rDNA Profiles Research Articles

First report of coconut lethal yellowing disease in Guatemala

The Atlantic coast of Guatemala runs between Belize and Honduras; two countries in which coconut lethal yellowing (LY) disease is highly active (Harrison et al., 2002). Coconut palms (Cocos nucifera, Atlantic tall ecotype) with symptoms indicative of LY (Harrison & Jones, 2003), including premature nutfall, necrosis of immature inflorescences, progressive frond yellowing and eventual death of the palm, were first observed at several sites along the coast during 2000. However, the syndrome differed from that reported for LY, as inflorescence necrosis was evident after, rather than before, frond yellowing. Tissues from trunks, young leaves or inflorescences were sampled from palms with and without symptoms at Estero Lagarto, Finca Baltimore, Santa María del Mar and Punta Manabique. Total DNA was extracted from tissues by a small-scale method and tested for phytoplasma DNA by a nested PCR using phytoplasma universal rRNA primer pair P1/P7, followed by LY group-specific primer pair LY16Sf/LY16Sr (Harrison et al., 2002). RFLP profiles obtained by digesting the nested PCR products with HinfI endonuclease (Harrison & Jones, 2003) were identical for all phytoplasma-positive Guatemalan palms and matched rDNA profiles of Honduran and Florida isolates of the LY phytoplasma included for comparison. LY phytoplasma was detected in all of the palms with symptoms (8/8), but none (0/8) of the symptomless palms tested positive. These results were confirmed later at the Centro de Investigación Científíca de Yucatán and the University of Florida using either LY group-specific or LY pathogen-specific PCR (Harrison et al., 1994). Following this initial survey, more LY-diseased coconut palms have been sighted at other locations along the Atlantic coast, but as of 2003, LY has not been seen during surveys of coconut populations along the Pacific coast of Guatemala. This is the first report of coconut lethal yellowing disease in Guatemala.

Changes in metabolic and structural diversity of a soil bacterial community in response to cadmium toxicity

Cadmium toxicity reduces the species diversity of soil microbial communities and their ability to metabolize different carbon substrates. However, it is not yet clear whether there is a relationship between loss of species diversity and the metabolic rates at which different substrates are used. In this research, the ability of a soil microbial community to metabolize 31 C substrates was examined using Biolog Ecoplates amended with Cd over a range of concentrations (0, 5, 10, 20, 50 μg ml−1). After 48 h growth, the bacterial communities that were produced on ten of the substrates were characterized in relation to their 16S rDNA profiles by PCR denaturing-gradient gel electrophoresis. Results showed that both the microbial growth rates and the numbers of substrates that were used were reduced with increasing Cd concentration. When examined across all substrates, approximately 30% of the 16S rDNA bands representing different bacterial species were eliminated by 5 μg Cd ml−1. However, there was considerable variation in the bacterial community responses with different C substrates. Both cluster analysis and discriminant analysis indicated that the bacterial community structures could be grouped into sensitive, moderately sensitive, and Cd-resistant communities.

Cereal/legume rotation effects on rhizosphere bacterial community structure in west african soils

The increased use of cereal/legume crop rotation has been advocated as a strategy to increase cereal yields of subsistence farmers in West Africa, and is believed to promote changes in the rhizosphere that enhance early plant growth. In this study we investigated the microbial diversity of the rhizoplane from seedlings grown in two soils previously planted to cereal or legume from experimental plots in Gaya, Niger, and Kaboli, Togo. Soils from these legume rotation and continuous cereal plots were placed into containers and sown in a growth chamber with maize (Zea mays L.), millet (Pennisetum glaucum L.), sorghum (Sorghum bicolor L. Moench.), cowpea (Vigna unguiculata L.) or groundnut (Arachis hypogaea L.). At 7 and 14 days after sowing, 16S rDNA profiles of the eubacterial and ammonia-oxidizing communities from the rhizoplane and bulk soil were generated using denaturing gradient gel electrophoresis (DGGE). Community profiles were subjected to peak fitting analyses to quantify the DNA band position and intensities, after which these data were compared using correspondence and principal components analysis. The data showed that cropping system had a highly significant effect on community structure (p <0.005), irrespective of plant species or sampling time. Continuous cereal-soil grown plants had highly similar rhizoplane communities across crop species and sites, whereas communities from the rotation soil showed greater variability and clustered with respect to plant species. Analyses of the ammonia-oxidizing communities provided no evidence of any effects of plant species or management history on ammonia oxidizers in soil from Kaboli, but there were large shifts with respect to this group of bacteria in soils from Gaya. The results of these analyses show that crop rotation can cause significant shifts in rhizosphere bacterial communities.

