Frequency and biodiversity of 2,4-diacetylphloroglucinol-producing bacteria isolated from the maize rhizosphere at different stages of plant growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Cyclopiazonic acid production by submerged cultures of Penicillium and Aspergillus strains

Abstract 62 isolates of Penicillium and Aspergillus were screened for cyclopiazonic acid (CPA) production by surface and submerged culture on different media. The production of this mycotoxin was restricted to Penicillium camembertii group II (and its domesticated form P. camembertii ), P. griseofulvum , and Aspergillus flavus (and its domesticated form A. oryzae ). The best yield of CPA was obtained by a strain of P. griseofulvum , but several strains of P. camembertii group II were also good producers. Propionic acid (500 and 1000 mg/l medium) did not enhance the production of CPA. The best yields of CPA were obtained in submerged culture, but in some cases growth and CPA production only occured in surface culture. A simplified procedure for isolation of CPA is described.     

Characteristics of selected virulence factors of Enterobacter cloacae strains isolated from clinical specimens

We demonstrated that Enterobacter cloacae possesses a selective haemolytic activity on sheep erythrocytes. All the screened strains showed a haemolytic activity on sheep erythrocytes when cultures were preincubated with beta-mercaptoethanol. The investigation circulation of the genes encoding extended spectrum beta-lactamases (ESBL) shows that beta-lactamase producers can be ascribed to specific patterns of plasmids. We also demonstrated that genetic material from E. coli can be transferred and established in selected Enterobacter cloacae strains. In a survival tests we demonstrated that similarly to Salmonella or Vibrio clinical isolates Enterobacter cloacae doesn't demonstrate acid tolerance.     

Isolation from food sources, of lactic acid bacteria that produced antimicrobials

The potential of lactic acid bacteria, isolated from a variety of foods, to inhibit indicators representative of spoilage and pathogenic bacteria associated with food products was examined. Fruit and vegetables were a poor source of lactic acid bacteria but large numbers were readily isolated on MRS agar from cheese, milk and meat samples. Approximately 1000 isolates from each of the food samples were examined by the deferred antagonism procedure to determine their ability to inhibit Staphylococcus aureus, Listeria innocua and Pseudomonas fragi. Listeria innocua was the bacterium predominantly inhibited by isolates from the cheese, milk and meats, but antagonism was also observed to a lesser extent against the other indicators. The only inhibition observed for isolates from vegetable material was directed against Staph. aureus. The majority of inhibitor producers were effective against only one of the indicators but a small number were isolated which inhibited two or three.     

Spatial and Temporal Patterns of Deforestation in Rio Cajarí Extrative Reserve,Amapá, Brazil

The Rio Cajarí Extractive Reserve (RCER) is a sustainable use protected area located in Southern Amapá state, Brazil. This protected area is home to traditional agro-extractive families, but has been increasingly invaded by commercial agriculture producers. In this work, we test the hypothesis that the RCER implementation has distinctly affected spatial patterns of deforestation and rates of bare soil and secondary forest formation by the social groups occupying the protected area and its surrounding area. Detailed maps of vegetation cover and deforestation were elaborated, based on Landsat TM images from 1991, 1998, 2007 and 2008 and Linear Spectral Mixture Models. Based on an extensive fieldwork, patches were classified according to the agents causing deforestation and characterized with ten explanatory variables. A discriminant function analysis was used to identify homogeneous groups based on the data. Results show increased rates and distinct spatial patterns of deforestation by three groups: extractivists, non traditional commercial agriculture producers, and a less representative group constituted of miners, cattle and timber producers. In all analyzed dates, clearings by the extrativist community presented the highest total area and smaller average sizes and were located in close proximity to villages. Deforestation patches by the non-traditional group were exclusively associated with ombrophilous forests; these presented higher average sizes and proximity indexes, and showed increased aggregation and large cluster formation. No significant differences were observed in deforestation patterns by the three groups inside or outside the reserve.     

Genetic heterogeneity in Bacillus sporothermodurans as demonstrated by ribotyping and repetitive extragenic palindromic-PCR fingerprinting

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.     

