16S-23S rRNA Intergenic Spacer Region Research Articles

An extremophile Microbacterium strain and its protease production under alkaline conditions

Extremophiles are potential resources for alkaline protease production. In order to search for alkaline protease producers, we isolated and screened alkaliphilic microorganisms from alkaline saline environments. The microorganism HSL10 was identified as a member of the genus Microbacterium by morphological observation, Gram staining and sequence analysis of the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region. By colony-forming unit counting under alkali or salt stress, it was further identified as an alkaliphilic microbe with mild halotolerance. In addition, it was capable of secreting alkaline proteases, evidenced by larger hydrolyzation zones in the skim milk-containing medium at pH 9.0 than at pH 7.0. Subsequently, we demonstrated that both NaCl and yeast extract significantly promoted protease production by HSL10. Finally, we established a sensitive colorimetric method for the detection of protease production by HSL10 under neutral and alkaline conditions, by using the Bradford reagent for substrate staining to improve the contrast between the hydrolyzation zone and the substrate background on agar plates. HSL10 was the first example of an alkaliphilic protease-producing member in Microbacterium, and its isolation and characterization have both academic and commercial importance.

Isolation, identification and characterisation of three novel probiotic strains (Lactobacillus paracaseiCNCM I-4034,Bifidobacterium breveCNCM I-4035 andLactobacillus rhamnosusCNCM I-4036) from the faeces of exclusively breast-fed infants

The aim of the present study was to isolate, identify and characterise novel strains of lactic acid bacteria and bifidobacteria with probiotic properties from the faeces of exclusively breast-fed infants. Of the 4680 isolated colonies, 758 exhibited resistance to low pH and tolerance to high concentrations of bile salts; of these, only forty-two exhibited a strong ability to adhere to enterocytes in vitro. The identities of the isolates were confirmed by 16S ribosomal RNA (rRNA) sequencing, which permitted the grouping of the forty-two bacteria into three different strains that showed more than 99% sequence identity with Lactobacillus paracasei, Lactobacillus rhamnosus and Bifidobacterium breve, respectively. The strain identification was confirmed by sequencing the 16S-23S rRNA intergenic spacer regions. Strains were assayed for enzymatic activity and carbohydrate utilisation, and they were deposited in the Collection Nationale de Cultures de Microorganismes (CNCM) of the Institute Pasteur and named L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036. The strains were susceptible to antibiotics and did not produce undesirable metabolites, and their safety was assessed by acute ingestion in immunocompetent and immunosuppressed BALB/c mouse models. The three novel strains inhibited in vitro the meningitis aetiological agent Listeria monocytogenes and human rotavirus infections. B. breve CNCM I-4035 led to a higher IgA concentration in faeces and plasma of mice. Overall, these results suggest that L. paracasei CNCM I-4034, B. breve CNCM I-4035 and L. rhamnosus CNCM I-4036 should be considered as probiotic strains, and their human health benefits should be further evaluated.

Characteristics of the two microbial markers in vaginal secretions in Chinese Han population

Distinguishing vaginal secretions from other forensically relevant biological fluids could significantly aid the forensic investigation, especially in cases of sexual assault. Recently, two microbial markers, Lactobacillus crispatus and Lactobacillus gasseri for identification of vaginal secretions, were reported. However, the relevant study was not performed in Chinese Han population. In this study, we investigated the characteristics of the two markers in Chinese Han population. We detected 16S–23S rRNA intergenic spacer region by reverse transcription polymerase chain reaction (RT-PCR) assay and characterized the species composition by sequencing 16S rRNA genes. The results revealed that the two microbial markers only expressed in vaginal secretions samples rather than in other body fluids, but they did not express in all female individuals. In conclusion, the inter-individual variation of these two markers should be considered, when the two markers were used for the identification of vaginal secretions in Chinese Han population.

MRNA profiling for vaginal fluid and menstrual blood identification

The detection and identification of human biological fluids, including vaginal secretions and menstrual blood, are highly important in forensic biology. Previous studies have proposed a few mRNA and bacterial markers for vaginal fluid detection, but they have not proven to be specific and reliable. The aim of this project was to develop, validate and evaluate a reliable, specific test for vaginal fluid identification that would combine detection of vaginal mRNAs and Lactobacilli. We have developed a hexaplex that detects HBD1 (human beta-defensin 1), MUC4 (mucin 4), menstrual blood marker MMP11 (matrix metalloproteinase 11), housekeeping gene G6PDH (glucose 6-phosphate dehydrogenase) and the 16S-23S rRNA intergenic spacer region of Lactobacillus crispatus and Lactobacillus gasseri/Lactobacillus johnsonii. We analysed the specificity of the markers and variations among women, as well as the sensitivity of the test and its ability to detect vaginal fluid in mixtures with semen and blood. This approach allows for the detection of vaginal fluid in stains that were up to 2 years old, if stored at room temperature and up to 18 years old if stored frozen. Through simultaneous analysis of 5 vaginal markers, the proposed hexaplex ensures high specificity and reliability in the detection of vaginal material.

