Major Ribotype Research Articles

Genome Variability in Artificial Allopolyploid Hybrids of Avena sativa L. and Avena macrostachya Balansa ex Coss. et Durieu Based on Marker Sequences of Satellite DNA and the ITS1-5.8S rDNA Region.

Artificial hybrids between cultivated Avena species and wild Avena macrostachya that possess genes for resistance to biotic and abiotic stresses can be important for oat breeding. For the first time, a comprehensive study of genomes of artificial fertile hybrids Avena sativa × Avena macrostachya and their parental species was carried out based on the chromosome FISH mapping of satellite DNA sequences (satDNAs) and also analysis of intragenomic polymorphism in the 18S-ITS1-5.8S rDNA region, using NGS data. Chromosome distribution patterns of marker satDNAs allowed us to identify all chromosomes in the studied karyotypes, determine their subgenomic affiliation, and detect several chromosome rearrangements. Based on the obtained cytogenomic data, we revealed differences between two A. macrostachya subgenomes and demonstrated that only one of them was inherited in the studied octoploid hybrids. Ribotype analyses showed that the second major ribotype of A. macrostachya was species-specific and was not represented in rDNA pools of the octoploids, which could be related to the allopolyploid origin of this species. Our results indicate that the use of marker satDNAs in cytogenomic studies can provide important data on genomic relationships within Avena allopolyploid species and hybrids, and also expand the potential for interspecific crosses for breeding.

Origin of Wild Polyploid Avena Species Inferred from Polymorphism of the ITS1 rDNA in Their Genomes

In this article, we analyzed the origin of wild polyploid oats (Avena L., Poaceae) using the region 18S rDNA (partially)–ITS1–5.8S rDNA obtained via NGS. There are six tetraploid (2n = 28) and four hexaploid (2n = 42) wild species differing by specific genome combinations: A. barbata, A. vaviloviana (AB), A. agadiriana (AB or BB), A. magna, A. murphyi, A. insularis (AC or CD), A. ludoviciana, A. sterilis, A. fatua, and A. occidentalis (ACD). We compared the pool of marker sequences of polyploid oats with those of their putative diploid ancestors: A. atlantica (As-genome), A. hirtula (As), A. canariensis (Ac), A. ventricosa (Cv), and A. clauda (paleopolyploid with Cp and A-related rDNA). We found 15 major ribotypes (more than 1000 reads per rDNA pool) in polyploid oats. Comparing them, we found that the AB-tetraploid oats possibly inherited their A-genome ribotypes from A. atlantica (As1-ribotype), whereas their B-genome ribotype is specific and can be a derivative of the A-genome family. Our data do not support the hypothesis of the CD-genome set in A. magna, A. murphyi, and A. insularis: they have an AC-genome ribotype constitution instead. The C-genome-related sequences could have been obtained from A. ventricosa. Hexaploids show a different ribotype pattern than tetraploids; the main ribotypes of A. fatua, A. ludoviciana, and A. sterilis probably belong to the D-group and are also shared with one of the major ribotypes of A. clauda.

High Prevalence of Clostridioides difficile Ribotype 176 in the University Hospital in Kosice.

Dysbiosis of the gut microbiota, caused by antibiotics, plays a key role in the establishment of Clostridioides difficile CD). Toxin-producing strains are involved in the pathogenesis of Clostridioides difficile infection (CDI), one of the most common hospital-acquired infections. We cultured a total of 84 C. difficile isolates from stool samples of patients hospitalized at Louis Pasteur University Hospital in Kosice, Slovakia, that were suspected of CDI and further characterized by molecular methods. The presence of genes encoding toxin A, toxin B, and binary toxin was assessed by toxin-specific PCR. CD ribotypes were detected using capillary-based electrophoresis ribotyping. A total of 96.4% of CD isolates carried genes encoding toxins A and B, and 54.8% of them were positive for the binary toxin. PCR ribotyping showed the presence of three major ribotypes: RT 176 (n = 40, 47.6%); RT 001 (n = 23, 27.4%); and RT 014 (n = 7, 8.3%). Ribotype 176 predominated among clinical CD isolates in our hospital. The proportion of RT 176 and RT 001 in four hospital departments with the highest incidence of CDI cases was very specific, pointing to local CDI outbreaks. Based on our data, previous use of antibiotics represents a significant risk factor for the development of CDI in patients over 65 years of age.

