ABSTRACT
Staphylococcus aureus is one of the most common microorganisms responsible for high morbidity and mortality in humans and animals. Methicillin-resistant S. aureus are responsible for several outbreaks worldwide and therapeutic arsenal has become scarce. The present investigation verified the epidemiological profile of S. aureus strains isolated from the veterinary hospital staff, from dairy cattle workers and also from milk samples of dairy cattle presenting mastitis. Samples were characterized phenotypically by antibiogram, catalase, and coagulase tests, and also by Voges-Proskauer test. The isolated strains were characterized genotypically by specific Polymerase Chain Reaction and Amplified Ribosomal DNA Restriction Analysis (ARDRA). From the 218 isolated strains, 27 were identified as S. aureus (12%), four of them were resistant to oxacillin and two of them were classified as MRSA (Methicillin-resistant S. aureus). The prevalence of isolated strains among animal personnel care was low (2%) but all MRSA isolates were found among the clinical staff. Results of ARDRA pointed out that S. aureus strains isolated from different animal care personnel were grouped in the same cluster when HindIII and HinfII restriction enzymes were used. When ARDRA was performed with HaeIII enzyme, the formation of two clusters was observed, but the isolated strains were not correlated. The prevalence of S. aureus strains isolated was higher in clinical staff and the biochemical and molecular assays of them presented 100% of correlation.(AU)
Staphylococcus aureus está entre os microrganismos que apresentam as maiores taxas de morbidade e mortalidade em seres humanos e animais. Linhagens de S. aureus resistentes a meticilina podem causar surtos de infecção em todo o mundo, o que contribui para a escassez de arsenal terapêutico. Este trabalho analisou o perfil epidemiológico de estirpes de S. aureus isoladas de pessoas que trabalham em contato com animais em um hospital veterinário com gado leiteiro e também em amostras de leite de vacas acometidas por mastite. As estirpes de S. aureus isoladas foram caracterizadas fenotipicamente por meio de antibiograma, testes de catalase e coagulase, e pelo teste de Voges-Proskauer. As amostras também foram caracterizadas genotipicamente pela técnica de Análise de Restrição de DNA Ribossômico Amplificado (ARDRA-PCR). Das 218 estirpes isoladas, 27 foram identificadas como S. aureus (12%). Entre essas, quatro estirpes foram resistentes à oxacilina e duas classificadas como SARM (S. aureus resistente à meticilina). A ocorrência de estirpes de S.aureus isoladas entre o pessoal que trabalha em contato com os animais foi baixa (2%), mas estirpes identificadas como SARM foram encontradas na equipe clínica. As análises de ARDRA realizadas com as enzimas de restrição HindIII e HinfII demonstraram que S. aureus isolados de diferentes indivíduos que trabalhavam com animais foram agrupados no mesmo cluster. Quando a ARDRA foi realizada com HaeIII foi observada formação de dois grupos, mas os isolados não se correlacionaram. Conclusão: a ocorrência de estirpes de S. aureus isoladas foi maior na equipe clínica, apresentando também correlação de 100% nos ensaios bioquímicos e moleculares.(AU)
Subject(s)
Animals , Cattle , Animal Technicians , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Milk/microbiology , Staphylococcus aureus/genetics , Staphylococcus aureus/isolation & purification , Mastitis, Bovine , Microbial Sensitivity Tests/veterinary , Polymerase Chain Reaction/methodsABSTRACT
Objeetive:To update the status of Gardnerella vaginalis (G.vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology,metronidazole resistance and virulence properties.Methods:It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur,Malaysia) prospective study with laboratory-based microbiological follow up and analyses.Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis,isolation and identification of G.vaginalis,metronidazole susceptibility testing,vaginolysin and sialidase gene PCR,Piot's biotyping and amplified ribosomal DNA restriction analysis genotyping.Results:Among the 207 patients suspected for BV,G.vaginalis was isolated from 47 subjects.G.vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples.Three G.vaginalis isolates were resistant to metronidazole.Biotyping revealed 1 and 7 as the common types.Amplified ribosomal DNA restriction analysis genotype Ⅱ was found to be more common (n =22;46％) than Ⅰ (n =12;25.53％) and Ⅲ (n =13;27.6％).All genotype Ⅰ and Ⅲ isolates carried the sialidase gene,while 91.6％ and 84.6％ contained the vaginolysin gene.Genotype Ⅰ was significantly associated with postgynaecological surgical complications and abortions (P =0.002).Conclusions:The existence of pathogenic G.vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.
