Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

Genetic and ecotypic structure of a fluorescent Pseudomonas population

Fluorescent pseudomonads are among the most numerous bacteria found on plant surfaces and the activity of certain isolates can affect plant growth. In 1993, 108 fluorescent Pseudomonas isolates were collected on a single sampling occasion from the leaves of sugar beet plants grown at the Oxford University Field Station, Wytham. Isolates were obtained from 54 different leaves, from nine plants, and characterized using 10 allozyme and 23 biotype markers. Statistical analysis of the combined data revealed five biotypic traits which permitted a rational classification of the sample. Analysis of the allozyme data showed that the population was in overall linkage disequilibrium. Clonality was also observed after subdivision of allozyme data along spatial and habitat levels. However, two genetically defined subgroups were in linkage equilibrium which suggests the possibility of frequent large-scale recombination among certain isolates. A significant correlation between isolate distribution and habitat (leaf type and plot) indicates that the population has ecotypic structure.     

Phenotypic and allozyme variation in Mediterranean and desert populations of wild barley,Hordeum spontaneum Koch

Populations of wild barley, Hordeum spontaneum Koch, were collected in two distinct climatic regions, desert and Mediterranean. Plants from five desert and five Mediterranean populations were compared and contrasted for extent and structure of phenotypic variation. These same 10 and one other population from each region were analyzed for allozyme variation. In a field trial of phenotypic diversity, two phenological and 14 morphological traits were examined. Study of allozyme variation was performed using eight enzyme systems encoding for 13 loci. Plants from the desert and Mediterranean regions were significantly different in seven of 16 phenotypic traits, exhibited a high (30%) interregional component of phenotypic variation, and showed a high degree of segregation on a principal component scattergram indicating ecotypic differentiation. Mediterranean populations were twice as variable as desert populations in reproductive growth parameters (stem and spike length) and grain filling (spikelet weight), but half as variable for onset of reproduction. The extent and structure of phenotypic and allozyme variation did not match. The Mediterranean and desert populations did not differ in amount of allozyme variation as estimated by mean number of alleles per locus, effective number of alleles, polymorphism, and gene diversity (n(a), n(e), P, and H(e)), did not segregate on the basis of population genetic distances, and exhibited a low proportion of interregion allozyme diversity (2%). No effect of selection on allozyme distribution was detected. Our results suggest that the adaptation of plants originating from desert and Mediterranean environments is reflected in phenotypic but not in allozyme variation.     

Unusual Antigenic and Genetic Characteristics of Human Respiratory Syncytial Viruses Isolated in Cuba

The G protein of 23 strains of human respiratory syncytial virus isolated in Havana, Cuba, between October 1994 and January 1995 was analyzed at the antigenic and genetic level. All viruses reacted with 10 of 11 antibodies specific for the Long strain. Moreover, the G protein gene of the Cuban isolates had only five nucleotide differences from the sequence of the Long gene. The homogeneity of the Cuban isolates and their resemblance to an ancient strain, such as Long, are at odds with previous findings for viruses isolated in countries with a temperate climate and different socioeconomic status. The G proteins of three of four other viruses isolated in Havana 2 years later (1996) were also identical to those of the 1994-to-1995 isolates, and the fourth virus had a single extra nucleotide difference. This, again, is unusual, since no identical viruses had been isolated in different epidemics previously. The singular characteristics of the Cuban isolates reported here are discussed in terms of the epidemiological, climatic, and socioeconomic characteristics of Cuba.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Associations of allozyme variation and behavior in the cowpea weevil (Callosobruchus maculatus)

The cowpea weevil (Callosobruchus maculatus (Fabré)) exhibits several behavioral traits that are stable within, but vary among, strains. These traits are heritable and quantitative. We used cellulose acetate gel electrophoresis to quantify allozyme variation within and among laboratory cultures of four weevil strains and determine whether allozyme variation correlates with behavioral traits. Significant variation exists at 8 of 11 loci assayed and gene frequencies are significantly different among strains. The South Indian strain (SI) is most variable and measures of genetic distance set it apart from the other strains. It is also behaviorally unique. The Brazilian strain (BC) is most different from SI in allozyme diversity and behavioral phenotype, while two African strains (IITA, CAM) are intermediate in allozyme diversity and phenotype. These results are consistent with the known history of these strains and the differences in the allozymes parallel the differences in behavioral traits.     