Functional and structural adaptations of bacterial communities growing on particulate substrates under stringent nutrient limitation.

Biomass recycle reactors (BRRs) were used as a model system to study the functional and structural adaptations of mixed bacterial communities in response to the imposition of increasingly severe nutrient limitation. BRRs were fed synthetic media containing either spinach homogenate or autoclaved yeast cells to simulate the complex mixtures of particulate carbon sources that are often present in nature. In the BRRs fed spinach homogenate, the biomass (measured as particulate protein) exhibited a physiological response similar to previous studies as detected by 40-80% reductions in respiratory potential and by relatively stable catabolic ectoenzyme activities. Concomitant adaptations in bacterial community structure were detected by PCR-DGGE and RT-PCR-DGGE of 16S rDNA and 16S rRNA fragments, respectively. The microbial community structure was dynamic even after the biomass had reached a quasi-steady state with respect to physiological measurements. In the BRRs fed yeast cells, respiratory potentials increased 2- to 5-fold during the initial portion of the BRR run and alpha-glucosidase and beta-glucosidase activities increased 2- to 4-fold. Substantial bacterial community shifts were also detected in both the rDNA and rRNA profiles, indicating that this community was also structurally dynamic. These experiments suggest that phylogenetically different bacteria sustained the functional activities in these ecosystems in response to increasingly stringent nutrient limitation.

Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE.

To evaluate the rpoB gene as a biomarker for PCR-DGGE microbial analyses using soil DNA from the Cerrado, Brazil. DNA extraction from soil was followed by Polymerase Chain Reaction (PCR) amplification of rpoB and 16S rRNA genes. PCR products were compared by Denaturing Gradient Gel Electrophoresis (DGGE) to compare gene/community profiles. The rpoB DGGE profiles comprised fewer bands than the 16S rDNA profiles and were easier to delineate and therefore to analyse. Comparison of the community profiles revealed that the methods were complementary. The gene for the beta subunit of the RNA polymerase, rpoB, is a single copy gene unlike 16S rDNA. Multiple copies of 16S rRNA genes in bacterial genomes complicate diversity assessments made from DGGE profiles. Using the rpoB gene offers a better alternative to the commonly used 16S rRNA gene for microbial community analyses based on DGGE.

Detection and Characterization of a Lethal Yellowing (16SrIV) Group Phytoplasma in Canary Island Date Palms Affected by Lethal Decline in Texas.

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in Canary Island date (Phoenix canariensis) palms displaying symptoms similar to lethal yellowing (LY) disease in Corpus Christi, TX. An rDNA product (1.8 kb) was amplified consistently from 10 of 11 palms by PCR employing phytoplasma universal rRNA primer pair P1/P7. Also, AluI endonu-clease digests and sequencing of P1/P7 products revealed that nontarget Bacillus megaterium-related rDNA sequences of similar size were co-amplified along with phytoplasma rDNA from 10 palms. A 1,402-bp product was obtained from all 11 symptomatic palms when initial P1/P7 products were reamplified by PCR employing nested LY phytoplasma group-specific 16S rRNA primer pair LY16Sf/LY16Sr. Restriction fragment length polymorphism (RFLP) analysis of nested PCR products revealed that palm-infecting phytoplasmas were uniform and most similar to strains composing the coconut lethal yellowing phytoplasma (16SrIV) group. Sequence analysis of 16S rDNA determined the Texas Phoenix palm decline (TPD) phytoplasma to be phylogenetically closest to the Carludovica palmata leaf yellowing (CPY) phytoplasma. rDNA profiles of strains TPD and CPY obtained with AluI were co-identical and distinct from other known 16SrIV group phytoplasmas. On this basis, both strains were classified as members of a new subgroup, 16SrIV-D.