Revisiting the Acanthamoeba species that form star-shaped cysts (genotypes T7, T8, T9, and T17): characterization of seven new Brazilian environmental isolates and phylogenetic inferences

Free-living amoebae of the genus Acanthamoeba are the agents of both opportunistic and non-opportunistic infections and are frequently isolated from the environment. Of the 17 genotypes (T1-T17) identified thus far, 4 (T7, T8, T9, and T17) accommodate the rarely investigated species of morphological group I, those that form large, star-shaped cysts. We report the isolation and characterization of 7 new Brazilian environmental Acanthamoeba isolates, all assigned to group I. Phylogenetic analyses based on partial (~1200 bp) SSU rRNA gene sequences placed the new isolates in the robustly supported clade composed of the species of morphological group I. One of the Brazilian isolates is closely related to A. comandoni (genotype T9), while the other 6, together with 2 isolates recently assigned to genotype T17, form a homogeneous, well-supported group (2·0% sequence divergence) that likely represents a new Acanthamoeba species. Thermotolerance, osmotolerance, and cytophatic effects, features often associated with pathogenic potential, were also examined. The results indicated that all 7 Brazilian isolates grow at temperatures up to 40°C, and resist under hyperosmotic conditions. Additionally, media conditioned by each of the new Acanthamoeba isolates induced the disruption of SIRC and HeLa cell monolayers.     

Typing Listeria monocytogenes isolates from fish products and human listeriosis cases.

Seventy-two Listeria monocytogenes isolates originating from 10 different fish products of 12 producers and 47 isolates from human listeriosis cases were typed by serotyping and multilocus enzyme electrophoresis. Seventy-five of these isolates were further subtyped by restriction analysis of genomic DNA with the enzyme XhoI and by pulsed-field gel electrophoresis using the enzymes ApaI and SmaI. The results show that several L. monocytogenes clones identified by multilocus enzyme electrophoresis are frequently found in fish products of different origins. One of these clones is the same as another previously shown to be frequently associated with meat and meat products. The epidemic-associated electrophoretic type 1 was only rarely found in fish products. No association was found between any type of fish product and a particular lineage of L. monocytogenes. Both long-term persistence of a strain and simultaneous presence of several clearly distinct strains in the products of single producers were observed. The comparison of L. monocytogenes isolates from human clinical listeriosis cases in Switzerland and those from imported fish products by use of multilocus enzyme electrophoresis showed that they do not form two clearly distinct lineages but nevertheless belong to two separate populations. None of the 48 subtypes distinguished by the combination of all four typing methods could be found in both populations of human origin and those of fish origin.     

Evaluation of Propionibacterium acnes Isolates Using Contour-clamped Homogeneous Electric Field Gel Electrophoresis

Contour-clamped homogeneous electric field electrophoresis was performed to compare strains ofPropionibacterium acnes isolated from patients with chronic postoperative endophthalmitis. Propionibacterium acnes isolates were obtained from the vitreous humor of nine patients with chronic postoperative endophthalmitis following cataract surgery. In two of the patients, P. acnes isolates were also obtained from the aqueous humor as well as from the vitreous humor. Bacterial DNA was digested using Not I and Spe I restriction endonucleases. The DNA fragments were then subjected to contour-clamped homogeneous electric field electrophoresis and the DNA banding patterns were analysed. Eight nonidentical banding patterns were identified among the nine vitreous isolates of P. acnes. In each of the two cases from which aqueous and vitreous isolates were recovered from the same eye, the banding patterns were identical. Contour-clamped homogeneous electric field electrophoresis is a powerful method to distinguish P. acnes isolates based on DNA banding patterns and could be used in the epidemiological study of clinical processes caused by this organism.     

Polyphasic characterization of a suite of bacterial isolates capable of degrading 2,4-D

To develop a better understanding of the ecological aspects of microbial biodegradation, it is important to assess the phenotypic and biochemical diversity of xenobiotic degrading organisms. Forty-six bacterial isolates capable of degrading 2,4-dichlorophenoxyacetic acid (2,4-D) and representing several geographically distinct locations were characterized and placed into taxonomic groups based on the results of several independent analyses. The isolates were characterized based on Gram's reaction, colony morphology, cell morphology, fatty acid methyl ester (FAME) fingerprints, carbon substrate oxidation patterns (BIOLOG), DNA homology to whole-plasmid probes and repetitive extragenic palindromic (REP) fingerprints. Attempts to group organisms taxonomically based on colony morphology and cell morphology were largely unsuccessful. Both FAME and BIOLOG analyses were generally unable to provide reliable genus or species identifications of these environmental isolates by comparison of fingerprints or substrate use patterns to existing data bases. Modification of the standard protocols for these analyses, however, allowed taxonomic grouping of the isolates and the construction of new data bases, comprised solely of 2,4-D-degrading organisms, against which future novel isolates can be compared. Independent cluster analysis of the FAME and BIOLOG data shows that the isolates can be segregated into five taxonomic classes. The collection of 2,4-D-degrading isolates was also separated into five classes based on DNA homology to whole-plasmid probes obtained from individual isolates. REP analysis allowed isolates that likely represent the same (or very similar) organism(s) to be identified and grouped. Each of the analyses used represents a mechanistically different means of classifying organisms, yet the taxonomic groupings obtained by several of the methods (FAME, BIOLOG, DNA homology, and to some degree, REP analysis) were in good agreement. This indicates that the features discriminated by these different methods represent fundamental characteristics that determine phylogenetic groups of bacteria. Correspondence to: W.E. Holben.     

