RESUMO
We present high-throughput amplicon sequence (HTS) datasets of the purified microbial metacommunity DNA of coastal surface sediments from Portersville Bay (PVB) (n = 3), Bayou La Batre (BLB) (n = 3), and Mobile Bay (MOB) (n = 3) of the U.S. Gulf of Mexico (U.S. Gulf Coast). The PVB samples were collected from the oyster aquaculture Shellevator™ system; the BLB samples were from locations on the shoreline adjacent to wild oysters attached to rocks and likely polluted from sewage and possibly chemical contamination from boats, shipyards, and seafood processing facilities; and MOB samples were adjacent to aquaculture oysters in bottom cages. The amplicons of the V4 hypervariable segment of the 16S rRNA gene from each sample were sequenced on an Illumina MiSeq to generate these HTS datasets. The raw sequences were quality-checked, demultiplexed into FASTQ files, denoised using DADA2, and subsampled. Then, the FASTA formatted sequences were assigned the taxonomic ids to amplicon sequence variants (ASVs) against the silva-138-99-nb-classifier using the Quantitative Insights Into Microbial Ecology (QIIME2 v2022.2). The applicability of the HTS datasets was confirmed by microbial taxa analysis at the phylum level using the "qiime taxa collapse" command. All HTS datasets are available through the BioSample Submission Portal under the BioProject ID PRJNA876773 (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA876773).
RESUMO
The data in this article includes the sequences of bacterial 16S rRNA gene from metagenome of Macondo oil (MC252)-treated and non-oil-treated sediment microcosms, collected from coastal Gulf of Mexico and Bayou La Batre, USA. Metacommunity DNA was PCR amplified with 341F and 907R oligonucleotide primers, targeting V3-V5 regions of the 16S rRNA gene. Data were generated by using bacterial tag-encoded FLX-amplicon pyrosequencing (bTEFAP) methodology and then processed using bioinformatics tools such as QIIME. The data information is deposited to NCBI׳s BioProject and BioSample and raw sequence files are available via NCBI׳s Sequence Read Archive (SRA) database.
RESUMO
In this study, we examined the responses by the indigenous bacterial communities in salt-marsh sediment microcosms in vitro following treatment with Mississippi Canyon Block 252 oil (MC252). Microcosms were constructed of sediment and seawater collected from Bayou La Batre located in coastal Alabama on the Gulf of Mexico. We used an amplicon pyrosequencing approach on microcosm sediment metagenome targeting the V3-V5 region of the 16S rRNA gene. Overall, we identified a shift in the bacterial community in three distinct groups. The first group was the early responders (orders Pseudomonadales and Oceanospirillales within class Gammaproteobacteria), which increased their relative abundance within 2 weeks and were maintained 3 weeks after oil treatment. The second group was identified as early, but transient responders (order Rhodobacterales within class Alphaproteobacteria; class Epsilonproteobacteria), which increased their population by 2 weeks, but returned to the basal level 3 weeks after oil treatment. The third group was the late responders (order Clostridiales within phylum Firmicutes; order Methylococcales within class Gammaproteobacteria; and phylum Tenericutes), which only increased 3 weeks after oil treatment. Furthermore, we identified oil-sensitive bacterial taxa (order Chromatiales within class Gammaproteobacteria; order Syntrophobacterales within class Deltaproteobacteria), which decreased in their population after 2 weeks of oil treatment. Detection of alkane (alkB), catechol (C2,3DO) and biphenyl (bph) biodegradation genes by PCR, particularly in oil-treated sediment metacommunity DNA, delineates proliferation of the hydrocarbon degrading bacterial community. Overall, the indigenous bacterial communities in our salt-marsh sediment in vitro microcosm study responded rapidly and shifted towards members of the taxonomic groups that are capable of surviving in an MC252 oil-contaminated environment.