Profiling of Bacterial Species Associated with Haddock Larviculture by PCR Amplification of 16S rDNA and Denaturing Gradient Gel Electrophoresis

Abstract A major limitation to marine finfish culture is the sudden and substantial increase in mortality frequently referred to as a “larval crash.” Contamination of the production system with opportunistic pathogenic bacteria has frequently been implicated as a cause of this mortality. In studies designed to test this hypothesis, 15 larval haddock from 16 tanks were sampled weekly between hatching and weaning in spring 2000 for their bacterial 16S rDNA profiles by polymerase chain reaction amplification combined with denaturing gradient gel electrophoresis (PCR−DGGE). The profiles obtained were generally dominated by fewer than five bands. A well-defined trend in bacterial succession was observed during the growth of the haddock larvae, the diversity of bacterial species being greater at the onset but invariably ending with a singular predominance by a species with 16S rDNA homology to various α-Proteobacteria such as Roseobacter. Sequence analysis of a larger fragment of 16S rDNA (1,437 nucleotides) of...

Impact of fumigants on soil microbial communities.

Agricultural soils are typically fumigated to provide effective control of nematodes, soilborne pathogens, and weeds in preparation for planting of high-value cash crops. The ability of soil microbial communities to recover after treatment with fumigants was examined using culture-dependent (Biolog) and culture-independent (phospholipid fatty acid [PLFA] analysis and denaturing gradient gel electrophoresis [DGGE] of 16S ribosomal DNA [rDNA] fragments amplified directly from soil DNA) approaches. Changes in soil microbial community structure were examined in a microcosm experiment following the application of methyl bromide (MeBr), methyl isothiocyanate, 1,3-dichloropropene (1,3-D), and chloropicrin. Variations among Biolog fingerprints showed that the effect of MeBr on heterotrophic microbial activities was most severe in the first week and that thereafter the effects of MeBr and the other fumigants were expressed at much lower levels. The results of PLFA analysis demonstrated a community shift in all treatments to a community dominated by gram-positive bacterial biomass. Different 16S rDNA profiles from fumigated soils were quantified by analyzing the DGGE band patterns. The Shannon-Weaver index of diversity, H, was calculated for each fumigated soil sample. High diversity indices were maintained between the control soil and the fumigant-treated soils, except for MeBr (H decreased from 1.14 to 0.13). After 12 weeks of incubation, H increased to 0.73 in the MeBr-treated samples. Sequence analysis of clones generated from unique bands showed the presence of taxonomically unique clones that had emerged from the MeBr-treated samples and were dominated by clones closely related to Bacillus spp. and Heliothrix oregonensis. Variations in the data were much higher in the Biolog assay than in the PLFA and DGGE assays, suggesting a high sensitivity of PLFA analysis and DGGE in monitoring the effects of fumigants on soil community composition and structure. Our results indicate that MeBr has the greatest impact on soil microbial communities and that 1,3-D has the least impact.