The chemotaxonomy ofBeauveria bassiana (Deuteromycotina: Hyphomycetes) isolates from the coffee berry borerHypothenemus hampei (Coleoptera: Scolytidae)

A preliminary chemotaxonomic analysis was carried out on 16 isolates ofBeauveria bassiana from adults of coffee berry borer (Hypothenemus hampei) from 10 countries in Latin America, Africa, Asia and the Pacific. Thirteen formed an homogeneous group with very similar electrophoretic and physiological profiles. Two isolates differed in esterase and acid phosphatase band patterns, one of which was also deficient in chitin-degrading enzyme production, but both were still relatively similar to the major group compared with the total variability within the species. A third isolate was abnormal and may have degenerated in storage. These results suggest a distinct population of the fungus on this host. The implications for selection of pathogenic isolates for development as biological pesticides are discussed.     

Genetic Heterogeneity in Bacillus sporothermodurans as Demonstrated by Ribotyping and Repetitive Extragenic Palindromic-PCR Fingerprinting

Thirty-eight strains of Bacillus sporothermodurans isolated from ultra-high-temperature (UHT)-treated milk or sterilized milk (UHT isolates) and from animal feed or raw milk (farm isolates) were characterized by automated ribotyping and by repetitive extragenic palindromic (REP)-PCR fingerprinting. By investigating the genetic relationships among isolates from these various sources, the relative importance of different contamination sources could be evaluated. The results of the separate clustering analyses of the PvuII and EcoRI ribopatterns and the REP-PCR patterns were largely consistent with each other and revealed the existence of two main clusters; there was one homogeneous group containing all (REP-PCR) or most (ribotyping) of the UHT isolates, and there was a second more diverse group comprising the farm isolates. A combined three-dimensional analysis of all data showed that three German UHT isolates did not belong to the compact group containing the majority of the UHT isolates. These results demonstrate that B. sporothermodurans is more heterogeneous than previously assumed and that most of the UHT isolates form a genetically distinct subgroup and are capable of producing highly heat-resistant spores. The close genetic relationship of these UHT isolates suggests a clonal origin of a few predominant strains of B. sporothermodurans that can be found in UHT-treated or sterilized milk products.     

Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.     

Sequence breakpoints in the aflatoxin biosynthesis gene cluster and flanking regions in nonaflatoxigenic Aspergillus flavus isolates

Aspergillus flavus populations are genetically diverse. Isolates that produce either, neither, or both aflatoxins and cyclopiazonic acid (CPA) are present in the field. We investigated defects in the aflatoxin gene cluster in 38 nonaflatoxigenic A. flavus isolates collected from southern United States. PCR assays using aflatoxin-gene-specific primers grouped these isolates into eight (A-H) deletion patterns. Patterns C, E, G, and H, which contain 40 kb deletions, were examined for their sequence breakpoints. Pattern C has one breakpoint in the cypA 3' untranslated region (UTR) and another in the verA coding region. Pattern E has a breakpoint in the amdA coding region and another in the ver1 5'UTR. Pattern G contains a deletion identical to the one found in pattern C and has another deletion that extends from the cypA coding region to one end of the chromosome as suggested by the presence of telomeric sequence repeats, CCCTAATGTTGA. Pattern H has a deletion of the entire aflatoxin gene cluster from the hexA coding region in the sugar utilization gene cluster to the telomeric region. Thus, deletions in the aflatoxin gene cluster among A. flavus isolates are not rare, and the patterns appear to be diverse. Genetic drift may be a driving force that is responsible for the loss of the entire aflatoxin gene cluster in nonaflatoxigenic A. flavus isolates when aflatoxins have lost their adaptive value in nature.     