ABSTRACT
Objective To update the status of Gardnerella vaginalis (G. vaginalis) as a causative agent of bacterial vaginosis (BV) in Malaysia and to define its epidemiology, metronidazole resistance and virulence properties. Methods It is a single-centre (Gynaecology clinic at the Hospital Kuala Lumpur, Malaysia) prospective study with laboratory-based microbiological follow up and analyses. Vaginal swabs collected from the patients suspected for BV were subjected to clinical BV diagnosis, isolation and identification of G. vaginalis, metronidazole susceptibility testing, vaginolysin and sialidase gene PCR, Piot's biotyping and amplified ribosomal DNA restriction analysis (ARDRA) genotyping. Results Among the 207 patients suspected for BV, G. vaginalis was isolated from 47 subjects. G. vaginalis coexisted with Trichomonas vaginalis and Candida albicans in 26 samples. Three G. vaginalis isolates were resistant to metronidazole. Biotyping revealed 1 and 7 as the common types. ARDRA genotype II was found to be more common (n = 22; 46%) than I (n = 12; 25.53%) and III (n = 13; 27.6%). All genotype I and III isolates carried the sialidase gene, while 91.6% and 84.6% contained the vaginolysin gene. Genotype I was significantly associated with post-gynaecological surgical complications and abortions (P = 0.002). Conclusions The existence of pathogenic G. vaginalis clones in Malaysia including drug resistant strains should not be taken lightly and needs to be monitored as these may bring more complications especially among women of child bearing age and pregnant women.
ABSTRACT
El objetivo de este estudio fue caracterizar bacterias halófilas con actividad amilolítica provenientes de las Salinas de San Blas-Junín, ubicadas en los Andes peruanos aproximadamente a 4100 m de altitud. Este estudio se realizó con 34 bacterias aisladas de muestras de suelos las cuales se cultivaron en agar agua de sales (SW) 5 % conteniendo extracto de levadura 0,5 % y almidón 1 %. El 41 % de bacterias mostró la capacidad de hidrolizar almidón, éstas fueron caracterizadas mediante pruebas fisiológicas y bioquímicas convencionales. Tres bacterias fueron Gram-negativas y once Gram-positivas. El 21 % (3/14) creció en un amplio rango de concentración de sales, entre 5 y 20 %. El 14 % (2/14) de las bacterias presentó actividad lipolítica, proteolítica y nucleolítica, y el 29 % (4/14), presentó actividad proteolítica y nucleolítica. Las bacterias se identificaron mediante los perfiles de restricción de los genes ribosómicos 16S amplificados, las enzimas usadas fueron Hae III, BstU I, Hinf I y Cfo I. Los genes ribosómicos 16S de siete bacterias que presentaron perfiles de ADN diferentes se amplificaron, secuenciaron y analizaron mediante programas bioinformáticos. Del análisis fenotípico y molecular de las 14 bacterias amilolíticas se obtuvieron dos grupos, uno perteneciente al género Halomonas (3) y el otro, al género Bacillus (11). Las bacterias amilolíticas caracterizadas podrían ser de potencial uso a nivel industrial.