Characterization of Ehrlichia species from Ixodes ovatus ticks at the foot of Mt. Fuji, Japan

A total of 390 adult ticks (288 Ixodes ovatus and 102 I. persulcatus ) collected at the foot of Mt. Fuji and two near cities in Shizuoka prefecture, Japan, were examined for Ehrlichia infection by isolation with laboratory mice from whole tick tissues. Ehrlichial DNAs were detected from the spleens of mice inoculated with tissues from I. ovatus, but not I. persulcatus. The prevalence of ehrlichiae in the ticks was estimated to be ca. 3%. The 16S rDNA analysis revealed that the sequences of 8 ehrlichial isolates (termed "Shizuoka" isolates) obtained were identical, and they were very similar, but not identical, to those of two Ehrlichia species strain variants recently isolated in Japan, followed by Ehrlichia chaffeensis in the US. Analysis of parts of the omp-1 multigene family specific for monocytic ehrlichiosis agents showed that the Shizuoka isolates were distinct from other ehrlichial organisms. The Shizuoka isolates caused death in immunocompetent laboratory mice, suggesting that they are highly pathogenic in mice. The data show that the Shizuoka isolates are likely to be a new strain variant of Ehrlichia species in Japan. Further characterization and surveillance will be required in Japan due to the presence of these human ehrlichiosis agent-like organisms.     

Tsukamurella spumae sp. nov., a novel actinomycete associated with foaming in activated sludge plants

A polyphasic taxonomic study was undertaken to establish the taxonomic position of six representative strains isolated from activated sewage sludge foam. The organisms were found to have chemical and morphological properties consistent with their assignment to the genus Tsukamurella. DNA:DNA relatedness studies showed that five out of the six isolates formed a distinct genomic species, the remaining strain was most closely associated with this taxon. The five isolates had a unique phenotypic profile that served to distinguish them from representatives of the validly described species of Tsukamurella. The combination of the genotypic and phenotypic data indicated that the five strains should be classified as a new species in the genus Tsukamurella. The name proposed for this taxon is Tsukamurella spumae, the type strain is N1171T (= DSM 44.113T = NCIMB 13947T). It was also shown that some of the reference strains were misclassified as Tsukamurella paurometabola.     

Pathogenicity of Beauveria bassiana,Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), and other entomopathogenic fungi against Lygus lineolaris (Hemiptera: Miridae)

The pathogenicity of 32 fungal isolates from the genera of Beauveria, Verticillium, Paecilomyces, Metarhizium, Mariannaea, and Hirsutella to second-instar tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was tested under laboratory conditions. These isolates originated from various insect hosts and substrates from France, Denmark, Austria, Italy, Turkey, Syria, and the United States. A single exposure concentration (1 x 10(7) conidia/ ml) assay for each isolate was first conducted by immersing the insects in 10 ml of a fungal suspension for 5s. These were followed by concentration-mortality assays on five of the most pathogenic isolates using four test concentrations ranging from 2 x 10(4) to 2 x 10(7) conidia/ml. B. bassiana 726 (Bemisia-passaged GHA strain) was used as a standard for comparison in all of the assays. Among the test isolates, three produced mortality not significantly different from the water control. Mortality ranged from 35 to 98% among the other 29 isolates. The LC50 values of the five most pathogenic isolates ranged from 0.8 to 5.0 x 10(5) conidia/ml. The LT50 values for these isolates ranged from 6.0 to 6.9, 3.1 to 5.1, and 2.5 to 4.0 d for concentrations of 2 x 10(5), 2 x 10(6), and 2 x 10(7) conidia/ml, respectively. Two strains of B. bassiana (ARSEF 1394,5665) and one M anisopliae (ARSEF 3540) were more pathogenic to the nymphs than the standard, having significantly lower LC50 and LT50, values. Our results demonstrated that several genera of entomopathogenic fungi have promise as microbial control agents against L. lineolaris.     