UPDATING AND EXTENDING GENETIC CHARACTERIZATION AND CLASSIFICATION OF PHYTOPLASMAS FROM WILD AND CULTIVATED PLANTS IN SOUTHERN ITALY

Restriction fragment length polymorphism (RFLP), sequence analysis of PCR-amplified ribosomal DNA and chromosome size determination by pulsed-field gel electrophoresis (PFGE) were used to characterize phytoplasmas associated with diseases of wild and cultivated plants in southern Italy. The diseases examined were known, but not the identity of the pathogens, their incidence in southern Italy, or their genome sizes. The phytoplasma detected in Dimorphotheca sinuata (Cape marigold) was identified by RFLP analysis as a member of the pigeon pea witches’-broom (PPWB) group and proved to be indistinguishable from the picris echiodes yellows (PEY) reference strain. However, it differed from PEY in genome size. Phytoplasmas infecting Cape marigold have not previously been characterized. The phytoplasma detected in diseased Taraxacum officinale (dandelion) showed the same RFLP profiles as the picris echioides phyllody (PEP) reference strain of the Italian alfalfa witches’-broom (IAWB) group. Members of this group were not previously known to occur in dandelion. The phytoplasmas detected in diseased tobacco, pepper, celery and grapevine were indistinguishable, on the basis of rDNA profiles, from the stolbur phytoplasma strain (STOL). The phytoplasmas in diseased tobacco and pepper were characterized for the first time in Italy. Sequence analysis of the 16S rDNA confirmed that the knautia phyllody phytoplasma (KAP) is a member PPWB group and is closely related to PEY. Chromosome sizes from 785 to 1020 kb were found for the phytoplasmas associated with diseases of Cape marigold, French hydrangea, onion, field bindweed and tomato.

16S rDNA fingerprinting of rhizosphere bacterial communities associated with healthy and Phytophthora infected avocado roots

Molecular techniques employing 16S rDNA profiles generated by PCR-DGGE were used to detect changes in bacterial community structures of the rhizosphere of avocado trees during infection by Phytophthora cinnamomi and during repeated bioaugmentation with a disease suppressive fluorescent pseudomonad. When the 16S rDNA profiles were analyzed by multivariate analysis procedures, distinct microbial communities were shown to occur on healthy and infected roots. Bacterial communities from healthy roots were represented by simple DNA banding profiles, suggestive of colonization by a few predominant species, and were approximately 80% similar in structure. In contrast, roots that were infected with Phytophthora, but which did not yet show visible symptoms of disease, were colonized by much more variable bacterial communities that had significantly different community structures from those of healthy roots. Root samples from trees receiving repeated applications of the disease suppressive bacterium Pseudomonas fluorescens st. 513 were free of Phytophthora infection, and had bacterial community structures that were similar to those of nontreated healthy roots. Sequence analysis of clones generated from four predominant bands cut from the DGGE gels revealed the presence of pseudomonads, as well as several previously unidentified bacteria. Differentiation of 16S rDNA profiles for healthy and infected roots suggests that rhizosphere bacterial community structure may serve as an integrative indicator of changes in chemical and biological conditions in the plant rhizosphere during the infection process.

16S rDNA fingerprinting of rhizosphere bacterial communities associated with healthy and Phytophthora infected avocado roots.

Molecular techniques employing 16S rDNA profiles generated by PCR-DGGE were used to detect changes in bacterial community structures of the rhizosphere of avocado trees during infection by Phytophthora cinnamomi and during repeated bioaugmentation with a disease suppressive fluorescent pseudomonad. When the 16S rDNA profiles were analyzed by multivariate analysis procedures, distinct microbial communities were shown to occur on healthy and infected roots. Bacterial communities from healthy roots were represented by simple DNA banding profiles, suggestive of colonization by a few predominant species, and were approximately 80% similar in structure. In contrast, roots that were infected with Phytophthora, but which did not yet show visible symptoms of disease, were colonized by much more variable bacterial communities that had significantly different community structures from those of healthy roots. Root samples from trees receiving repeated applications of the disease suppressive bacterium Pseudomonas fluorescens st. 513 were free of Phytophthora infection, and had bacterial community structures that were similar to those of nontreated healthy roots. Sequence analysis of clones generated from four predominant bands cut from the DGGE gels revealed the presence of pseudomonads, as well as several previously unidentified bacteria. Differentiation of 16S rDNA profiles for healthy and infected roots suggests that rhizosphere bacterial community structure may serve as an integrative indicator of changes in chemical and biological conditions in the plant rhizosphere during the infection process.