The aim of this study was to characterize halophilic amylolytic bacteria from San Blas Salterns-Junin, located in the Peruvian Andes at approximately 4 100 m of altitude. This study was conducted with 34 bacteria isolated from soil samples which were cultured in salt water medium (SW) 5 % containing 0,5 % yeast extract and 1 % starch. It was found that 41 % were starch-degrading bacteria, which were further characterized with conventional physiological and biochemical tests. Three bacteria were Gram-negative and eleven Gram-positive. Also, 21 % (3/14) was able to grow in a wide range of saltconcentration from 5 to 20 %. We reported that 14 % (2/14) of bacteria had all lipolytic, proteolytic and nucleolytic activity, and 29 % (4/14) had both proteolytic and nucleolytic activity. Bacteria were identified by restriction 16S ribosomal genes profiles, enzymes used were Hae III, BstU I, Hinf I and Cfo I. 16S ribosomal genes of seven isolated wich showed different DNA profiles were amplified, partial sequenced and analyzed using bioinformatic programs. By both phenotypic and molecular analysis of 14 amylolytic bacteria two groups were obtained, one belonged to the genus Halomonas (3) and the other, to the genus Bacillus (11). The characterized amylolytic bacteria could have a potential industrial use.
ABSTRACT
The mangrove's sediments from the coastal areas under human activities may contain significant contaminations by hydrocarbons, even when there are no visual evidences of it. The microorganisms are essential to these ecosystems, especially in the control of their chemical environment. Sediment samples were collected in two regions under different environment conditions (pristine and contaminated) of the Paranaguá Estuarine Complex (Paranaguá Bay and Laranjeiras Bay), Brazil. Aliphatic hydrocarbons were determined by the GC-FID to assess the status of contamination of the studied areas. The total DNA was extracted from these samples. The 16S rRNA gene was amplified by the PCR reactions with the pair of primers 21F and 958R for the archaeal domain, and 27F and 1492R for the bacterial domain. Comparisons of communities were made by the ARDRA technique, using the HinfI restriction enzyme. The phosphate concentration showed significant differences between the two regions. The aliphatic hydrocarbons analysis showed the presence of unresolved complex mixture (UCM), an indicator of oil contamination, in the samples from the Paranaguá Bay, which was corroborated by the concentration of total aliphatic hydrocarbons. The ARDRA profile indicated that the structure of archaeal and bacterial communities of the sampled areas was very similar. Therefore, the anthropogenic influences in the Paranaguá Bay showed to be not sufficient to produce disturbances in the prokaryotic dominant groups.
ABSTRACT
The performance of an anaerobic sequencing-batch biofilm reactor (ASBBR- laboratory scale- 14L )containing biomass immobilized on coal was evaluated for the removal of elevated concentrations of sulfate (between 200 and 3,000 mg SO4-2·L-1) from industrial wastewater effluents. The ASBBR was shown to be efficient for removal of organic material (between 90% and 45%) and sulfate (between 95% and 85%). The microbiota adhering to the support medium was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). The ARDRA profiles for the Bacteria and Archaea domains proved to be sensitive for the determination of microbial diversity and were consistent with the physical-chemical monitoring analysis of the reactor. At 3,000 mg SO4-2·L-1, there was a reduction in the microbial diversity of both domains and also in the removal efficiencies of organic material and sulfate.
ABSTRACT
The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.
Subject(s)
Base Sequence , Bradyrhizobium/growth & development , Bradyrhizobium/isolation & purification , Fabaceae/growth & development , Gene Amplification , In Vitro Techniques , Polymerase Chain Reaction/methods , Rhizobium/growth & development , Rhizobium/isolation & purification , MethodsABSTRACT
Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ¡Ü 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.