Origin of the Rare Hybrid Genus ×Trisetokoeleria Tzvelev (Poaceae) According to Molecular Phylogenetic Data.

In our article, we analyzed new data on the origin of the hybrid genus ×Trisetokoeleria. According to the morphological criteria ×T. jurtzevii is a hybrid between Koeleria asiatica s. l. and Trisetum spicatum, ×T. taimyrica, and originated from Koeleria asiatica s. l. and Trisetum subalpestre, ×T. gorodkowii, a hybrid between Koeleria asiatica and Trisetum ruprechtianum. Later ×T. taimyrica was transferred to Koeleria. Parental taxa are prone to active hybridization themselves, thus, new methods of next-generation sequencing (NGS) were needed to clarify the relationships of these genera. For NGS we used the fragment 18S rDNA (part)-ITS1-5.8S rDNA (totally 441 accessions). We analyzed ITS1-5.8S rDNA-ITS2 region, trnL-trnF and trnK-rps16 from eight samples of the five species, using the Sanger method: ×Trisetokoeleria jurtzevii, ×T. taimyrica, Koeleria asiatica, Sibirotrisetum sibiricum (=Trisetum sibiricum), and Trisetum spicatum. We also studied the pollen fertility of ×Trisetokoeleria and its possible progenitors. Our data partly contradicted previous assumptions, based on morphological grounds, and showed us a picture of developed introgression within and between Koeleria and Trisetum. ×T. jurtzevii, a totally sterile hybrid formed rather recently. We can suppose that ×T. jurtzevii is a hybrid between K. asiatica and some Trisetum s. str. Species, but not T. spicatum. ×T. gorodkowii, a hybrid in the stage of primary stabilization; it has one unique ribotype related to T. spicatum s. l. The second parental species is unrelated to Trisetum ruprechtianum. ×T. taimyrica and is a stabilized hybrid species; it shares major ribotypes with the T. spicatum/T. wrangelense group and has a minor fraction of rDNA related to genus Deyeuxia s. l.

Hybridization and diversity of aquatic macrophyte Sparganium L. (Typhaceae) as revealed by high-throughput nrDNA sequencing

Sparganium is an emergent aquatic macrophyte widely spread in temperate and subtropical zones. Taxa of this genus feature high phenotypic plasticity and can produce interspecific hybrids. By means of high-throughput sequencing of the internal transcribed spacer (ITS1) of 35S rDNA, the status of 15 Eurasian Sparganium species and subspecies was clarified and the role of hybridization events in the recent evolution of the genus was investigated. It has been shown that a number of species such as S. angustifolium, S. fallax and S. subglobosum have homogenized rDNA represented by one major ribotype. The rDNA of other taxa is represented by two or more major ribotypes. Species with high rDNA heterogeneity are apparently of hybrid origin. Based on the differences in rDNA patterns, intraspecific diversity was identified in S. probatovae and S. emersum. Thus, we have concluded that Sparganium has extensive interspecific hybridization at the subgenus level, and there may also be occasional hybridization between species from different subgenera.

Acne and Stress: Impact of Catecholamines on Cutibacterium acnes.