RESUMO
The indigenous bacterial communities in sediment microcosms from Dauphin Island (DI), Petit Bois Island (PB) and Perdido Pass (PP) of the coastal Gulf of Mexico were compared following treatment with Macondo oil (MC252) using pyrosequencing and culture-based approaches. After quality-based trimming, 28,991 partial 16S rRNA sequence reads were analyzed by rarefaction, confirming that analyses of bacterial communities were saturated with respect to species diversity. Changes in the relative abundances of Proteobacteria, Bacteroidetes and Firmicutes played an important role in structuring bacterial communities in oil-treated sediments. Proteobacteria were dominant in oil-treated samples, whereas Firmicutes and Bacteroidetes were either the second or the third most abundant taxa. Tenericutes, members of which are known for oil biodegradation, were detected shortly after treatment, and continued to increase in DI and PP sediments. Multivariate statistical analyses (ADONIS) revealed significant dissimilarity of bacterial communities between oil-treated and untreated samples and among locations. In addition, a similarity percentage analysis showed the contribution of each species to the contrast between untreated and oil-treated samples. PCR amplification using DNA from pure cultures of Exiguobacterium, Pseudoalteromonas, Halomonas and Dyadobacter, isolated from oil-treated microcosm sediments, produced amplicons similar to polycyclic aromatic hydrocarbon-degrading genes. In the context of the 2010 Macondo blowout, the results from our study demonstrated that the indigenous bacterial communities in coastal Gulf of Mexico sediment microcosms responded to the MC252 oil with altered community structure and species composition. The rapid proliferation of hydrocarbonoclastic bacteria suggests their involvement in the degradation of the spilt oil in the Gulf of Mexico ecosystem.
Assuntos
Biota/efeitos dos fármacos , Sedimentos Geológicos/microbiologia , Biotransformação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Golfo do México , Redes e Vias Metabólicas/genética , Metagenômica , Dados de Sequência Molecular , Óleos/metabolismo , Filogenia , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
In this study, we have developed a SYBR Green I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The C(t) values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (approximately 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.
Assuntos
Proteínas de Bactérias/genética , Proteínas Hemolisinas/genética , Ostreidae/microbiologia , Água do Mar/microbiologia , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Oceano Atlântico , Proteínas de Bactérias/isolamento & purificação , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Benzotiazóis , Sondas de DNA , DNA Bacteriano/análise , Diaminas , Microbiologia de Alimentos , Genes Bacterianos , Proteínas Hemolisinas/isolamento & purificação , Humanos , Hibridização In Situ , Compostos Orgânicos , Reação em Cadeia da Polimerase/métodos , Quinolinas , Sensibilidade e Especificidade , Vibrio parahaemolyticus/genéticaRESUMO
A Gram-positive, rod-shaped, non-spore-forming bacterium (strain K362(T)) was isolated from a deep-water marine sponge collected off the coast of Curaçao in the Netherlands Antilles. On the basis of 16S rRNA gene sequence similarities, strain K362(T) was shown to belong to the genus Tsukamurella, being most closely related to Tsukamurella pulmonis (99.2 %), Tsukamurella tyrosinosolvens (98.9 %), Tsukamurella strandjordii (98.8 %), Tsukamurella pseudospumae (98.8 %) and Tsukamurella spumae (98.8 %). A combination of the substrate utilization patterns, the fatty acid and mycolic acid profiles and the DNA-DNA hybridization results supported the affiliation of strain K362(T) to the genus Tsukamurella and enabled the genotypic and phenotypic differentiation of strain K362(T) from the seven recognized Tsukamurella species. Strain K362(T) therefore represents a novel species of the genus Tsukamurella, for which the name Tsukamurella spongiae sp. nov. is proposed. The type strain is K362(T) (=DSM 44990(T)=NRRL B-24467(T)).