Subject(s)
Gram-Positive Endospore-Forming Rods/genetics , Gram-Positive Endospore-Forming Rods/isolation & purification , DNA Repair Enzymes , RNA , Environmental MicrobiologyABSTRACT
With the purpose of isolating and characterizing free nitrogen fixing bacteria (FNFB) of the genus Azotobacter, soil samples were collected randomly from different vegetable organic cultures with neutral pH in different zones of Boyacá-Colombia. Isolations were done in selective free nitrogen Ashby-Sucrose agar obtaining a recovery of 40 percent. Twenty four isolates were evaluated for colony and cellular morphology, pigment production and metabolic activities. Molecular characterization was carried out using amplified ribosomal DNA restriction analysis (ARDRA). After digestion of 16S rDNA Y1-Y3 PCR products (1487pb) with AluI, HpaII and RsaI endonucleases, a polymorphism of 16 percent was obtained. Cluster analysis showed three main groups based on DNA fingerprints. Comparison between ribotypes generated by isolates and in silico restriction of 16S rDNA partial sequences with same restriction enzymes was done with Gen Workbench v.2.2.4 software. Nevertheless, Y1-Y2 PCR products were analysed using BLASTn. Isolate C5T from tomato (Lycopersicon esculentum) grown soils presented the same in silico restriction patterns with A. chroococcum (AY353708) and 99 percent of similarity with the same sequence. Isolate C5CO from cauliflower (Brassica oleracea var. botrytis) grown soils showed black pigmentation in Ashby-Benzoate agar and high similarity (91 percent) with A. nigricans (AB175651) sequence. In this work we demonstrated the utility of molecular techniques and bioinformatics tools as a support to conventional techniques in characterization of the genus Azotobacter from vegetable-grown soils.
Subject(s)
Agar/isolation & purification , Base Sequence , DNA, Ribosomal , Genetics, Microbial , In Vitro Techniques , Nitrogen Fixation , Polymerase Chain Reaction , Ribosomes/genetics , Soil Microbiology , Methods , Soil , MethodsABSTRACT
The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100 percent similarity, according to ARDRA results.
Subject(s)
Enzyme Activation/genetics , In Vitro Techniques , Rhizobiaceae/cytology , Rhizobiaceae/genetics , DNA Repair Enzymes/genetics , DNA Repair Enzymes/isolation & purification , Genes, Plant , Genetic Variation , Polymerase Chain Reaction , Rhizobiaceae/growth & developmentABSTRACT
Communities of Actynomicetes fungy in three vegetation types of the Colombian Amazon: abundance, morphotypes and the 16s rDNA gene. Among soil microorganisms, Actinomycetes play an important role in the sustainability of natural and agricultural systems: decomposition of organic matter; degradation of recalcitrant compounds like lignin; nitrogen fixation; degradation of agricultural chemicals and biological control in plants and animals. We evaluated their diversity in soils under three different vegetation covers (pasture, tropical primary forest and stubble) at two depths in the Southern Colombian Amazon border. We collected five replicates per vegetation type (in each, three samples at 0-20cm and three at 20-30cm; for a total of 30 samples). Abundance and phenotypic diversity were determined by plate counting. Genomic DNA was extracted from the isolates: the 16s rDNA gene was amplified with specific primers, and its genetic diversity was estimated by means of an amplified restriction analysis (ARDRA). Actynomicetes abundance varied with vegetation and depth, possibly reflecting presence of earthworms, macro-fauna and physico-chemical characteristics associated to fertility, as well as organic matter, total bases, and optimal capacity to cationic interchange. Primary forests had the highest diversity. Sixteen morpho-types (six genera) were identified; Streptomyces was the most abundant everywhere. The heterogeneity of ARDRA patterns prevented species identification because of the intra-species variability in sequences of 16s rDNA operons. This community is a biological indicator of landscape alteration and could include new bio-active compounds of pharmaceutical interest. Rev. Biol. Trop. 57 (4): 1119-1139. Epub 2009 December 01.
Los actinomicetos son importantes en la sostenibilidad de sistemas naturales. Su diversidad fue evaluada en suelos de bosque, pastizal y rastrojo, y dos profundidades en el Sur del Trapecio Amazónico Colombiano. Se analizaron suelos de cinco repeticiones por cobertura para un total de 15 unidades. Se tomaron seis muestras en cada unidad y dos profundidades, para un total de 30. Los actinomicetos cultivables se determinaron por recuento en placa, se extrajo ADN, se amplificó el gen ADNr 16s y su diversidad genética se estimó por ARDRA. Hubo diferencias de abundancia entre coberturas y profundidades, relacionadas con la vegetación, presencia de lombrices, macrofauna, altos niveles de materia orgánica, y bases totales. Se obtuvieron valores de diversidad fenotípica similares para las tres coberturas, pero los bosques son más diversos. Se identificaron 16 morfotipos, agrupados en séis géneros, siendo Streptomyces el más abundante. La heterogeneidad de los patrones ARDRA no permitió la asignación de especies, reflejándose variaciones en las secuencias de diferentes operones ADNr 16s en un mismo organismo. Las perturbaciones en la cobertura influyen sobre los actinomicetos, generando cambios en su abundancia y diversidad. Su importancia ecológica permite proponerlos como indicadores biológicos de alteración del paisaje.