Identification and virulence of Chryseobacterium indologenes isolated from diseased yellow perch (Perca flavescens )

To identify pathogen of diseased yellow perch and determine their virulence. Fifteen Gram-negative bacterial isolates were recovered from the skin lesions of diseased yellow perch (Perca flavescens). Based on API 20NE test, ten isolates were found to share 67.2-99.9% homologies with Chryseobactertium indologenes. Based on fatty acid methyl ester analysis, 13 isolates were found to share similarities with C. indologenes and other species of Chryseobacterium. Based on sequencing results of partial 16S rRNA gene, 13 isolates shared 99% identities (e value = 2e-50) with the 16S rRNA sequence of C. indologenes (GenBank HQ259684). Based on the 16S-23S rRNA intergenic spacer region (ISR) sequence, the 13 isolates shared 88% identity (e value = 1e-165) with the 16S-23S ISR sequence of C. indologenes (GenBank EU014570). T-coffee multiple sequence alignment revealed that the partial 16S rRNA or the 16S-23S ISR sequence of the 13 isolates shared 100% identity with each other. When healthy yellow perch were exposed to the 15 isolates by bath immersion (c. 6 × 10(7) CFU ml(-1) for 1 h), only C. indologenes isolates killed 10-20% of fish, whereas other isolates were avirulent. When yellow perch were exposed to C. indologenes by intraperitoneal injection, mortality was dose dependent, with LD(50) and LD(95) values of 1.5 × 10(8) and 3.2 × 10(8) CFU per fish, respectively. Chryseobactertium indologenes could be pathogenic to yellow perch. This is the first report on the isolation of C. indologenes from diseased yellow perch. Virulence studies suggested that C. indologenes could become pathogenic to yellow perch.

Development of a 16S-23S rRNA intergenic spacer-based quantitative PCR assay for improved detection and enumeration ofLactococcus garvieae

Lactococcus garvieae is an important foodborne pathogen causing lactococcosis associated with hemorrhagic septicemia in fish worldwide. A real-time quantitative polymerase chain reaction (qPCR) protocol targeting the 16S-23S rRNA intergenic spacer (ITS) region was developed for the detection and enum-eration of L.garvieae. The specificity was evaluated using genomic DNAs extracted from 66 cocci strains. Fourteen L.garvieae strains tested were positive, whereas 52 other strains including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. hordniae and Lactococcus lactis ssp. cremoris did not show a specific signal. The minimal limit of detection was 2.63fg of purified genomic DNA, equivalent to 1 genome of L.garvieae. The optimized protocol was applied for the survey of L.garvieae in naturally contaminated fish samples. Our results suggest that the qPCR protocol using ITS is a sensitive and efficient tool for the rapid detection and enumeration of L.garvieae in fish and fish-containing foods.

Two Genotypes among ‘<i>Candidatus</i> Mycoplasma haemobos’ Strains Based on the 16S-23S rRNA Intergenic Spacer Sequences

'Candidatus Mycoplasma haemobos', sometimes causative of bovine infectious anemia at various extents, has been demonstrated throughout the world. Here, we show two distinct types of 'Ca. M. haemobos' are distributed among cattle in Japan, by examining the primary and secondary structures of the 16S-23S rRNA intergenic spacer region that has been shown to be a stable genetic marker for mycoplasma species. Our results may explain differences in severity of anemic condition as well as provide a genetic marker for an epidemiological study of bovine hemoplasma infections.

Associations of passerine birds, rabbits, and ticks with Borrelia miyamotoi and Borrelia andersonii in Michigan, U.S.A.

