root-associated bacteria of common reed and narrow-leaved cattail growing in the Beijing Cuihu Wetland, China, were investigated using 16S rDNA library and PCR-denaturing gradient gel electrophoresis methods. Root-associated bacterial diversity was higher in common reed than in narrow-leaved cattail. In both plant species, the dominant root-associated bacterial species were Alpha, Beta and Gamma Proteobacteria, including the genera Aeromonas, Hydrogenophaga, Ideonella, Uliginosibacterium and Vogesella. Acidobacteria, Actinobacteria, Nitrospirae and Spirochaetes were only found in the roots of common reed. Comparing the root-associated bacterial communities of reed and cattail in our system, many more species of bacteria related involved in the total nitrogen cycle were observed in reed versus cattail, while species involved in total phosphorus and organic matter removal were mainly found in cattail. Although we cannot determine their nutrient removal capacity separately, differences in the root-associated bacterial communities may be an important factor contributing to the differing water purification effects mediated by T. angustifolia and P. australis wetlands. Thus, further work describing the ecosystem functions of these bacterial species is needed, in order to fully understand how effective common reed- and narrow-leaved cattail-dominated wetlands are for phytoremediation.

Response of Intestinal Microbiota to Antibiotic Growth Promoters in Chickens

Antibiotic growth promoters (AGPs) have been used as feed additives to improve average daily weight gain and feed efficiency in food animals for more than five decades. However, use of AGPs is associated with the emergence of antibiotic-resistant human pathogens of animal origin, posing a significant threat to food safety and public health. Thus, development of novel alternatives to AGPs is important to mitigate antimicrobial resistance in foodborne pathogens. To achieve this goal, the mode of action of AGPs should be elucidated. In this study, the response of the chicken gut microbiota to AGPs was examined using two culture-independent approaches: phospholipid fatty acid (PLFA) biomarker analysis and 16S rDNA clone library sequencing. PLFA analysis showed that AGP tylosin treatment changed composition of the microbiota in various intestinal sites; however, total viable bacterial biomass in intestine was not affected by tylosin treatment. PLFA analysis also revealed an abundant viable fungal population in chicken microbiota. Eight 16S rDNA libraries (96 clones per library) were constructed using ileal samples from chickens that received either antibiotic-free or medicated feed. The 16S rDNA clone analysis of the growth-relevant samples showed the AGP treatment influenced the diversity of ileum microbiota in the chickens primarily in the Firmicutes division. In particular, Lactobacillus spp. populations in the ileum of AGP-treated chickens were significantly lower than those from chickens receiving antibiotic-free feed. Together, this study revealed novel features of the intestinal microbiota in response to AGP treatment and suggested approach to develop potential alternatives to AGPs for mitigation of antimicrobial resistance in foodborne pathogens.