Subject(s)
Actinobacteria/genetics , DNA, Ribosomal/genetics , Poaceae/microbiology , /genetics , Soil Microbiology/standards , Trees/microbiology , Actinobacteria/classification , Actinobacteria/isolation & purification , Colombia , Genetic Variation , PhenotypeABSTRACT
The genetic diversity of siderophore-producing bacteria of tobacco rhizosphere was studied by amplifiedribosomal DNA restriction analysis (ARDRA), 16S rRNA sequence homology and phylogenetics analysis methods. Studies demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium. A total of 28 ARDRA patterns were identified among the 299 siderophore-producing bacterial isolates.The 28 ARDRA patterns represented bacteria of 14 different genera belonging to six bacterial divisions, namely β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli, and Actinobacteria. Especially, γ- Proteobacteria consisting of Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia and Stenotrophomonas genus encountered 18 different ARDRA groups. Results also showed a greater siderophore-producing bacterial diversity than previous researches. For example, Sphingobacterium (isolates G-2-21-1 and G-2-27-2), Pseudomonas poae (isolate G-2-1-1), Enterobacter endosymbiont (isolates G-2-10-2 and N-5-10), Delftia acidovorans (isolate G-1-15), and Achromobacter xylosoxidans (isolates N-46-11HH and N-5-20) were reported to be able to produce siderophores under low-iron conditions for the first time. Gram-negative isolates were more frequently encountered, with more than 95% total frequency. For Gram-positive bacteria, the Bacillus and Rhodococcus were the only two genera, with 1.7% total frequency. Furthermore, the Pseudomonas and Enterobacter were dominant in this environment, with 44.5% and 24.7% total frequency, respectively. It was also found that 75 percent of the isolates that had the high percentages of siderophore units (% between 40 and 60) belonged to Pseudomonas. Pseudomonas sp. G-229-21 screened out in this study may have potential to apply to low-iron soil to prevent plant soil-borne fungal pathogen diseases.
A diversidade genética de bactérias de rizosfera de tabaco produtoras de sideróforos foi estudada por meio da técnica de análise de restrição do DNA ribossomal amplificado (ARDRA), homologia de seqüência de 16s rRNA e métodos de análise filogenética. Observou-se que 85% do total de 354 isolados produziram sideróforos em meio liquido com restrição de ferro.Entre os 299 isolados produtores de sideróforos identificou-se 28 padrões ARDRA, que representaram 14 gêneros bacterianos diferentes, pertencentes a seis divisões bacterianas: β-, γ-, α-Proteobacteria, Sphingobacteria, Bacilli e Actinobacteria. γ- Proteobacteria, consistindo de Pseudomonas, Enterobacter, Serratia, Pantoea, Erwinia e Stenotrophomonas, pertenceram a 18 grupos ARDRA. Os resultados também mostraram uma diversidade maior de bactérias produtoras de sideróforos doque a relatada em outros estudos. Por exemplo, Sphingobacterium (isolados G-2-21-1 e G-2-27-2), Pseudomonaspoae (isolado G-2-1-1), Enterobacter endosymbiont (isolados G-2-10-2 e N-5-10), Delftia acidovorans (isolado G-1-15) e Achromobacter xylosoxidans (isolados N-46-1HH e N-5-20), capazes de produzir sideróforos em condições de baixa disponibilidade de ferro, foram relatados pela primeira vez. Isolados Gram negativos foram encontrados com maior freqüência, correspondendo a mais de 95% da freqüência total. Entre as bactérias Gram positivas, foram encontrados apenas os gêneros Bacillus e Rhodococcus, com 1,7% da freqüência total. Além disso, neste ambiente houve predominância de Pseudomonas e Enterobacter, com 44,5% e 24,7% da freqüência total, respectivamente. Verificou-se também que 75% dos isolados com alta porcentagem de unidades de sideróforos (% entre 40 e 60) pertenceram a Pseudomonas. Pseudomonas sp. G- 229-21, selecionado neste estudo, apresenta potencial de aplicação em solos com baixo teor de ferro para prevenção dedoenças fúngicas em plantas.