COMPARISON OF CHLOROPLAST DNA AND ALLOZYME VARIATION IN WINTER STRAINS OF THE MARINE DIATOM SKELETONEMA COSTATUM(BACILLARIOPHYTA)1

Restriction fragment length polymorphisms (RFLPs) were examined in 12 winter strains of the marine diatom Skeletonema costatum (Grev.) Cleve using homologous chloroplast gene probes. The winter strains included eight different allozyme genotypes exhibiting physiological differences. These 12 winter strains were representative of the least diverse genetic group present in Narragansett Bay populations. Five chloroplast DNA probes and four different restriction enzymes were used to analyze the 12 Narragansett Bay strains and a reference strain “Skel.” A total of 46 restriction fragments were identified. All 12 of the winter strains had identical patterns. Strain Skel exhibited two RFLPs in comparison to the Narragansett Bay strains. Calculated diversity within the winter strain group was 0.0 and 0.85 for the chloroplast DNA and allozyme data, respectively. The chloroplast DNA polymorphisms revealed by this study are expected to represent a minimum level of the chloroplast DNA diversity present in Narragansett Bay seasonal populations.     

Diversity and differentiation among fluorescent pseudomonads in crop rhizospheres with whole-cell protein profiles

Fluorescent pseudomonads from rhizospheres of four different crops, grown under similar soil and climatic conditions were phenotypically characterized to differentiate them into biovars. In protein electrophenogram studies of relationships among fluorescent pseudomonads by UPGMA cluster analysis based on DICE similarity index, the isolates were mainly discerned into three major clusters representing four different biovars. The biovars generally matched the delineated phenotypic clusters with the exception of a strain belonging to biovar II. However, the isolates representing similar rhizospheres and geographic locations were generally distributed into different phenotypic clusters as influenced by factors yet to be determined. The studies reinstated the importance of whole-cell protein analyses in characterizing pseudomonads and assessing their diversity.     

Diversity of the Phytophthora infestans population in Flanders, Belgium

A total of 94 isolates of Phytophthora infestans were collected from disease outbreaks in commercial potato crops and private gardens in 2002 and 2003. The isolates were recovered successfully from single lesions of diseased potato foliage. Not from all isolates pure cultures were obtained due to contaminations with Fusarium species and bacteria. The structure of the population was analysed phenotypically. Characteristics of the isolates included in vitro growth rate, mating type, in vitro sensitivity to the phenylamide fungicide metalaxyl-M and allozyme genotype at glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) loci. Significant differences in in vitro growth rate were observed among the 52 isolates by comparing the main radial growth of the isolates after 7 days. Forty seven from the isolates tested were the Al mating type. Only one isolate was characterized as A2 mating type. Isolates with sensitive, intermediate and resistant responses to metalaxyl-M were detected in the populations. Forty isolates had a growth of less then 40 % at 5 ppm metalaxyl-M. Three isolates had a growth of less then 40 % at 100 ppm metalaxyl-M. Eight isolates had a growth of more then 40 % at 5 and 100 ppm metalaxyl-M. Cellulose acetate electrophoresis was used to examine Gpi and Pep banding pattern of the population of P. infestans attacking potato in Flanders. All the isolates tested produced the 100/100 Gpi isozyme electromorph. Five different allozyme genotypes of the Pep loci were identified: 92/92, 96/96, 100/100, 92/100, 83/100.     