Glomus proliferum sp. nov.: A Description Based on Morphological, Biochemical, Molecular and Monoxenic Cultivation Data

A new arbuscular mycorrhizal fungus, Glomus proliferum (Glomales, Zygomycetes) is described. The description, based on a monoxenic culture established in association with a Ri T-DNA transformed carrot root, combines sequencing of the small subunit (SSU) rDNA, spore sterols and fatty acid profiles with more classical taxonomic tools such as optical and electron microscopy. The fungus forms clusters containing hundreds of small, hyaline, four-layered spores. The necessity to use different tools for identification of arbuscular mycorrhizal fungi is discussed.

Glomus proliferum sp. nov.: a description based on morphological, biochemical, molecular and monoxenic cultivation data

A new arbuscular mycorrhizal fungus, Glomus proliferum (Glomales, Zygomycetes) is described. The description, based on a monoxenic culture established in association with a Ri T-DNA transformed carrot root, combines sequencing of the small subunit (SSU) rDNA, spore sterols and fatty acid profiles with more classical taxonomic tools such as optical and electron microscopy. The fungus forms clusters containing hundreds of small, hyaline, four-layered spores. The necessity to use different tools for identification of arbuscular mycorrhizal fungi is discussed.

Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences.

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA profiles were objectively digitized using an image analyzer; the individual microbial species in a community can thus be precisely quantified. The similarity between various microbial communities was compared to the digitized DGGE profiles using the cluster analyses technique. The microbial community in a biofilm was considerably different from that in suspended sludge obtained from the same system.

Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles.

The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides.

Detection and Differentiation of Phytoplasmas Associated with Diseases of Strawberry in Florida.

Strawberry (Fragaria × ananassa Duchesne) plants with symptoms suggestive of phytoplasmal disease were identified in commercial fields and a breeder's plot in west central Florida during the 1995 to 1996 winter growing season. Affected plants were all conspicuously stunted and unproductive. Primary symptoms on cvs. Rosa Linda and Carlsbad and on a breeder's accession resembled those of strawberry green petal (SGP). Plants displayed sparse clusters of virescent flowers with enlarged sepals and phylloid receptacles that failed to develop fully into fleshy structures or redden on ripening. Symptoms on cv. Oso Grande were more typical of multiplier disease and included a proliferation of branch crowns producing numerous small leaves with spindly petioles. Oso Grande and Carlsbad originated as transplants from a nursery in Montreal, Canada, whereas Rosa Linda transplants were from Nova Scotia. Plants were assessed for phytoplasma infection by polymerase chain reaction with total DNAs from leaves and petioles as template and phytoplasma-specific ribosomal RNA primers P1 and P7 (3), or mollicute-specific ribosomal protein (rp) gene primers rpF1 and rpR4 (2). Amplification of a 1.8-kb rDNA or 1.2-kb rp gene product, respectively, confirmed infection of Rosa Linda (7 of 7 plants), Carlsbad (3 of 7), Oso Grande (4 of 4), and a single breeder's accession. No products were amplified from DNAs of healthy plants. Restriction fragment length polymorphism patterns of rDNA digested with AluI, EcoRI, HaeIII, HhaI, HpaII, KpnI, ScaI, or Tru9I endonucleases, or of rp gene products digested with AluI, DraI, RsaI, TaqI, or Tru9I, revealed no differences among phytoplasma strains affecting both Rosa Linda and Carlsbad. Collectively, patterns were comparable to those of clover phyllody and SGP phytoplasmas, two Canadian strains previously classified as members of phytoplasma 16S rRNA (rr)-ribosomal protein (rp) group 16S rI, subgroup C (16S rI-C (rr-rp)) (1). Similarly, no differences were evident among phytoplasmas associated with all four diseased Oso Grande plants. Both rDNA and rp fragment profiles associated with this cultivar were characteristic of strains such as tomato big bud and eastern aster yellows delineated as 16S rI-A (rr-rp) subgroup members (1). However, AluI rDNA and TaqI rp fragment patterns were unique, identifying Oso Grande-infecting strains as representatives of a new subgroup within the larger 16S rI (rr-rp) group. Cumulative rDNA and rp fragment profiles of the phytoplasma associated with the breeder's accession matched those of the Mexican periwinkle virescence phytoplasma, identifying this strain as a 16S rI-I (rr-rp) subgroup member (1) and a second possible etiological agent of SGP. This is the first report of phytoplasmas infecting strawberry in Florida.