Subject(s)
Base Sequence , Genes, Bacterial , Genetic Variation , In Vitro Techniques , RNA , Siderophores/genetics , Siderophores/isolation & purification , Tobacco/genetics , Methods , Plants , MethodsABSTRACT
Se utilizaron 62 cepas identificadas inicialmente como bacilos gramnegativos no fermentadores (BGNNF), aisladas de pacientes con diagnóstico de infección nosocomial, con el objeto de determinar su género y especie. Cuarenta y cinco cepas fueron identificadas como Acinetobacter baumannii, 10 como Pseudomonas aeruginosa, 3 como Stenotrophomonas maltophilia, 3 como Comamonas acidovorans y 1 como Achromobacter xylosoxidans subsp. xylosoxidans, mediante el API 20NE. Con respecto a las cepas de Acinetobacter, el ARDRA permitió identificar 20 cepas como A. baumannii y 23 como Acinetobacter genoespecie 13TU, pero 2 cepas no fueron identificadas por este método. La secuencia de la subunidad 16S del ADNr de todas las cepas incluidas en este estudio permitió identificar 20 cepas como A. baumannii, 23 cepas como Acinetobacter RUH1139, 10 como P. aeruginosa, 4 como A. xylosoxidans subsp. xylosoxidans (2 de estas cuatro cepas habían sido identificadas como A. baumannii mediante API20NE), 3 como S. maltophilia, 1 como C. acidovorans y 1 como β-Proteubacterium. Las discrepancias entre la identificación bioquímica por API 20NE y por ARDRA, para diferenciar las genoespecies de Acinetobacter, fue resuelta por la secuenciación de la subunidad 16S del ADNr, indicando que la identificación de los aislados de Acinetobacter, entre otros BGNNF, mediante API 20NE, debe ser confirmada por técnicas genéticas.
We used 62 strains initially identified as non fermenting gram-negative bacilli (NFGNB) isolated from patients with a nosocomial infection diagnosis with the purpose of identifying their genus and species. Forty five strains were identified as Acinetobacter baumannii, 10 as Pseudomonas aeruginosa, 3 as Stenotrophomonas maltophilia, 3 as Comamonas acidovorans and 1 as Achromobacter xylosoxidans subspecies xylosoxidans through the API 20NE. Regarding the Acinetobacter strains, the ARDRA allowed to identify 20 strains as A. baumannii and 23 as Acinetobacter genospecies 13TU, but 2 strains were not identified with this method. The ADNr 16S sequence of all the strains included in this study allowed to identify 20 strains as A. baumannii, 23 strains as Acinetobacter RUH1139, 10 as P. aeruginosa, 4 as A. xylosoxidans subsp. xylosoxidans (2 of these four strains strains had been identified as A. baumannii through API20NE), 3 as S. maltophilia, and 1 as C. acidovorans and 1 as β-Proteubacterium. The discrepancies between the biochemical identification by API 20NE and by ARDRA to differentiate the Acinetobacter genospecies was resolved by the ADNr16S sequencing, indicating that the identification of the Acinetobacter isolates, among other NFGNB, through API 20NE, should be confirmed through genetic techniques.