Electrophoresis karyotype and chromosome-length polymorphism of Histoplasma capsulatum clinical isolates from Latin America

Intact chromosomes of 19 clinical isolates of Histoplasma capsulatum recently obtained in Argentina, Mexico and Guatemala and the laboratory reference strain G186B from Panama were analyzed using pulsed-field gel electrophoresis. Chromosomal banding patterns of the human isolates revealed 5-7 bands, ranging from 1.3 to 10 Mbp in size. Strain G186B showed five bands of approximately 1.1, 2.8, 3.3, 5.4 and 9.7 Mbp. Thirteen different electrokaryotypes were identified, indicating that the genome of H. capsulatum varies widely in nature, as observed previously in laboratory strains. No definite association was found between electrokaryotype and geographical or clinical source.     

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in southwest China from 2007 to 2009

To gain insight into the molecular epidemiology and possible mechanisms of genetic variation of porcine reproductive and respiratory syndrome (PRRS) in Yunnan Province of China, the ORF5 gene of 32 PRRSV isolates from clinical samples collected from 2007 to 2009 were sequenced and analyzed. Nucleotide and amino acid analyses were carried out on 32 isolates and representative strains of the North American genotype, European genotype and two representative Chinese isolates. Results revealed that these isolates share 86.9-99.0% nucleotide and 87.5-98.0% amino acid identity with VR-2332 the prototypical North American PRRSV, 61.7-62.9% and 54.3-57.8% with Lelystad virus (LV) the representative strain of European genotype, 91.2-95.4% and 90.0-94.5% with CH-1a that was isolated in mainland China in 1996, 88.1-99.3% and 85.5-99.0% with JX-A1 the representative strain of High pathogenic PRRSV in China, and 86.2-99.8% and 85.5-100.0% between isolated strains of different years, respectively. Phylogenetic analysis revealed that all 32 PRRSV isolates belonged to the North American genotype and were further divided into two different subgenotypes. Subgenotype 1 comprised twenty two Yunnan isolates which divided into two branches. Subgenotype 2 comprised ten isolates which closely related to the RespPRRS vaccine and its parent strain VR-2332. The functional domains of GP5 such as the signal peptide, ectodomain, transmembrane regions and endodomain were identified and some motifs in GP5 with known functions, such as primary neutralizing epitope (PNE) and decoy epitope were also further analyzed. Our study shown the great genetic diversity of PRRSV in southwest China, rendering the guide for control and prevention of this disease.     

Polysaccharide-hydrolyzing enzymes ofFrankia (Actinomycetales)

Studies were made of the polysaccharide-hydrolyzing activity inFrankia (Actinomycetales) grown in synthetic media using modifications of three standard assay procedures. In screening five different strains ofFrankia for cellulase activity, based on the method of utilization of cellulose in liquid culture, only one strain, CcI3, degraded filter paper cellulose to complete disintegration and only under very specific conditions of pH and primary carbon source. When carboxymethylcellulose (CMC) at 1% was used as substrate, all five strains showed the capacity to produce reducing sugars as hydrolytic products. Microcystalline cellulose, xylans and gum arabic were hydrolyzed to a lesser extent. Optimum activity depended upon pH and primary carbon source with pH 5.0 and pyruvate or propionate producing highest activities. In fractionation studies of culturedFrankia, assays for hydrolysis of 1% CMC in liquid medium showed that highest activity was in the enzyme preparation supernatant with lesser activity in the cell-free extract and cell wall fractions.Frankia strain CpI1 showed the greatest total hydrolytic activity against CMC after 2 weeks of culture. Strains ArI3 and CcI3 also showed good activity. The agar plate method for direct dye-polysaccharide interaction proved to be the least sensitive assay method with only ArI3 showing significant activity using CMC as substrate. It appears that theFranka strains grown in synthetic media all showed hydrolytic activity but the degree of hydrolysis of polysaccharides to reducing sugars depends upon strain of bacteria and very specific cultural conditions.     