Cutibacterium acnes (former Propionibacterium acnes), is a bacterium characterized by high genomic variability, consisting of four subtypes and six major ribotypes. Skin is the largest neuroendocrine organ of the human body and many cutaneous hormones and neurohormones can modulate bacterial physiology. Here, we investigated the effect of catecholamines, i.e., epinephrine and norepinephrine, on two representative strains of C. acnes, of which the genome has been fully sequenced, identified as RT4 acneic and RT6 non-acneic strains. Epinephrine and norepinephrine (10−6 M) had no impact on the growth of C. acnes but epinephrine increased RT4 and RT6 biofilm formation, as measured by crystal violet staining, whereas norepinephrine was only active on the RT4 strain. We obtained the same results by confocal microscopy with the RT4 strain, whereas there was no effect of either catecholamine on the RT6 strain. However, this strain was also sensitive to catecholamines, as shown by MATs tests, as epinephrine and norepinephrine affected its surface polarity. Flow cytometry studies revealed that epinephrine and norepinephrine are unable to induce major changes of bacterial surface properties and membrane integrity. Exposure of sebocytes to control or catecholamine-treated bacteria showed epinephrine and norepinephrine to have no effect on the cytotoxic or inflammatory potential of either C. acnes strains but to stimulate their effect on sebocyte lipid synthesis. Uriage thermal spring water was previously shown to inhibit biofilm production by C. acnes. We thus tested its effect after exposure of the bacteria to epinephrine and norepinephrine. The effect of the thermal water on the response of C. acnes to catecholamines depended on the surface on which the biofilm was grown. Finally, an in-silico study revealed the presence of a protein in the genome of C. acnes that shows homology with the catecholamine receptor of Escherichia coli and eukaryotes. This study suggests that C. acnes may play a role as a relay between stress mediators (catecholamines) and acne.

Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays

While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r2≥0.995) were established between Ct and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve.

Contrasting Patterns of Evolution of 45S and 5S rDNA Families Uncover New Aspects in the Genome Constitution of the Agronomically Important Grass Thinopyrum intermedium (Triticeae)

We employed sequencing of clones and in situ hybridization (genomic and fluorescent in situ hybridization [GISH and rDNA-FISH]) to characterize both the sequence variation and genomic organization of 45S (herein ITS1-5.8S-ITS2 region) and 5S (5S gene + nontranscribed spacer) ribosomal DNA (rDNA) families in the allohexaploid grass Thinopyrum intermedium. Both rDNA families are organized within several rDNA loci within all three subgenomes of the allohexaploid species. Both families have undergone different patterns of evolution. The 45S rDNA family has evolved in a concerted manner: internal transcribed spacer (ITS) sequences residing within the arrays of two subgenomes out of three got homogenized toward one major ribotype, whereas the third subgenome contained a minor proportion of distinct unhomogenized copies. Homogenization mechanisms such as unequal crossover and/or gene conversion were coupled with the loss of certain 45S rDNA loci. Unlike in the 45S family, the data suggest that neither interlocus homogenization among homeologous chromosomes nor locus loss occurred in 5S rDNA. Consistently with other Triticeae, the 5S rDNA family in intermediate wheatgrass comprised two distinct array types-the long- and short-spacer unit classes. Within the long and short units, we distinguished five and three different types, respectively, likely representing homeologous unit classes donated by putative parental species. Although the major ITS ribotype corresponds in our phylogenetic analysis to the E-genome species, the minor ribotype corresponds to Dasypyrum. 5S sequences suggested the contributions from Pseudoroegneria, Dasypyrum, and Aegilops. The contribution from Aegilops to the intermediate wheatgrass' genome is a new finding with implications in wheat improvement. We discuss rDNA evolution and potential origin of intermediate wheatgrass.

Allopolyploid origin of highly invasive Centaurea stoebe s.l. (Asteraceae)

Spotted knapweed (Centaurea stoebe) occurs from Western Asia to Western Europe both as diploid and tetraploid cytotypes, predominantly in single-cytotype populations with higher frequency of diploid populations. Interestingly, only tetraploids have been recorded so far from its introduced range in North America where they became highly invasive.We performed phylogenetic and network analyses of more than 40 accessions of the C. stoebe and C. paniculata groups and other related taxa using cloned internal transcribed spacer (ITS) and sequences of the chloroplast trnT–trnL and atpBrbcL regions to (i) assess the evolutionary origin of tetraploid C. stoebe s.l., and (ii) uncover the phylogeny of the C. stoebe group. Both issues have not been studied so far and thus remained controversial.Cloned ITS sequences showed the presence of two slightly divergent ribotypes occurring in tetraploid cytotype, while only one major ribotype was present in diploid C. stoebe s.str. This pattern suggests an allopolyploid origin of tetraploids with contribution of the diploid C. stoebe s.str. genome. Although we were not able to detect the second parental taxon, we hypothesize that hybridization might have triggered important changes in morphology and life history traits, which in turn may explain the colonization success of the tetraploid taxon. Bayesian relaxed clock estimations indicate a relatively recent – Pleistocene origin of the tetraploid C. stoebe s.l. Furthermore, our analyses showed a deep split between the C. paniculata and C. stoebe groups, and a young diversification of the taxa within the C. stoebe group. In contrast to nrDNA analyses, the observed pattern based on two cpDNA regions was inconclusive with respect to the origin and phylogeny of the studied taxa, most likely due to shared ancient polymorphism and frequent homoplasies.

Analysis of the microbial community of the biocathode of a hydrogen-producing microbial electrolysis cell

The microbial electrolysis cell (MEC) is a promising system for hydrogen production. Still, expensive catalysts such as platinum are needed for efficient hydrogen evolution at the cathode. Recently, the possibility to use a biocathode as an alternative for platinum was shown. The microorganisms involved in hydrogen evolution in such systems are not yet identified. We analyzed the microbial community of a mixed culture biocathode that was enriched in an MEC bioanode. This biocathode produced 1.1 A m−2 and 0.63 m3 H2 m−3 cathode liquid volume per day. The bacterial population consisted of 46% Proteobacteria, 25% Firmicutes, 17% Bacteroidetes, and 12% related to other phyla. The dominant ribotype belonged to the species Desulfovibrio vulgaris. The second major ribotype cluster constituted a novel taxonomic group at the genus level, clustering within uncultured Firmicutes. The third cluster belonged to uncultured Bacteroidetes and grouped in a taxonomic group from which only clones were described before; most of these clones originated from soil samples. The identified novel taxonomic groups developed under environmentally unusual conditions, and this may point to properties that have not been considered before. A pure culture of Desulfovibrio strain G11 inoculated in a cathode of an MEC led to a current development from 0.17 to 0.76 A m−2 in 9 days, and hydrogen gas formation was observed. On the basis of the known characteristics of Desulfovibrio spp., including its ability to produce hydrogen, we propose a mechanism for hydrogen evolution through Desulfovibrio spp. in a biocathode system.

Molecular analysis of bacterial communities in uranium ores and surrounding soils from Banduhurang open cast uranium mine, India: A comparative study

Bacterial community structure of heavy metal rich- uranium ores and surrounding soils was explored using 16S rRNA gene based clone library analysis and denaturing gradient gel electrophoresis (DGGE) to provide baseline microbial diversity data on autochthonous communities. Sequence analysis of major ribotypes and/or DGGE bands revealed Proteobacteria and Acidobacteria as the two most frequently present bacterial phyla across the samples, although relative abundance of each phyla and identity of their members at lower taxonomic level showed marked difference. Gammaproteobacteria (Pseudomonas and Escherichia) was most abundant in U-ore samples along with the lineages of β-Proteobacteria (Burkholderia and Janthinobacterium), α-Proteobacteria (Brevundimonas), Bacteroidetes (Spingobacterium), Firmicutes (Peptoniphilus), Actinobacteria (Corynebacterium), uncultured -Acidobacteria, -Chloroflexi and -Cyanobacterium. In contrast to this soil communities were represented by mixed populations predominated by uncultured Acidobacteria along with Gammaproteobacteria (Succinivibrio, Cellovibrio and Legionella), β-Proteobacteria (Rhodocyclus), α-Proteobacteria (Methylocystis and Phenylobacterium), δ-Proteobacteria, unclassified bacteria, uncultured Bacteroidetes, Firmicutes (Bacillus), Cyanobacteria (Scytonema), Actinobacteria (Actinomadura) and candidate division TM7. Principle Component Analyis (PCA) of geochemical data and UPGMA cluster analysis of DGGE profiles were in close agreement showing characteristic relatedness of samples obtained from either ores or soils. Our analysis indicated that soils surrounding the ore deposit bear specific geochemical as well as microbiologial characteristics distinct from the ore deposit and therefore these data obtained at the onset of mining could serve as a baseline of information to gauge the subsequent environmnetal impact of U-mining.

Genetic diversity of porcine Pasteurella multocida strains from the respiratory tract of healthy and diseased swine

A total of 382 porcine Pasteurella multocida strains, isolated from cases of pneumonia and progressive atrophic rhinitis (PAR) as well as from clinically healthy pigs of more than 150 German husbandries were characterized by detection of virulence-associated genes (VAGs) and ribotyping to understand the relationships between “commensal” and “pathogenic” strains, enabling a rational choice of vaccine strains. The diversity of the strains according to VAGs was low and mainly limited to capsular type genes ( capA: 53.4%; capD: 45.8%; capF: 0.3%; cap-negative: 0.5%; hssB: 95.3%), dermonecrotoxin gene toxA (3.4%), as well as adhesion-related genes pfhaB (20.9%) and hgbB (84.3%). Ribotyping identified 13 patterns, but the vast majority of strains (95.8%) clustered in only three of these, namely IA-1 (45.5%), IA-7 (30.1%), and IIA-1 (20.2%). Pattern IA-1 was associated with capD + strains (93.6%) and harboured the majority of toxA + strains (84.6%). Pattern IA-7 mostly contained pfhaB −, toxA − capA + strains (93.9%), while pattern IIA-1 was predominantly composed of pfhaB +, toxA − capA + strains (87.0%). Clinical strains associated with pneumonia or PAR shared the above mentioned major ribotypes in comparable proportions with strains derived from healthy pigs, suggesting P. multocida to act more as an opportunistic than as an obligate pathogen in pigs. The limited number of subpopulations may either reflect a recent evolution of P. multocida in pigs or a selection by means of horizontal transfer of capsular genes, toxA or pfhaB. These data enforce further phylogenetic and epidemiological studies, examining the properties of different subpopulations of porcine P. multocida strains as well as factors of the porcine hosts themselves, which might be involved in disease susceptibility.

Annual patterns of presence and activity of marine bacteria monitored by 16S rDNA–16S rRNA fingerprints in the coastal NW Mediterranean Sea

The annual dynamics of the bacterial community structure and its activity remain poorly studied in marine environments. Our goal was to gain new insights into the year-long bacterial dynamics and activity in correlation with environmental changes in the NW Mediterranean Sea. To accomplish this goal, we combined 16S rDNA versus 16S rRNA fingerprints and environmental vari- ables using multivariate analyses. A clone library was constructed to determine to which bacterial taxa the major ribotypes were related. Our results revealed similar environmental controls of bacter- ial community structure at both the DNA and RNA levels. The 16S rRNA signal of several ribotypes differed relative to their 16S rDNA counterpart, suggesting that the members of the community have either a higher or lower activity than their relative abundance would suggest. Such differences included the dominant ribotypes observed in the fingerprints, such as Roseobacter, Synechococcus, SAR11 and SAR116. All these results give support to the 16S rDNA and 16S rRNA fingerprinting approach to monitor bacterial community structure and activity in marine environments.

Isolation and Characterization of Clostridium difficile From 2 Geographically Separate Hospitals in Japan

Background The epidemiology of Clostridium difficile is not well known in Japan because C. difficile cultures are not routinely performed. Methods We analyzed the toxigenicity and the polymerase chain reaction (PCR) ribotype of C. difficile strains isolated from 2 geographically separate hospitals in Japan. Results The results of PCR ribotyping of toxigenic C. difficile strains isolated from these 2 hospitals indicated that a single type (type smz) was predominant at hospitals located in diverse areas of Japan. Toxin A-negative, toxin B-positive (A-B+) variant C. difficile comprised 8 (18.6%) of 43 and 11 (16.4%) of 67 toxigenic C. difficile isolates from 2 hospitals, respectively. Of the 19 A-B+ strains, 18 (94.7%) showed the same PCR ribotype pattern (type z), which was identical to that of previously reported outbreak strains. Only 1 A+B+ strain possessed the binary toxin genes. Conclusion PCR ribotype smz, which has been reported as a responsible ribotype for multiple outbreaks at Japanese hospitals in the late 1990s, is still the major epidemic ribotype in Japan. PCR ribotype z strains of A-B+ variant C. difficile also have disseminated within Japan and represent a substantial proportion of C. difficile isolates.

RmtD 16S RNA Methylase in Epidemiologically Unrelated SPM-1-Producing Pseudomonas aeruginosa Isolates from Brazil

We read with interest the article by Doi et al. (2) suggesting that the coproduction of the metallo-β-lactamase SPM-1 and the RmtD 16S RNA methylase is highly prevalent among imipenem-resistant Pseudomonas aeruginosa isolates from Brazil. SPM-1 is widespread among Brazilian carbapenem-resistant P. aeruginosa isolates, and isolates carrying blaSPM-1 have been observed in distinct regions of this country (3). The majority of SPM-1-producing isolates belong to a unique molecular clone identified by pulsed-field gel electrophoresis and automated ribotyping (3). The findings of Doi et al. (2) led us to test a very broad collection of epidemiologically unrelated blaSPM-1-carrying P. aeruginosa strains recovered from diverse Brazilian regions for the presence of rmtD. We evaluated 26 epidemiologically unrelated SPM-1-producing P. aeruginosa clinical isolates collected from different patients during 2002 in 10 Brazilian cities located in nine states (Table ​(Table1).1). These isolates belonged to 16 different ribogroups, and six isolates belonged to the major ribotype spread throughout the country (ribotype 72-3). Screening for rmtD was performed by PCR using the primers rmtD-F (5′-GAG CGA ACT GAA GGA AAA AC-3′) and rmtD-R (5′-CAG CAC GTA AAA CAG CTC-3′), which produce a 730-bp product. Amplicons were sequenced on both strands by using the ABIPrism 377 system (Applied Biosystems, Foster City, CA). The nucleotide sequences were analyzed using the Lasergene software package (DNASTAR, Madison, WI), and the sequences obtained were compared to sequences available over the Internet (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). TABLE 1. Characteristics of the 26 epidemiologically unrelated SPM-1-producing P. aeruginosa clinical isolates from different regions in Brazil Among the 26 isolates, rmtD was detected in only 4 (15.4%) strains. This rate is much lower than the results obtained previously (51.0%), implying that the production of RmtD is not prevalent among SPM-1-producing isolates with distinct genetic backgrounds and/or from different geographic areas in Brazil. blaSPM-1 is different from other metallo-β-lactamase genes that are carried in class 1 integrons also harboring aminoglycoside resistance genes as part of other gene cassettes (5) in that blaSPM-1 is embedded in a distinct genetic element (4) where the correlation of β-lactam and aminoglycoside resistance is not as evident. However, many SPM-1-producing isolates are resistant to other antimicrobial agents, including aminoglycosides. Recently, Carvalho et al. (1) reported that all 13 SPM-1-producing P. aeruginosa isolates found in another Brazilian state carried a class 1 integron, named In163, harboring two aminoglycoside resistance genes, aacA4 and aadA7, showing that aminoglycoside resistance among SPM-1-producing isolates can be also encoded by distinct resistance mechanisms other than the RmtD 16S RNA methylase gene.

Genetic surveillance of endemic bovine Salmonella Infantis infection

BackgroundSalmonella serovar Infantis is endemic in Finnish food-producing animals since the 1970s. The purpose of this study was to describe the molecular epidemiology of the infection in cattle during 1985–2005, to follow the persistence of the feed-related outbreak strain from 1995 in the cattle population, and to analyse the stability of XbaI-banding patterns in individual herds during long-lasting infections.MethodsSalmonella Infantis isolates from 478 cattle herds (n = 588), covering 73% of the subclinically or clinically infected herds, were typed by pulsed-field gel electrophoresis (PFGE) using XbaI. DNA fragments larger than 125 kb were counted in PFGE types because of high plasmid background. Ribotyping and IS200-typing with BanI-digested DNA were done on 57 selected isolates.ResultsThe isolates associated with the infection consisted of 51 PFGE types with genetic similarity (F value) between 0.58 and 0.95. From 1985 to 2003, the major type appeared on 68% of the farms. The three most common types, with F values of 0.90 to 0.95, accounted for 80% of the isolates. Only 17% of the isolates had F values below 0.80, and 1% below 0.70. Ribotyping and IS200-typing classified 89% of the analysed isolates into the major ribotype and IS200 type combination, and the rest fell into closely related types. Analysis of successive isolates from 142 herds revealed changes in XbaI-banding patterns in 21% of the herds with two analysed isolates and in 38% of the herds from which three or more isolates were analysed. The feed-related S. Infantis genotype from the 1995 outbreak had disappeared by 1999, at the time when the incidence of bovine salmonella, and S. Infantis in particular, strongly decreased.ConclusionThe study showed how genetic surveillance, as part of salmonella control, provides tools to follow the persistence of particular infections, and to assess the efficacy of control measures. Testing of several isolates from a herd in outbreak investigations is advisable, because minor changes in PFGE banding patterns frequently occur during long-lasting infections.