ABSTRACT
El objetivo principal de esta investigación fue determinar la diversidad bacteriana del proceso de biorremediación de agua contaminada con nafta en un biorreactor de lecho fluidificado en el Recinto Universitario de Mayagüez, de la Universidad de Puerto Rico. El aislamiento y la caracterización de las colonias bacterianas del sistema de biorremediación fueron realizados en medio R2A. Las pruebas morfológicas incluyeron la determinación de la morfología celular y de las colonias, y la reacción frente a la coloración de Gram. Las propiedades fisiológicas se determinaron usando el sistema Biolog® y sobre la base de la habilidad para desarrollar en medio mínimo con nafta como única fuente de carbono. La caracterización molecular se llevó a cabo por BOX-PCR y por análisis de secuencia del ADNr 16S mediante la técnica de ARDRA (amplified ribosomal DNA restriction analysis). De los 162 morfotipos de colonias aislados, 75% fueron bacilos gram-negativos, 19% bacilos gram-positivos, 5% cocos gram-negativos y 1% cocos gram-positivos. Según el análisis ARDRA, estos morfotipos se distribuyeron en 90 grupos genéticos, de los cuales 53% incluyeron cepas con crecimiento en nafta. Las 86 cepas que crecieron en nafta presentaron 52 patrones de amplificación, los que a través de BOX-PCR se agruparon en 50 grupos metabólicamente no relacionados. El alto nivel de diversidad microbiana observado en el reactor permitió la remoción del contaminante y, al parecer, fue importante para la operación estable y eficiente del sistema.
The main objective of this research project was to determine the bacterial diversity during the process of bioremediation of water contaminated with gasoline in a fluidized bed reactor at Mayagüez, PR. Isolation and characterization of bacterial populations from the bioremediation system was performed on R2A medium. Morphological tests included cellular and colonial shape and reaction to Gram coloration. Physiological properties were determined by using carbon utilization profiles (Biolog®) and by the ability of axenic cultures to use gasoline as the sole carbon source. Molecular characterization was performed by BOX-PCR and 16S rDNA sequence analysis (ARDRA). From a total of 162 distinctive isolates, 75% were gram-negative bacilli, 19% gram-positive bacilli, 5% gram-negative cocci and 1% gram-positive cocci. The 162 axenic cultures corresponded to 90 different genetic groups; 53% of which included strains with growth in gasoline as sole carbon source. The 86 strains capable of growing in gasoline corresponded to 52 different amplification patterns in BOX-PCR; which were not metabolically related (Biolog® system). The high degree of microbial diversity in the FBR allowed efficient and stable hydrocarbon removal throughout the operation of the system.
Subject(s)
Bioreactors/microbiology , Fresh Water/microbiology , Gasoline , Gram-Negative Bacteria/isolation & purification , Gram-Positive Bacteria/isolation & purification , Water Pollutants, Chemical/metabolism , Bacterial Typing Techniques , Biodegradation, Environmental , Carbon/metabolism , DNA, Bacterial/analysis , Gram-Negative Bacteria/growth & development , Gram-Negative Bacteria/metabolism , Gram-Positive Bacteria/growth & development , Gram-Positive Bacteria/metabolism , Gram-Positive Cocci/growth & development , Gram-Positive Cocci/isolation & purification , Gram-Positive Cocci/metabolism , Polymerase Chain Reaction , Puerto Rico , Ribotyping , RNA, Bacterial/analysis , Species SpecificityABSTRACT
BACKGROUND: The glucose -acidifying genomic species 1, 2, 3 and 13 of the genus Acinetobacter are highly related genetically and may be difficult to differentiate by phenotypic identification schemes using biochemical tests. The aim of this study was to explore the brief restriction enzyme profiles of amplified ribosomal DNA restriction analysis (ARDRA) to identify medically important species of Acinetobacter. Using ARDRA analysis, we evaluated the ID 32 GN system (bioMerieux, Lyon, France) for the identification of A. baumannii (genospecies 2). METHODS: A collection of 78 A. baumannii stains initially identified by the ID 32 GN system was used to determine its accuracy by ARDRA analysis. ARDRA was performed with 10 different restriction enzymes, AluI (AGCT), CfoI (GCGC), HaeIII (GGCC), HinfI (GANTC), MboI (GATC), MspI (CCGG), NciI (CCGG), RsaI (GTAC), ScrFI (CCNGG) and TaqI (TCGA). RESULTS: The combination of restriction patterns obtained with respective enzymes AluI, CfoI and MboI allowed for the discriminatory value for the identification of medically important genospecies such as genospecies 2 (A. baumannii), 3 and 13. By comparing ARDRA results of the 78 strains previously identified by ID 32 GN system, we found the correlation rate between the two systems to be 88.5% (69/78). Nine strains were identified as Acinetobacter genospecies 13 by ARDRA. CONCLUSIONS: This result suggests that the ID 32 GN system may have difficulty in discriminating A. baumannii from genospecies 13. This revised ARDRA method gives a relatively rapid and definitive result for the identification of medically important genospecies of Acinetobacter.