A genetic approach to species criteria in the amoeba genus Naegleria using allozyme electrophoresis

The present study employs allozyme electrophoresis to characterize and inter-relate 61 isolates of Naegleria. Diploidy was confirmed, with heterozygotes observed at 29 of the 33 loci established and in all but two isolates. With a single exception, isolates clustered at two levels of similarity, either below 21% or above 52%. It is argued that such a major discontinuity provides a sound biological basis for a species concept in Naegleria. On this basis the present species-level taxonomy does not reflect the genetic diversity of the genus. The study recognized 18 genetic groups of species rank. The subspecies N. australiensis italica deserves specific rank; additional thermophilic species not closely related to N. fowleri and N. lovaniensis are recognized; and N. gruberi as currently conceived is a complex of 10 species, at least five of which are represented in the formal culture collections. Most species are genetically too different for relationships to be elucidated by allozyme electrophoresis, supporting the view that some of the times of divergence within the genus are extremely ancient.     

Molecular profiling demonstrates limited diversity amongst geographically separate strains of Ustilago scitaminea

Intraspecies diversity within Ustilago scitaminea isolates from South Africa, Reunion Island, Hawaii and Guadeloupe was assessed by RAPDs, bE mating-type gene detection, rDNA sequence analysis, microscopy and germination and morphological studies. Except for sequence data, the other analyses yielded no differences in the isolates that could be used in a phylogenetic separation. Mycelial DNA of the SA isolate shared 100% sequence identity with that of mycelial DNA cultured from in vitro produced teliospores of the parent cultivar. Overall the ITS1 and ITS2 regions were found to have 96.1% and 96.9% sequence identity with a total of 17 and 21 base changes, respectively, amongst the isolates. The Reunion Island isolate was shown to be most distantly related by 3.6% to the other isolates, indicating a single clonal lineage. The lack of germination in teliospores from Guadeloupe may be attributed to changes in temperature and humidity during transportation.     

Degradation of natural and synthetic polyesters under anaerobic conditions

Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).     

Epidemiology of Rhodococcus equi strains on Thoroughbred horse farms

Pulsed-field gel electrophoresis of restriction endonuclease-digested genomic DNA from a large collection of clinical isolates of Rhodococcus equi, an important pathogen of foals, was used to compare strain distribution between farms and over time. Forty-four strains were found among 209 isolates, with 5 of these accounting for over half the isolates and the 22 strains isolated more than once accounting for 90% of the isolates. The average genotypic diversity on each farm and in each year was found to be less than the genotypic diversity of the isolates taken as a whole, with 5.2% of the total diversity being due to differences between farms and 5.5% to differences between years. A small number of strains on each farm were found to have caused at least half the clinical cases of disease, and these varied between farms and, to a lesser extent, years. Most strains were found on more than one farm, and some very similar restriction patterns were found among isolates from different continents, indicating that strains can be very widespread. Multiple strains were isolated in five of the six cases in which more than one isolate from a single foal was examined, indicating that disease may commonly be caused by simultaneous infection with multiple strains. It was concluded that there are a number of different strains of R. equi which carry the vapA gene, and these strains tend to be widespread, but individual farms tend to have particular strains associated with them.     

Genetic diversity among dairy lactococcal strains investigated by polymerase chain reaction with three arbitrary primers

Randomly amplified polymorphic DNA (RAPD) has been used for the rapid typing of Lactococcus lactis strains isolated from raw milk from the Camembert region of Normandy. It is thought that the diversity and perhaps the area strain specificity due to climatic and geographical factors of such wild-type lactococcal strains could contribute to the flavour differences and specific features detected for the same product in different areas. The patterns from 58 isolates were analysed by UPGMA dendrograms. At a similarity level of 50%, four RAPD clusters were distinguished. Clusters 1 and 2 contained strains of subspecies lactis and cluster 3 contained strains related to the C2 strain which is genetically cremoris but phenotypically lactis. The type strain of cremoris subspecies was significantly differentiated from these strains with primers P2 and P3. Thus, there was a real genetic diversity in pattern, making it possible to detect potential typical RAPD fragments.