BackgroundWild birds contribute to maintenance and dissemination of vectors and microbes, including those that impact human, domestic animal, and wildlife health. Here we elucidate roles of wild passerine birds, eastern cottontail rabbits (Sylvilagus floridanus), and Ixodes dentatus ticks in enzootic cycles of two spirochetes, Borrelia miyamotoi and B. andersonii in a region of Michigan where the zoonotic pathogen B. burgdorferi co-circulates.MethodsOver a four-year period, wild birds (n = 19,631) and rabbits (n = 20) were inspected for tick presence and ear tissue was obtained from rabbits. Samples were tested for Borrelia spirochetes using nested PCR of the 16S-23S rRNA intergenic spacer region (IGS) and bidirectional DNA sequencing. Natural xenodiagnosis was used to implicate wildlife reservoirs.ResultsIxodes dentatus, a tick that specializes on birds and rabbits and rarely bites humans, was the most common tick found, comprising 86.5% of the 12,432 ticks collected in the study. The relapsing fever group spirochete B. miyamotoi was documented for the first time in ticks removed from wild birds (0.7% minimum infection prevalence; MIP, in I. dentatus), and included two IGS strains. The majority of B. miyamotoi-positive ticks were removed from Northern Cardinals (Cardinalis cardinalis). Borrelia andersonii infected ticks removed from birds (1.6% MIP), ticks removed from rabbits (5.3% MIP), and rabbit ear biopsies (5%) comprised twelve novel IGS strains. Six species of wild birds were implicated as reservoirs for B. andersonii. Frequency of I. dentatus larval and nymphal co-feeding on birds was ten times greater than expected by chance. The relatively well-studied ecology of I. scapularis and the Lyme disease pathogen provides a context for understanding how the phenology of bird ticks may impact B. miyamotoi and B. andersonii prevalence and host associations.ConclusionsGiven the current invasion of I. scapularis, a human biting species that serves as a bridge vector for Borrelia spirochetes, human exposure to B. miyamotoi and B. andersonii in this region may increase. The presence of these spirochetes underscores the ecological complexity within which Borrelia organisms are maintained and the need for diagnostic tests to differentiate among these organisms.

Potential use of molecular-typing methods for the identification and characterization of salmonella enterica serotypes isolated in the poultry production chain

Salmonella is widespread in nature and can be found in all links of the poultry production chain. Due to its high impact on meat processing, techniques for the rapid detection and reproducible characterization of Salmonella serotypes in foods are needed. The present study investigated the potential of molecular profiling to identify and differentiate 15 Salmonella serotypes isolated from the poultry production chain, based on 5 primers by random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC-PCR), amplification of rDNA internal spacer analysis (RISA), and amplified ribosomal DNA restriction analysis (ARDRA) of 16S-23S rRNA internal spacer region (ISR) cleaved with Alu I and Hha I restriction enzymes. Three isolates of each serotype were analyzed for the identification of similar and different profiles. Dendrograms were constructed from molecular profiles using the UPGMA method (unweighted pair-group method for the arithmetic averages) and the software program WinBoot. The present study indicates the usefulness of RISA and ARDRA of the 16S-23S rRNA intergenic spacer region (ISR) for systematic, epidemiological, and diagnostic purposes. Since these techniques can be used for the differentiation of serotypes, they are highly promising for the characterization of Salmonella serotypes and intra-serotypes. Data indicate that these techniques may be used to produce more consistent, reliable, and reproducible results in the identification and epidemiological study (traceability) of Salmonella in the poultry industry.

Bacterial Stripe of Hog Millet Caused by Acidovorax avenae subsp. avenae, a New Disease in Korea

In July 2011, bacterial stripe was observed on a commercial field of hog millet (Panicum miliaceum L.) in Chuncheon, Korea, with a disease incidence of 37% in the field. Symptoms on leaves included reddish-brown, long, narrow stripes that varied in length and were sharply delineated by uninfected adjacent vascular bundles. Eleven bacterial isolates (BC3107, BC3214 to BC3223) were recovered on trypticase soy agar from lesions surface sterilized in 70% ethanol for 1 min. The isolates, all obtained from different plants, were gram negative, oxidase positive, aerobic rods with two to four flagella. The isolates produced circular, cream-colored, nonfluorescent, butyrous colonies with entire margins on King's B medium. Using the Biolog Microbial Identification System, Version 4.2 (Biolog Inc., Hayward, CA), the isolates were identified as Acidovorax avenae subsp. avenae with Biolog similarity indices ranging from 0.52 to 0.72 after 24 hr. Characters for differentiating between Acidovorax spp. were tested according to Schaad et al. (2). The isolates were positive for gelatin liquefaction, nitrate reduction, lipase production, utilization of D-mannitol, sodium citrate, and alkaline in litmus milk. The isolates were negative for utilization of D-arabitol and did not amplify with PCR primer sets Aaaf5, Aaaf3/Aaar2, and Aacf2/Aacr2. Colonies were V-, V+, and V+ for utilization of D-fucose, maltose, and ethanol, respectively. Regions of the 16S rRNA (rrs) and the IGS were sequenced to aid in the identification of the isolates using reported PCR primer sets (1,4). A 1,426 bp fragment of the rrs region shared 100% similarity with all strains of A. avenae available in GenBank. Pathogenicity tests were separately performed for the 11 isolates in different greenhouses located in Suwon (National Academy of Agricultural Science), and Chuncheon (Gangwondo Agricultural Research and Extension Services) in Korea. Pathogenicity was confirmed by clip inoculation with sterilized scissors dipped into cell suspensions containing 105 CFU/ml on three 8-day-old leaves of hog millet (two plants per isolate), rice (Oryza sativa L. cv. Hopyeong), and sweet corn (Zea mays L. cv. Daehak) in a greenhouse maintained at 28 to 32°C and 90% relative humidity. The isolates induced similar symptoms as those originally observed on hog millet 5 days after inoculation. No symptoms were observed on the control plants (hog millet, rice, and sweet corn), which were clipped with scissors dipped in sterilized distilled water. The identity of bacteria reisolated from the stripes on inoculated leaves was confirmed by analyzing sequences of the 16S-23S rRNA intergenic spacer region (IGS) (1). On the basis of physiological, pathological, and sequence data, the isolates were identified as A. avenae subsp. avenae. To our knowledge, this is the first report of bacterial stripe of hog millet caused by A. avenae subsp. avenae in Korea. The spread of the bacterial disease is expected to have a significant economic impact on hog millet culture in the fields of Gangwon Province in Korea. Nucleotide sequence data reported are available under accession numbers JQ743877 to JQ743887 for rrs of BC 3207 and BC3214 to BC3223, and JQ743877 to JQ743887 for IGS of BC3207 and BC3214 to BC3223.