Subject(s)
Acinetobacter , Acinetobacter baumannii , Coloring Agents , DNA, Ribosomal , GlucoseABSTRACT
It is used the method of pure culture,Selected 32 strains,which were obvious difference in the shape,color and so on common characteristic,From the chilled beef with no packing and cling film on sale in this research;and it was included 12 strains from the chilled beef sample packed with cling film;20 strains from the chilled beef sample with no packing.Simultaneously selected 4 strains which were predominant in each bacterium from the two samples to conduct the further research,8 strains serial numbers are:S01～S08,S01～S04 from the chilled beef sample with no packing;S05～S08 from the chilled beef sample packed with cling film.Through ARDRA(Amplified ribosomal DNA restriction analysis) as well as 16S rDNA to clarify the bacterium's classified status.The physiological and chemical tests were done to determine the various bacteria respective genus.The experiment indicated:S01 is Pseudomonas putida;S02 is Shewanella cincia stain;S03 and S05 are the same Shewanella putrefaciens;S04 is Stenotrophomonas mal-tophilia;S06 is Psychrobacter;S07 is Staphylococcus sciuri;S08 is Microbacterium-laevaniformans.It was proved that two samples altogether have the same predominant bacterium.It can provide certain theory basis for the chilled meat processing craft as the preliminary investigation in the cultured microorganism situation in two samples.
ABSTRACT
In this study, three methods for identification of E.coli were compared. The conventional method was employed to select and identify the suspicious E.coli isolates from a fecal sample. PCR based ARDRA analysis was then carried out to distinguish these E.coli isolates, E.coli MG1655 and other bacterial species. All the potential E.coli isolates and E.coli MG1655 had the identical ARDRA banding pattern while the other bacterial species showed the different patterns.The result indicated that the ARDRA analysis was consistent with the traditional method for identification of E.coli and could be the practical method for distinguishing E.coli from other intestinal bacterial species. The ERIC-PCR analysis provided abundant polymorphism between different E.coli isolates, and might be a powerful approach for elucidating the genetic diversity among isolates of the same species.
ABSTRACT
Marine bacteria from the samples of sea sediments and seawater were directly plated on isolation media and the biodiversity of isolates was examined with DNA fingerprinting.542 single colonies were obtained from the media.ARDRA with enzyme Hinf I revealed 16 operational taxonomic units(OTU) which were dominated by OTU5 group which accounts for 19 isolates,and OTU7 group which accounts for 11 isolates.The biodiversity of isolates from these two dominant OTU groups was further investigated by a genomic fingerprinting technique, ERIC-PCR.The results indicated that there were 12 different ERIC-PCR types present among the OTU5 group while only 4 among the OTU7.The data indicated rich diversity profiles of marine microorganisms were presented in the East China Sea.
ABSTRACT
A number of strains were isolated from the wastewater by direct plating.PCR with specific primer of phenol hydroxylase gene was used to identify the strains as phenol degrading bacteria.By this,a total of 87 phenol degrading strains were purified and divided into populations based on enterobacterial repetitive intergenic consensus sequence PCR(ERIC-PCR) genomic fingerprints.The results indicated that there were 15 different ERIC-PCR types present among the isolates.ARDRA with enzymes AluI revealed 4 operational taxonomic units(OTU) among 15 strains that exhibited different ERIC-PCR types.Moreover,the results indicate that there were 4 species at least.