Pseudomonas aeruginosa ribotyping: Stability and interpretation of ribosomal operon restriction patterns

Fifty-one Pseudomonas aeruginosa isolates were differentiated into 21 types by ribotyping. Several enzyme combinations, including the best ones proposed in literature, were utilized and the highest discrimination was reached by individual digestion with PvuII, HindIII, and EcoRI or BamHI. Clinical isolates from outbreaks were clonally related as identified by this molecular approach. Restriction rDNA profiles were composed of strong and weak bands. Using 6 μg DNA we were able to demonstrate that PvuII, HindIII, and BamHI weak bands were reproducible. These weak bands should be considered not only to accomplish the highest discrimination but also to correctly assign isolate clonality. Conversely, we found that EcoRI weak bands were not reproducible and, therefore, are not recommended for ribotype analysis. Finally, profiles differing in one single band actually represented isolates of different genotype, as confirmed by further analysis using other molecular methods. In this report on P. aeruginosa ribotyping of clinical isolates, criteria for band pattern interpretation are established.

Classification of a world‐wide collection of isolates of Pseudocercosporella herpotrichoides by RFLP analysis of mitochondrial and ribosomal DNA and host range

Two profiles of mitochondrial DNA (mtDNA) were detected among 59 isolates of Pseudocercosporella herpotrichoides from diverse geographical sources. One profile (type II) was present only among isolates pathogenic on rye (R‐type), and a second profile (type I) was present in W, C and S‐pathotypes of the fungus. Mitochondrial DNA from P. herpotrichoides hybridized strongly to DNA of P. anguioides, but very weakly to DNA from P. aestiva, suggesting that the latter is not closely related to P. herpotrichoides. Fifteen profiles of ribosomal DNA (rDNA) were observed among isolates with type I mtDNA, and five of these were only found among seven isolates from Ireland. The other profiles present among type‐I isolates appeared to be largely independent of geographic origin. Three rDNA profiles were observed among R‐type (type II mtDNA) isolates.

Typing of methicillin resistant and susceptibleStaphylococcus aureus strains by ribosomal RNA gene restriction patterns using a biotinylated probe

Nine methicillin-sensitive (MSSA) and 37 methicillin-resistant (MRSA) Staphylococcus aureus isolates of various phage types and resistotypes from seven countries were investigated. Chromosomal DNA was restricted with HindIII or EcoRI, Southern blotted and hybridised with a cDNA probe to 16S+23S rRNA derived from MRSA NCTC 10442. Resulting rDNA profiles could be differentiated on the basis of 22 patterns which were unaffected by changes in plasmid, transposon, enterotoxin A or phage content. Percentage similarity values were calculated using the Dice coefficient and UPGA clustering. Australian and epidemic (EMRSA-1) isolates from the UK showed a high degree of similarity, but the pattern was not unique and was also found in MSSA and other MRSA, e.g. NCTC 10442. An MSSA of phage group II was the most distinct isolate. The method shows potential as an additional tool in a complex typing system, types non-phage typable strains and may provide clues to the clonal evolution of MRSA and MSSA.