Assuntos
Actinobacteria/classificação , Actinobacteria/isolamento & purificação , Poríferos/microbiologia , Actinobacteria/química , Actinobacteria/genética , Animais , Metabolismo dos Carboidratos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Genes de RNAr , Dados de Sequência Molecular , Ácidos Micólicos/análise , Antilhas Holandesas , Hibridização de Ácido Nucleico , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Água do Mar/microbiologia , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
A novel, extremely psychrotolerant, facultative anaerobe, strain PmagG1(T), was isolated from guano of Magellanic penguins (Spheniscus magellanicus) collected in Chilean Patagonia. Gram-variable, motile cocci with a diameter of 1.3-2.0 mum were observed singularly or in pairs, short chains and irregular conglomerates. Growth occurred within the pH range 6.0-10.0, with optimum growth at pH 8.5. The temperature range for growth of the novel isolate was from -5 to 35 degrees C, with optimum growth at 28-30 degrees C. Strain PmagG1(T) did not require NaCl, as growth was observed in the presence of 0-6.5 % NaCl with optimum growth at 0.5 % (w/v). Strain PmagG1(T) was a catalase-negative chemo-organoheterotroph that used sugars and some organic acids as substrates. The metabolic end products were lactate, formate, acetate, ethanol and CO(2). Strain PmagG1(T) was sensitive to ampicillin, tetracycline, chloramphenicol, rifampicin, kanamycin and gentamicin. The G+C content of its genomic DNA was 45.8 mol%. 16S rRNA gene sequence analysis showed 100 % similarity of strain PmagG1(T) with Trichococcus collinsii ATCC BAA-296(T), but DNA-DNA hybridization between them demonstrated relatedness values of <45+/-1 %. Another phylogenetically closely related species, Trichococcus pasteurii, showed 99.85 % similarity by 16S rRNA sequencing and DNA-DNA hybridization showed relatedness values of 47+/-1.5 %. Based on genotypic and phenotypic characteristics, the novel species Trichococcus patagoniensis sp. nov. is proposed, with strain PmagG1(T) (=ATCC BAA-756(T)=JCM 12176(T)=CIP 108035(T)) as the type strain.
Assuntos
Bactérias Anaeróbias/isolamento & purificação , Spheniscidae/microbiologia , Animais , Bactérias Anaeróbias/classificação , Bactérias Anaeróbias/genética , Técnicas de Tipagem Bacteriana , Chile , Dados de Sequência Molecular , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genéticaRESUMO
We describe a real-time multiplexed PCR method using Taqman probes for the detection of total and pandemic Vibrio parahaemolyticus O3:K6 serovar in oysters and Gulf of Mexico water (gulf water). The specificity of these primers and probes was tested for amplification of a 450 bp thermolabile hemolysin (tlh) and a 369 bp ORF8 amplicon representing all V. parahaemolyticus and post-1996 clinical isolates of pandemic serovar O3:K6, respectively. The sensitivity of detection was 10 pg purified DNA or 10(3) CFU in 1 mL pure culture. Enrichment of this pathogen in oyster tissue homogenate or gulf water for 5 or 8 h resulted in the detection of an initial inoculum of 1 CFU in 1 mL or 1 g of samples. Application of the Taqman PCR assay on natural oysters exhibited a positive detection of V. parahaemolyticus, ranging from 16% to 100% of the samples collected primarily during the summer months. None of the samples exhibited a positive detection of O3:K6 serovar. Rapid and sensitive detection of this pathogen will help shellfish industry and Interstate Shellfish Sanitation Conference (ISSC) undertake appropriate measures to monitor this pathogen in oysters and oyster-growing waters, thereby preventing disease outbreaks and consequently protecting consumer health.
Assuntos
Ostreidae/microbiologia , Reação em Cadeia da Polimerase/métodos , Água do Mar/microbiologia , Frutos do Mar/microbiologia , Vibrioses/epidemiologia , Vibrio parahaemolyticus/isolamento & purificação , Animais , Oceano Atlântico , Sondas de DNA , Surtos de Doenças , Gastroenterite/epidemiologia , Gastroenterite/microbiologia , Humanos , Sensibilidade e Especificidade , Sorotipagem , Vibrioses/microbiologia , Vibrio parahaemolyticus/classificação , Vibrio parahaemolyticus/genéticaRESUMO
In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87 degrees C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10(2) V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.
Assuntos
Ostreidae/microbiologia , Reação em Cadeia da Polimerase/métodos , Água do Mar/microbiologia , Frutos do Mar/microbiologia , Vibrio vulnificus/isolamento & purificação , Animais , Benzotiazóis , DNA Bacteriano/análise , Diaminas , Corantes Fluorescentes , Proteínas Hemolisinas/genética , Humanos , Compostos Orgânicos , Quinolinas , Sensibilidade e Especificidade , Fatores de Tempo , Vibrio vulnificus/genéticaRESUMO
This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10(2) ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10(3) cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.