Comparison of Two Capillary Gel Electrophoresis Systems for Clostridium difficile Ribotyping, Using a Panel of Ribotype 027 Isolates and Whole-Genome Sequences as a Reference Standard

PCR ribotyping is the most commonly used Clostridium difficile genotyping method, but its utility is limited by lack of standardization. In this study, we analyzed four published whole genomes and tested an international collection of 21 well-characterized C. difficile ribotype 027 isolates as the basis for comparison of two capillary gel electrophoresis (CGE)-based ribotyping methods. There were unexpected differences between the 16S-23S rRNA intergenic spacer region (ISR) allelic profiles of the four ribotype 027 genomes, but six bands were identified in all four and a seventh in three genomes. All seven bands and another, not identified in any of the whole genomes, were found in all 21 isolates. We compared sequencer-based CGE (SCGE) with three different primer pairs to the Qiagen QIAxcel CGE (QCGE) platform. Deviations from individual reference/consensus band sizes were smaller for SCGE (0 to 0.2 bp) than for QCGE (4.2 to 9.5 bp). Compared with QCGE, SCGE more readily distinguished bands of similar length (more discriminatory), detected bands of larger size and lower intensity (more sensitive), and assigned band sizes more accurately and reproducibly, making it more suitable for standardization. Specifically, QCGE failed to identify the largest ISR amplicon. Based on several criteria, we recommend the primer set 16S-USA/23S-USA for use in a proposed standard SCGE method. Similar differences between SCGE and QCGE were found on testing of 14 isolates of four other C. difficile ribotypes. Based on our results, ISR profiles based on accurate sequencer-based band lengths would be preferable to agarose gel-based banding patterns for the assignment of ribotypes.

Genetic Diversity of Streptococcus equi subsp. zooepidemicus and Doxycycline Resistance in Kennelled Dogs

The genetic diversity and antibiotic resistance profiles of 38 Streptococcus equi subsp. zooepidemicus isolates were determined from a kennelled canine population during two outbreaks of hemorrhagic pneumonia (1999 to 2002 and 2007 to 2010). Analysis of the szp gene hypervariable region and the 16S-23S rRNA intergenic spacer region and multilocus sequence typing (MLST) indicated a predominant tetO-positive, doxycycline-resistant ST-10 strain during 1999 to 2002 and a predominant tetM-positive doxycycline-resistant ST-62 strain during 2007 to 2010.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines

The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

Molecular Detection of Bartonella Sp. in Wild Ruminants and Analysis of its Genetic Diversity on the Basis of 16S–23S Rrna Intergenic Spacer (ITS)