Characterization of IS999, an unstable genetic element in Mycobacterium avium

An IS3-family insertion element, IS999, was identified in the opportunistic pathogen Mycobacterium avium. The 1347 bp element has 29 bp inverted repeats and two overlapping open reading frames coding for putative transposases. It was detected in the genomes of ten of 12 M. avium isolates examined. Copy numbers ranged from four to 16. IS999 is less stable than IS1245, the most commonly-used marker for typing M. avium isolates. Among 60 colonies picked from a single patient isolate, there were two distinct IS1245 restriction fragment length polymorphism banding patterns compared to eight distinct IS999 patterns (five in one IS1245 group and three in the other). In view of its instability, we asked whether transposition of IS999 might have phenotypic consequences. Nucleotide sequence analysis of insertion sites in four isolates revealed 16 putative structural genes that were variably disrupted by IS999. Insertions into hdhA, a gene that codes for a putative short chain alcohol dehydrogenase, were distributed non-randomly between colony type variants, consistent with phenotypic consequences that exert selective pressure. These observations illustrate the genetic heterogeneity that can exist within populations of M. avium that appear to be homogeneous by IS1245 analysis. IS999 may be a useful marker for tracking, at the sub-strain level, the rapid genetic drift that M. avium isolates undergo in nature and in the laboratory.     

Molecular relationship between two groups of the genus Leptospirillum and the finding that Leptospirillum ferriphilum sp. nov. dominates South African commercial biooxidation tanks that operate at 40 degrees C

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40 degrees C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45 degrees C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.     

Occurrence of extended-spectrum β-lactamases among<Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from hospitalized and healthy children

The prevalence of extended-spectrum beta-lactamases (ESBL) was determined among isolates of Escherichia coli (n = 63) isolated from hospitalized (43) and healthy (20) children. Ten isolates (21%) were ESBL-positive for two screening tests, the double disk-synergy test and the Oxoid Combination Disk method. One ESBL-positive isolate came from a healthy child. The transfer frequency of oxyimino-beta-lactam resistance from ESBL-producing isolates to E. coli K12 C600 recipient strain ranged from 10(-8) to 10(-5) per donor cell. Donor strains and transconjugants displayed susceptibility patterns typical of ESBL producers. They were resistant to oxyimino-beta-lactams but susceptible to clavulanic acid and carbapenems. Seven out of the 10 ESBL-positive isolates were found to produce MR/MS fimbria, which may play an important role in the colonization of the human intestinal mucosa.     

Investigation of persistent colonization by Pseudomonas aeruginosa-like strains in a spring water bottling plant.

Ninety-seven strains, producing a fluorescent pigment under UV light and/or a green diffusive pigment on cetrimide-naladixic acid agar, were isolated from a spring water bottling plant. These strains were presumptively identified as Pseudomonas aeruginosa, but they could not be confirmed as strains of this species nor identified by the API 20NE identification system. The isolates and reference strains were clustered by computer-assisted whole-cell protein sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The numerical analysis of the protein electrophoregrams resulted in the formation of four clusters at a similarity level of 80% and two unclustered type strains. One cluster included strains isolated during a 4-month period and reference strains of several biotypes of P. fluorescens. The remaining isolates formed another cluster with a very high similarity of level, which included two groups of strains based on biochemical characterization by the API 20NE Test System. Strains were typed by random amplified polymorphic DNA (RAPD)-PCR and two different RAPD patterns were obtained, corresponding to each biochemical profile. This persistent colonization seems to be caused by a single species present in the bottling system, with two clonal origins, not related to P. aeruginosa or to any of the other type strains tested. Partial 16S rDNA sequence of a representative strain of one cluster of isolates had a level of similarity of 99.3% with P. alcaligenes. This study shows that characteristics similar to P. aeruginosa on cetrimide-naladixic acid agar can be exhibited by several groups of fluorescent pseudomonads that do not belong to this species, clearly showing that confirmation tests must be performed before a decision regarding the water quality is made.     

Biotypes and randomly amplified polymorphic DNA (RAPD) profiles of subgingival Candida albicans isolates in HIV infection

A group of subgingival isolates of C. albicans recovered from Italian HIV-positive (HIV+) subjects were characterized both phenotypically and genotypically. Phenotyping of the isolates was carried out by a biotyping method based on the enzyme profiles, carbohydrate assimilation patterns and boric acid resistance of the yeasts. Genotyping was performed through randomly amplified polymorphic DNA (RAPD) analysis. Five biotypes were found among the 29 subgingival C. albicans strains examined. The predominant biotypes were A1R (55.17%), A1S (24.14%), and A2R (13.79%), while the biotypes A11R and A13R were represented by a single isolate each. RAPD profiles identified 15 genotypes among the 29 isolates. Almost every individual harboured his/her own specific isolate and in three out of the six subjects with multiple isolates (two to six each) more than one genotype (two to six) was found. The biotype distribution we found is consistent with previous reports on C. albicans isolates from other oral sources, whereas the resistance to boric acid was highly frequent in subgingival strains. RAPD analysis showed high genetic heterogeneity within subgingival isolates, also when isolates were phenotypically identical. These findings, obtained from HIV+ subjects living in Southern Italy, may be useful as baseline information on subgingival C. albicans colonization in the Mediterranean area.