Ribotyping of Indian Isolates of Pasteurella multocida Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.

Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities. To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter(-1) day(-1)) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter(-1) day(-1) (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.

Long-term predominance of two pan-European clones among multi-resistant Acinetobacter baumannii strains in the Czech Republic.

In a recent study, a large proportion of multi-drug-resistant (MDR) Acinetobacter baumannii strains that were isolated from hospitalized patients in the Czech Republic was found to belong to two major groups (A and B). These groups appeared to be similar to epidemic clones I and II, respectively, which were identified previously among outbreak strains from north-western European hospitals. The aim of the present study was to assess in detail the genetic relatedness of Czech A. baumannii strains and those of epidemic clones I and II by using ribotyping with HindIII and HincII and by AFLP fingerprinting. The study collection included 70 MDR strains that were isolated in 30 Czech hospitals in 1991-2001, 15 susceptible Czech strains from 1991 to 1996 and 13 reference strains of clones I and II from 1982 to 1990. One major HindIII/HincIII ribotype (R1-1) was observed in 38 MDR Czech strains and eight reference strains of clone I, whereas another major ribotype (R2-2) was observed in 11 MDR Czech strains and in three reference strains of clone II. A selection of 59 Czech strains (representative of all ribotypes) and the 13 reference strains were investigated by AFLP fingerprinting. At a clustering level of 83%, two large clusters could be distinguished: cluster 1 included all reference strains of clone I and 25 MDR Czech strains, whilst cluster 2 contained all reference strains of clone II and 11 MDR Czech strains. There was a clear correlation between the groupings by AFLP analysis and by ribotyping, as all strains with ribotype R1-1 and four strains with slightly different ribotypes were found in AFLP cluster 1, whereas all strains with ribotype R2-2 and seven strains with similar ribotypes were in AFLP cluster 2. Thus, 41 and 21 MDR Czech strains could be classified as belonging to clones I and II, respectively. The remaining eight MDR and 15 susceptible strains were highly heterogeneous and were distinct from clones I and II by both AFLP fingerprinting and ribotyping. These results indicate that the two predominant groups observed among MDR Czech A. baumannii strains from the 1990s are genetically congruent with the north-western European epidemic clones that were found in the 1980s. Recognition of these clinically relevant, widespread clones is important in infection prevention and control; they are also interesting subjects to study genetic mechanisms that give rise to their antibiotic resistance and epidemic behaviour.

Evaluation of the polymorphisms associated with tandem repeats for Pseudomonas aeruginosa strain typing.

We report on the development of a scheme for the typing of Pseudomonas aeruginosa, multiple-locus variable number of tandem repeat (VNTR) analysis (MLVA). We first evaluated the polymorphisms of 201 tandem repeat loci selected from more than 3,000 such sequences present in strain PAO1 with a test collection of 12 genotypically distinct clinical strains. Seven VNTR loci which can be easily scored with the technology used here were identified and used to genotype a collection of 89 clinical isolates that had previously been classified into 46 ribotypes, including 2 widespread ribotypes. Seventy-one different MLVA genotypes could be distinguished. With only two exceptions, strains with identical ribotypes were grouped together upon cluster analysis of the MLVA data. The 27 isolates with the most frequent ribotype were divided into 14 MLVA types, and the 18 isolates with the second most frequent ribotype were divided into 15 MLVA types. Analysis of a subset of 17 strains belonging to the major ribotype by pulsed-field gel electrophoresis with the enzyme SpeI distinguished seven types, identical to the number of MLVA types in this subset. Our data show that MLVA typing of P. aeruginosa based on the first set of loci has a high discriminatory power. Because MLVA is highly reproducible and easily portable among laboratories, it represents a very promising tool for the molecular surveillance of P. aeruginosa. A free, online strain identification service based on the genotyping data produced herein has been developed.