Abstract The aim of this study was to describe the state of infection of roe deer (Capreolus capreolus) and red deer (Cervus elaphus) by Bartonella sp. in North-Western Poland through PCR detection of Bartonella 16S-23S rRNA ITS region in isolates of animal tissues, and also to describe the genetic diversity of detected Bartonella species based on molecular analysis of ITS. The multiple alignment analysis of ITS sequences was carried out, and homology matrices and phylogenetic trees were constructed. The DNA of Bartonella sp. was detected in tissues of 45.6% (36/79) C. capreolus and of 50% (15/30) C. elaphus. Products of two different sizes were detected: 317 bp, characteristic for B. schoenbuchensis, and 198 bp, characteristic for B. bovis. The obtained results suggest that roe and red deer are potential reservoirs of Bartonella sp. Most of the analysed ITS sequences was not specific for one host species. In constructed phylogenetic trees, sequences obtained from roe and red deer clustered together. These results suggest a lack of host specificity of most detected B. schoenbuchensis and B. bovis intraspecies strains

Characterization of rhizobia from legumes of agronomic interest grown in semi‐arid areas of Central Spain relates genetic differences to soil properties

A study of symbiotic bacteria from traditional agricultural legumes from Central Spain was performed to create a collection of rhizobia from soils differing in physicochemical, analytical and/or agroecological properties which could be well-adapted to the environmental conditions of this region, and be used for sustainable agricultural practices. Thirty-six isolates were obtained from root-nodules of fifteen legume species (including Cicer arietinum, Lathyrus sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum sativum, and Vicia spp.) from three agriculture areas with soils of different pHs and from a forest area with undisturbed soils. Phenotypical characterization revealed uniformity across the thirty-six isolates, with important exceptions in terms of environmental tolerance (three isolates survived at high temperatures, three at high salinity and three at acid pH). The molecular analysis of 16S rRNA gene showed a close relationship of twenty-nine isolates to Rhizobium leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The sequence analysis of a symbiosis-specific gene, nod C, showed a correlation with the plant host and grouped twenty-six isolates with Rhizobium leguminosarum bv. viciae, establishing the diversity in relation to legume-host. The 16S-23S rRNA intergenic spacer (IGS) region allowed for intraspecific differentiation, so that strains with equal 16S rRNA were grouped by means of their soil origin. These results indicated that phenotypical and genetically related strains may be widely distributed in this region and that soil abiotic characteristics could have a substantial bearing on the selection of the strains living in each environment.

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S.parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S-23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype-specific biochemical characteristics and genetic characteristics of 16S-23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

For the rapid detection of Laribacter hongkongensis, which is associated with human community-acquired gastroenteritis and traveller's diarrhoea, we developed a duplex species-specific PCR assay. Full-length of the 16S-23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR-based sequencing. Two species-specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species-specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2.1 × 10(-2) ng μl(-1) genomic DNA which corresponds to 5000 CFU ml(-1). The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. This species-specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.

Molecular Characterization of Mycoplasma gallisepticum Isolates from Jordan

Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.

Phytoplasma classification: taxonomy based on 16S ribosomal gene, is it enough?

The discovery in 1967, of a new group of plant pathogens related to bacteria led to the finding of pleomorphic, wall-less prokaryotes in the phloem of many plant species affected by yellows-type diseases. Following the application of molecular technologies the phylogeny of these prokaryotes was resolved and led to the new trivial name of “phytoplasma” and to the designation of a new taxon named ‘Candidatus phytoplasma’. The first comprehensive phytoplasma classification scheme was based on RFLP analysis of PCR-amplified 16S rDNA, providing a reliable means for the differentiation of a broad array of phytoplasmas and has become the most comprehensive and widely accepted phytoplasma classification system. This approach using RFLP analyses of PCR amplified 16S rDNA provides a simple, reliable and rapid mean for differentiation and identification of known phytoplasmas. A consensus for naming novel phytoplasmas was recommended by the IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group that a ‘Candidatus Phytoplasma’ species description should refer to a single, unique 16S rRNA gene sequence that has <97.5% similarity to that of any previously described ‘Ca. Phytoplasma’ species. Because of the highly conserved nature of the 16S rDNA, many biologically or ecologically distinct phytoplasma strains, which may warrant designation of a new taxon may fail to meet the requirement. In this case, additional unique biological properties such as antibody specificity, host range and vector transmission specificity as well as other molecular criteria (gene) need to be included for speciation. However, because of the highly conserved nature of the 16S rDNA and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, the classification based on ‘Candidatus’ or on 16S ribosomal group does not always provide the molecular distinction necessary for phytoplasma strain characterization. Moreover, some additional tools for phylogenetic analyses and finer strain differentiation of phytoplasmas such as rp, secY, tuf, groEL genes, and the 16S-23S rRNA intergenic spacer region sequences have been used as supplementary tools selecting those providing the most useful and reliable taxonomic information in combination with 16SrDNA.

Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S–23S rRNA intergenic spacer region (16S–23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S–23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S–23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S–23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S–23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNAAla gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S–23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination.