Restriction Fragment Length Polymorphism Patterns Research Articles

Prevalence and characterization of hybrid blaCTX-M among Escherichia coli isolates from livestock and other animals

This study investigated 248 extended-spectrum β-lactamase-producing Escherichia coli isolates from 2012 to 2013 for hybrid blaCTX-M genes. blaCTX-M genes were detected in 228 isolates of which 14 isolates were hybrid blaCTX-M positive (6 blaCTX-M-123, 6 blaCTX-M-64, and 2 blaCTX-M-132). The 14 hybrid blaCTX-M–carrying isolates (8 from chickens, 2 each from pigs and cattle, 1 each from dog and rodent) were genetically diverse. All but 2 hybrid blaCTX-M were carried on IncI1 (5 blaCTX-M-123) and IncI2 (6 blaCTX-M-64 and one blaCTX-M-132) plasmids. Our IncI1 and IncI2 plasmids had pHNAH4-1–like and pHN1122-1–like restriction fragment length polymorphism patterns, respectively. Genetic relatedness of the plasmids to pHNAH4-1 and pHN1122-1 were confirmed by complete sequencing of 3 plasmids, pCTXM123_C0996, pCTXM64_C0967, and pCTXM132_P0421. Plasmids closely related to pHNAH4-1 and pHN1122-1 and carrying different blaCTX-M alleles have been reported from multiple geographic areas in China previously. The findings highlighted the wide dissemination of hybrid blaCTX-M variants in different parts of China.

Molecular Identification of a 16SrII‐A Group‐Related Phytoplasma Associated with Cinnamon Yellow Leaf Disease in China

AbstractChinese cinnamon (Cinnamomum cassia Presl), an evergreen tree native to China, is a multifaceted medicinal plant. The stem bark of cinnamon is used worldwide in traditional and modern medicines and is one of the most popular cooking spices. In recent years, cinnamon with pronounced yellow leaf symptoms has been observed in their natural habitat in Hainan, China. Phytoplasmas were detected from symptomatic cinnamon trees via polymerase chain reaction using phytoplasma universal primers P1/P7 followed by R16F2n/R16R2. No amplification products were obtained from templates of asymptomatic cinnamon trees. These results indicated a direct association between phytoplasma infection and the cinnamon yellow leaf (CYL) disease. Sequence analysis of the CYL phytoplasma 16S rRNA gene determined that CYL phytoplasma is a ‘Candidatus Phytoplasma australasiae’‐related strain. Furthermore, virtual restriction fragment length polymorphism pattern analysis and phylogenetic studies showed that CYL phytoplasma belongs to the peanut witches’‐broom (16SrII) group, subgroup A. This is the first report of a 16SrII group phytoplasma infecting cinnamon under natural conditions.

Fungal malignant otitis externa caused by Alternaria chlamydospora: first case report

Introduction: Malignant otitis externa (MOE) is a rare clinical entity, usually observed in diabetic or immunosuppressed patients, with serious morbidity due to associated osteomyelitis, cranial nerve palsies and intracranial infections. Pseudomonas aeruginosa is the main pathogen in over 95 % of cases; Aspergillus species and Candida albicans have also been implicated in fungal MOE. Case presentation: Α 79‐year‐old male with type 2 diabetes with otalgia, otorrhoea and granulation tissue occupying the right external ear canal was diagnosed with MOE. Direct microscopy of a tissue biopsy specimen taken from the granulation tissue revealed septate branching hyphae and chlamydospores. An olivaceous‐black colony cultured within 3 days at 30 °C was identified microscopically as Alternaria sp. and molecularly as Alternaria chlamydospora using the restriction fragment length polymorphism pattern of the internal transcribed spacer (ITS) region on the basis of the 570 bp ITS amplicon, a BstUI largest band of 578 bp and absence of the TaqI 114 bp band. The patient was unresponsive to ciprofloxacin, whereas the pain was relieved after 2 weeks of voriconazole treatment followed by surgical debridement. Conclusion: We present, we believe, the first reported case of MOE for which Alternaria sp. seems to be the causative pathogen. A high index of suspicion was needed in order to reach the diagnosis. We recommend taking tissue cultures when a high‐risk patient is not responsive to the initial antibiotic treatment, as fungal MOE could be a repercussion of unsuccessfully treated bacterial otitis externa or it could represent a de novo presentation of fungal disease.

Dianthus barbatus-A New Host of Stolbur Phytoplasma in Serbia.

Sweet William (Dianthus barbatus, Caryophyllaceae) is a biennial or short-lived perennial plant native to southern Europe, from the Pyrenees to the Carpathians and the Balkans. During the summers of 2012 and 2013, phytoplasma-like symptoms were observed on D. barbatus plants on a Serbian plantation (Pancevo, 44°51'49″ N, 20°39'33″ E, 80 m ASL). Only seven symptomatic plants were observed in the summer of 2012. Disease incidence in 2013 was estimated to be less than 1% but increased during 2014 to 4%. Affected plants, showing symptoms of leaf reddening, malformation, and proliferation; flower bud deficiency; and abnormal shoot production, were tested for phytoplasmas. Samples were collected from seven symptomatic and three symptomless plants each year (20 samples), and total nucleic acid was extracted from midrib tissue using a method that includes a phytoplasma enrichment step and DNA purification by chloroform/phenol (3). Oligonucleotide primers specific to the phytoplasma 16S to 23S rRNA intergenic spacer region were used in polymerase chain reaction (PCR) assays on DNA extracted from Sweet William plants (1,3). Using phytoplasma universal primer pairs P1/P7 and P1/16S-Sr, phytoplasma-specific 1.8- and 1.5-kb amplicons were obtained from four and six symptomatic plants collected in 2012 and 2013, respectively. Nested PCR with R16F2n/R2 primers yielded ~1.2-kb amplicons from DNAs of all symptomatic plants (1). No amplicon was generated in PCRs conducted with DNA templates from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of amplified 1.2-kb fragments was performed using four endonucleases (AluI, Tru1I, HhaI, and HpaII). Comparative analysis was done using RFLP patterns of Stolbur (Stol), Aster Yellows (AY), Flavescence Doree-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas. PCR-RFLP patterns from tested samples were identical to those of the Stol reference strain, indicating that diseased Sweet William was affected by phytoplasma belonging to the 16SrXII-A (Stolbur) group. The sequence of a 1.2-kb rDNA PCR product derived from sample Tk9 (deposited under accession number KM401436 in NCBI GenBank) showed the closest identity (100%) to those of Bulgarian corn (KF907506.1), Iranian 'Bois Noir' (KJ637208.1), and two Serbian phytoplasmas (KJ174507.1 from Calendula officinalis and KF614623.1 from Paeonia tenuifolia), all belonging to the 'Candidatus Phytoplasma solani' Stolbur subgroup. Previously, Aster Yellows Phytoplasma (16SrI) had been detected in two Dianthus species: D. barbatus (Sweet William) and D. caryophyllus (carnation) (2). This is the first record of the 16SrXII-A phytoplasma subgroup being associated with yellowing and reddening of D. barbatus in Serbia. The Stolbur phytoplasma occurrence on Sweet William is significant for the management of the disease in Serbia.

Restriction fragment length polymorphism analysis of rotavirus VP7-encoding gene from humans and animals of Northeast India: a relative study of Indian and global isolates.

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic relationship between 67 (29 Indian, 38 global) rotavirus isolates of human, bovine and porcine neonates. The assay involved direct digestion of RT-PCR amplified VP7 cDNAs with three restriction enzymes (VspI, HaeIII, NlaIV) independently. Forty-eight RFLP patterns were identified for all 67 strains, and of these 20 patterns were associated with Indian isolates. A correlation between the restriction patterns and G type was apparent through deduction of enzyme restriction sites from known sequences. Major G serotypes (G1, G2, G6, G8) with a few mixed types could be differentiated where there was a positive assortment of intrinsic serotypes from multiple host origin, and certain single or combined enzyme profiles were highly dominant in the population. Significant genetic variations were established between global and Indian isolates and none of the RFLP patterns were shared between them. These data suggest that the Indian wild-type rotavirus population is distinguishable based on the VP7 gene, and co-circulation of distinct strains in different hosts is foremost, indicating the possible likelihood of inter-species transmission.

Prevalence of VKORC1 polymorphism in population of Hyderabad India

Introduction: Warfarin/Acitrom administration dosage varies based on patient genotype with respect to the gene: Vitamin K epoxide reductase complex 1 (VKORC1). Ethnic diversity contributes to genotype variation. The frequency of polymorphism at VKORC1 in population of Hyderabad has not been reported in literature; hence the present study was conducted. Material & Methods: Genomic DNA from peripheral blood leukocytes of patients on warfarin/acitrom as well as normal control population without any thrombotic disease was extracted and Restriction Fragment Length Polymorphism (RFLP) pattern was established for 1173C>T transition by standard protocols. Result: The incidence of homozygote dominants (CC) genotypes were 74.63% and that of heterozygotes (CT) were 25.37 %. No recessives were found in the study group. Conclusion: Presently genotype based dosage is not implemented. This study through its collaboration with practising cardiologist while establishing the test at our diagnostic centre has also enabled awareness and consequently if followed will improve patient safety.

Molecular Characterization of Klebsiella Isolates from Enteral Diets

To evaluate the genotyping of Klebsiella isolates from enteral diets in public hospitals, as high-resolution support within epidemiological surveillance for controlling nosocomial infections. Methods: Klebsiella isolates were obtained from enteral diets at two public hospitals in the state of Minas Gerais, Brazil. These isolates were identified and serotyped. Total DNA of Klebsiella was extracted and subjected to three genotyping methods, to evaluate polymorphism between the strains and genetic diversity. Results: Twenty-one isolates of Klebsiella were obtained from the hospital enteral formulae; fifteen were identified as K. pneumoniae and six as K. oxytoca. The results from random amplified polymorphic DNA (RAPD) and 16S-23S rDNA analysis revealed high polymorphism among the K. pneumoniae isolates and a low level of polymorphism among the K. oxytoca isolates. The 1420 base pairs of DNA fragments generated through amplification of the 16S rDNA region and digestion with eight restriction endonucleases resulted in identical restriction fragment length polymorphism (RFLP) patterns for all isolates. Conclusion: Our data demonstrate that RAPD and 16S-23S rDNA analyses provide more reliable estimates of the genetic diversity among Klebsiella isolates than does amplification of the 16S rDNA gene. Therefore, we recommend that RAPD typing should be the preliminary method for fast and inexpensive investigation and that 16S-23S rDNA typing should be a confirmatory method if needed.

First Report on the Molecular Identification of Phytoplasma (16SrII-D) Associated with Witches' Broom of Kalmegh (Andrographis paniculata) in India.

Andrographis paniculata (family Acanthaceae), also known as "King of Bitters" or Kalmegh, is an important medicinal plant used for the treatment of various diseases. It has antimicrobial, antiviral, anti-inflammatory, hepatoprotective, antidiabetic, antihyperglycemic, and antioxidant properties (1). During June 2014, while performing a routine survey of the commercial trial fields of Kalmegh at Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, typical phytoplasma disease symptoms such as virescence, proliferation, and witches' broom along with little leaf and stunted growth were observed. The disease incidence was estimated to be approximately 7 to 10%. To ascertain the presence of phytoplasma, 16 samples of leaves were collected from nine different field sites, and total genomic DNA was extracted from the symptomatic and symptomless Kalmegh plants by the CTAB method. Direct and nested PCR assays were performed targeting the 16S rDNA using generic phytoplasma primer pairs P1/P6 followed by R16F2n/R16R2 (2). Resulting bands of the expected size (1.5 kb and 1.2 kb, respectively) were amplified from symptomatic plants. No amplification was observed with DNA from asymptomatic plant samples. The purified nested PCR products were cloned into E. coli DH5α, using the pGEM-T Easy vector (Promega, United States) and sequenced with primers M13For/M13Rev using an automatic sequencer (ABI Prism, Perkin Elmer) at CIMAP. The sequence was analyzed by BLASTn and found to share 99% similarity with Echinacea witches'-broom phytoplasma and Sesame phyllody phytoplasma strain (GenBank Accession Nos. JF340080 and KF612966, respectively), which belong to the 16SrII-D group. The sequence was deposited in NCBI as GenBank Accession No. KM359410. A phylogenetic tree using MEGA v5.0 (4) was constructed with 16S rDNA; consensus sequences of phytoplasmas belonging to distinct groups revealed that the present phytoplasma clustered with the 16SrII group. iPhyClassifier software was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile (5). On the basis of iPhyClassifier, the 16S rDNA sequence analysis of our isolate showed 99.2% similarity with that of the 'Candidatus Phytoplasma australasiae' reference strain (GenBank Accession No. Y10097), which belongs to 16Sr group II. The virtual RFLP pattern of F2n/R2 fragment was most similar to the 16SrII-D subgroup (similarity coefficient of 0.91) but showed a difference in profile with HpaI, HhaI, and MseI enzymes. Several bacterial/fungal and viral diseases have been reported on A. paniculata (3); however, to our knowledge, this is the first report of witches' broom disease in India and the first record of a 16SrII-D group phytoplasma on Kalmegh. Its presence in Kalmegh is of great significance due to its commercial interest.

Detection of Airborne Bacteria in a Duck Production Facility with Two Different Personal Air Sampling Devices for an Exposure Assessment

Prevalent airborne microorganisms are not well characterized in industrial animal production buildings with respect to their quantity or quality. To investigate the work-related microbial exposure, personal bioaerosol sampling during the whole working day is recommended. Therefore, bioaerosol sampling in a duck hatchery and a duck house with two personal air sampling devices, a filter-based PGP and a NIOSH particle size separator, was performed. Subsequent, quantitative and qualitative analyses were carried out with” culture independent methods. Total cell concentrations (TCC) determined via fluorescence microscopy showed no difference between the two devices. In average, 8 × 106 cells/m3 were determined in the air of the duck hatchery and 5 × 107 cells/m3 in the air of the duck house. A Generated Restriction Fragment Length Polymorphism (RFLP) pattern revealed deviant bacterial compositions comparing samples collected with both devices. Clone library analyses based on 16S rRNA gene sequence analysis from the hatchery's air showed 65% similarity between the two sampling devices. Detailed 16S rRNA gene sequence analyses showed the occurrence of bacterial species like Acinetobacter baumannii, Enterococcus faecalis, Escherichia sp., and Shigella sp.; and a group of Staphylococcus delphini, S. intermedius, and S. pseudintermedius that provided the evidence of potential exposure to risk group 2 bacteria at the hatchery workplace. Size fractionated sampling with the developed by the Institute for Occupational Safety and Health of the German Social Accident Insurance (IFA) device revealed that pathogenic bacteria would deposit in the inhalable, the thorax, and possibly alveolar dust fraction according to EN481. TCC analysis showed the deposition of bacterial cells in the third stage (< 1μm) at the NIOSH device which implies that bacteria can reach deep into the lungs and contaminate the alveolus after inhalation. Nevertheless, both personal sampling devices could be recommended for exposure assessment at agricultural workplaces.

Multiple gene analyses identify distinct “bois noir” phytoplasma genotypes in the Republic of Macedonia

“Bois noir” (BN) is a grapevine yellows disease, associated with phytoplasma strains related to ‘ Candidatus Phytoplasma solani’, that causes severe losses to viticulture in the Euro-Mediterranean basin. Due to the complex ecological cycle of its etiological agent, BN epidemiology is only partially known, and no effective control strategies have been developed. Numerous studies have focused on molecular characterization of BN phytoplasma strains, to identify molecular markers useful to accurately describe their genetic diversity, geographic distribution and host range. In the present study, a multiple gene analysess were carried out on 16S rRNA , tuf , vmp1 , and stamp genes to study the genetic variability among 18 BN phytoplasma strains detected in diverse regions of the Republic of Macedonia. Restriction fragment length polymorphism (RFLP) assays showed the presence of one 16S rRNA (16SrXII-A), two tuf ( tuf -type a, tuf -type b), five vmp1 (V2-TA, V3, V4, V14, V18), and three stamp (S1, S2, S3) gene patterns among the examined strains. Based on the collective RFLP patterns, seven genotypes (Mac1 to Mac7) were described as evidence for genetic heterogeneity, and highlighting their prevalence and distribution in the investigated regions. Phylogenetic analyses on vmp1 and stamp genes underlined the affiliation of Macedonian BN phytoplasma strains to clusters associated with distinct ecologies.

First Report of Candidatus Phytoplasma solani Associated with Potato Plants in Greece.

In August 2013, potato plants (Solanum tuberosum) cv. Banba displaying symptoms resembling those caused by Candidatus Phytoplasma solani (potato stolbur phytoplasma) were observed in a 2-ha field in the area of the Peripheral Unit of Drama (northern Greece). The plants were 10 weeks old and their symptoms included reddening and upward rolling of leaflets, reduced size of leaves, shortened internodes, and aerial tuber formation. Incidence of affected plants was estimated to be 40% in the field. Four symptomatic potato plants were collected for laboratory testing of possible phytoplasma infection. From each of these four plants, total DNA was extracted from mid veins of reddish leaflets from apical shoot parts and of leaflets emerging from aerial tubers, using a phytoplasma enrichment procedure (1). A nested PCR using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (3) amplified the expected ~1.2-kb 16S rDNA fragment in all four symptomatic potato plants. No amplification was observed with DNA similarly extracted from leaflets of asymptomatic potato plants of the same variety collected from an apparently healthy crop. One of the four 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced by Beckman Coulter Genomics (United Kingdom). At least twofold coverage per base position of the cloned PCR product was achieved. BLAST analysis showed that the obtained sequence of the PCR 16S rDNA product was: i) 100% identical to several GenBank sequences of Ca. P. solani strains, including strains detected previously in Greece infecting tomato (GenBank Accession No. JX311953) and Datura stramonium (HE598778 and HE598779), and ii) 99.7% similar to that of the Ca. P. solani reference strain STOL11 (AF248959). Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup (AF248959). The sequence of this PCR product was deposited in NCBI GenBank database under the accession no. KJ810575. The presence of the stolbur phytoplasma in all four symptomatic potato plants examined was further confirmed by nested PCR using the stolbur-specific STOL11 primers (3) targeting non-ribosomal DNA. Based on the observed symptoms in the field and laboratory molecular examinations, we concluded that the potato plants were infected by a Ca. P. solani related strain. The stolbur disease has been previously reported in Greece affecting tomato (2,5) and varieties of D. stramonium (4). To our knowledge, this is the first report of a Ca. P. solani related strain infecting a potato crop in Greece. As northern Greece is a center of potato production, the source of this pathogen is to be investigated.

Polymorphism in mtDNA control region of Mizo-Mongloid Breast Cancer samples as revealed by PCR-RFLP analysis

Mutations in mitochondrial D-loop region of DNA (mtDNA) may serve as a potential sensor for cellular DNA damage and marker for cancer development. We investigated the restriction fragment length polymorphism (RFLP) pattern of the D-loop region in the blood samples of breast cancer patients among Mizoram population. Significant differences were observed among breast cancer and healthy blood samples in the RFLP pattern using AluI, HaeIII and RsaI enzymes. Polymorphic information content (PIC – 0.258), band informativeness (∑Ib – 3.283) and marker index (MI – 0.006) were highest in the case of RsaI enzyme. Our data suggest that the RsaI polymorphic site in the mitochondrial control region is an informative marker for breast cancer development in Mizo population.

Application of dna techniques for the identification of two entomopathogenic nematode genera Steinernema and Heterorhabditis from Vietnam

Entomopathogenic nematodes (epn), e.g. Steinernema Travassos, 1927 and Heterorhabditis Poinar, 1975 belong to one of the most important groups among biological agents. Their morphological identification to species level is very difficult. Since they are often homomorphic in the adult stage of the first generation, but they are strongly polymorphic and variable among different generations that are usually occurred even in the same species. Therefore, the molecular techniques have become useful tools for the identification and taxonomy of these nematodes. The DNA techniques such as polymerise chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing have been employed for the molecular characterization of entomopathogenic nematode isolates collected from Vietnam. Based on the morphological diagnoses, morphometrics and genetic analyses of 18S-rDNA of collected isolates from Vietnam were revealed 7 species of two genera Steinernema and Heterorhabditis so far. Of these, four species viz. Steinernema tami Pham et al., 2000, S. sangi Phan et al., 2001, S. loci Phan et al., 2001, and S. thanhi Phan et al., 2001 were described as new species for science. The results of the rDNA digestion with seventeen endoenzymes, e.g. Alu I, Mva I, Dde I, EcoR I, Hae III, Cfo I, Hind III, Hinf I, Msp I, Kpn I, Pst, Pvu II, Rsa I, Sal I, Nde II, Bsiz I and Xba I RFLP shown a significant difference in RFLP pattern between three isolates S-TK10, S-TG10, S-TX1 and that of S. Kraussei (Steiner, 1923) Travassos, 1927. Apparently, the complete differences which were occurred in six RFLP patterns digested by seven enzymes Alu I, Dde I, Cfo I, Hinf I, Msp I, Rsa I, Nde II. were not only by the band number but are also by the size (bp) of the DNA fragment lengths, inspite of five other enzymes were not digested any more. These might be documented the genetic similarly within the S. kraussei group. Along with some morphological characters and morphometrics, these genetic characterizations were strongly support to reveal three new species of three correspondent isolates. Based on the sequencing of 18S according ITS (Internal Transcribed Spacer) of DNA ribosome, the paper gives also the phylogenic tree that shown the related relationship and degree of genetic distance of species among each genus of Steinernema and Heterorhabditis and that were also significantly distinguished to Meloidogyne chitwoodi Golden, O’ Bannon, Santo &amp; Finley, 1980.

A comparative study on morphological versus molecular identification of dermatophyte isolates

Dermatophytes are taxonomically classified in the genera Trichophyton, Microsporum, and Epidermophyton. Pleomorphism, cultural variability, slow growth and sporulation, and the need for additional physiological tests make dermatophytes notoriously difficult to identify. The present study aimed to compare the results of morphological and molecular identification of certain groups of clinical isolates of dermatophytes with a view to evaluating the accuracy of molecular methods. For each sample, the ITS1-5.8S-ITS2 rDNA region was amplified using the primers ITS1 and ITS4. PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using the enzyme MvaI and isolate identification was performed by comparing the electrophoretic RFLP patterns with reference profiles obtained previously. Finally, paired comparative analyses of molecular and conventional methods were performed. While morphology results from routine daily reports of the laboratories indicated that 18 (6.8%) and 136 (52.10%) of the isolates were T.rubrum and T.interdigitale, respectively, PCR-RFLP results suggested that T.rubrum was the most common etiological agent of ringworm accounting for 94 (36.01%), followed by T.interdigitale accounting for 71 (27.20%). Interestingly, 80.8% out of the 94 isolates identified as T.rubrum by molecular testing had been identified by morphological examination as belonging to different species, such as T.interdigitale (75.5%), E.floccosum (2.1%) and M.canis, T.verrucosum, and T.tonsurans (each 1.06%). Ten strains out of 261 (T.interdigitale, n=8; E.floccosum, n=2) had been defined as unknown species by morphological tests. An unexpected high percent of isolates identified as T.interdigitale by conventional methods were in effect T.rubrum shown by PCR-RFLP, and regarding the necessity of correct identification of dermatophytes recovered from different clinical forms of the infection, we highly recommend ITS-sequencing or ITS-RFLP of the isolates, particularly for epidemiological research studies.

Genotypic characterization of Malaysian human isolates of Streptococcus pneumoniae from carriage and clinical sources.

This study characterized carriage and clinical pneumococcal isolates for serotypes, penicillin susceptibility, virulence genes and restriction fragment length polymorphism (RFLP) pattern of penicillin binding protein (PBP) genes. DNA fingerprint of isolates was generated by BOX-PCR. Majority of serotypes were 23F followed by 19F, 19A and 6A. Twenty-four percent of isolates were penicillin non-susceptible (PNSP). All of the targeted virulence genes were detected in all isolates with the exception of pili; 20.6% (n=22) for PI-1 and 14.0% (n=15) for PI-2. Of the 13 isolates which carried both PI-1 and PI-2, 10 were of clinical origin. Digested pbp-DNA produced three PBP-RFLP profiles for pbp1a (A1 to A3), six profiles for pbp2b (B1 to B6) and seven for pbp2x (X1 to X7) mostly in PNSPs. Based on BOX-PCR analysis, the majority of isolates were genetically diverse with a small number of potentially related isolates carrying pili genes. No obvious genotypic association was observed pertaining to carriage and clinical origin of isolates.

Recovery of plasmid pEMB1, whose toxin-antitoxin system stabilizes an ampicillin resistance-conferring β-lactamase gene in Escherichia coli, from natural environments.

Non-culture-based procedures were used to investigate plasmids showing ampicillin resistance properties in two different environments: remote mountain soil (Mt. Jeombong) and sludge (Tancheon wastewater treatment plant). Total DNA extracted from the environmental samples was directly transformed into Escherichia coli TOP10, and a single and three different plasmids were obtained from the mountain soil and sludge samples, respectively. Interestingly, the restriction fragment length polymorphism pattern of the plasmid from the mountain soil sample, designated pEMB1, was identical to the pattern of one of the three plasmids from the sludge sample. Complete DNA sequencing of plasmid pEMB1 (8,744 bp) showed the presence of six open reading frames, including a β-lactamase gene. Using BLASTX, the orf5 and orf6 genes were suggested to encode a CopG family transcriptional regulator and a plasmid stabilization system, respectively. Functional characterization of these genes using a knockout orf5 plasmid (pEMB1ΔparD) and the cloning and expression of orf6 (pET21bparE) indicated that these genes were antitoxin (parD) and toxin (parE) genes. Plasmid stability tests using pEMB1 and pEMB1ΔparDE in E. coli revealed that the orf5 and orf6 genes enhanced plasmid maintenance in the absence of ampicillin. Using a PCR-based survey, pEMB1-like plasmids were additionally detected in samples from other human-impacted sites (sludge samples) and two other remote mountain soil samples, suggesting that plasmids harboring a β-lactamase gene with a ParD-ParE toxin-antitoxin system occurs broadly in the environment. This study extends knowledge about the dissemination and persistence of antibiotic resistance genes in naturally occurring microbial populations.

Understanding the haplotypic diversity and distribution of malaria-protective β-globin mutations

Background Sickle cell (HbS) and b-thalassaemia, two common malaria-protective b-globin mutations, represent impressive examples of human adaptation to infectious disease. Highly prevalent in Sub-Saharan Africa and the Mediterranean, respectively, these mutations display contrasting patterns in their haplotype structure and distribution. The majority of HbS is associated with five major restriction fragment length polymorphism patterns that exhibit remarkable geographical specificity. By contrast, b-thalassaemia, which can result from several different mutations, is found on many more haplotypes whose distributions considerably overlap. Here, we shed light on the evolutionary and demographic factors that likely gave rise to such differences, despite malaria being a common selective force.

The First Report of Stolbur Phytoplasma Associated with Phyllody of Calendula officinalis in Serbia.

Pot marigold (Calendula officinalis L.) is native to southern Europe. Compounds of marigold flowers exhibit anti-inflammatory, anti-tumor-promoting, and cytotoxic activities (4). In Serbia, pot marigold is cultivated as an important medicinal and ornamental plant. Typical phyllody, virescence, proliferation of axillary buds, and witches' broom symptoms were sporadically observed in 2011 in Pancevo plantation, Serbia (44°51'49″ N, 20°39'33″ E, 80 m above sea level). Until 2013, the number of uniformly distributed affected pot marigold plants reached 20% in the field. Due to the lack of seed production, profitability of the cultivation was seriously affected. Leaf samples from 10 symptomatic and 4 symptomless marigold plants were collected and total nucleic acid was extracted from midrib tissue (3). Direct PCR and nested PCR were carried out with primer pairs P1/16S-SR and R16F2n/R16R2n, respectively (3). Amplicons 1.5 and 1.2 kb in length, specific for the 16S rRNA gene, were amplified in all symptomatic plants. No PCR products were obtained when DNA isolated from symptomless plants was used. Restriction fragment length polymorphism (RFLP) patterns of the 1.2-kb fragments of 16S rDNA were determined by digestion with four endonucleases separately (TruI1, AluI, HpaII, and HhaI) and compared with those of Stolbur (Stol), Aster Yellows (AY), Flavescence dorée-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas (2). RFLP patterns from all symptomatic pot marigold plants were identical to the Stol pattern, indicating Stolbur phytoplasma presence in affected plants. The 1.2-kb amplicon of representative Nv8 strain was sequenced and the data were submitted to GenBank (accession no. KJ174507). BLASTn analysis of the sequence was compared with sequences available in GenBank, showing 100% identity with 16S rRNA gene of strains from Paeonia tenuifolia (KF614623) and corn (JQ730750) from Serbia, and peach (KF263684) from Iran. All of these are members of the 16SrXII 'Candidatus Phytoplasma solani' group, subgroup A (Stolbur). Phytoplasmas belonging to aster yellows (16SrI) (Italy and Canada) and peanut witches' broom related phytoplasma (16SrII) group (Iran) have been identified in diseased pot marigold plants (1). To our knowledge, this is the first report of natural infection of pot marigold by Stolbur phytoplasma in Serbia.

A New Disease of Cherry Plum Tree with Yellow Leaf Symptoms Associated with a Novel Phytoplasma in the Aster Yellows Group

A novel phytoplasma was detected in a cherry plum (Prunus cerasifera Ehrh) tree that mainly showed yellow leaf symptom. The tree was growing in an orchard located in Yangling District, Shaanxi Province, China. The leaves started as chlorotic and yellowing along leaf minor veins and leaf tips. Chlorosis rapidly developed to inter-veinal areas with the whole leaf becoming pale yellow in about 1-4 wk. Large numbers of phytoplasma-like bodies (PLBs) were seen under transmission electron microscopy. The majority of the PLBs was spherical or elliptical vesicles, with diameters in range of 0.1-0.6 µm, and distributed in the phloem cells of the infected tissues. A 1 246-bp 16S ribosomal RNA (rRNA) gene fragment was amplified from DNA samples extracted from the yellow leaf tissues using two phytoplasma universal primer pairs R16mF2/R16mR1 and R16F2n/R16R2. Phylogenetic analysis using the 16S rRNA gene sequence suggested that the phytoplasma associated with the yellow leaf symptoms belongs to a novel subclade in the aster yellows (AY) group (16SrI group). Virtual and actual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene fragment revealed that the phytoplasma was distinguishable from all existing 19 subgroups in the AY group (16SrI) by four restriction sites, Hinf I, Mse I, Sau3A I and Taq I. The similarity coefficients of comparing the RFLP pattern of the 16S rRNA gene fragment of this phytoplasma to each of the 19 reported subgroups ranged from 0.73 to 0.87, which indicates the phytoplasma associated with the cherry plum yellow leaf (CPYL) symptoms is probably a distinct and novel subgroup lineage in the AY group (16SrI). In addition, the novel phytoplasma was experimentally transmitted to periwinkle (Catharanthus roseus) plants from the tree with CPYL symptoms and then back to a healthy 1-yr-old cherry plum tree via dodder (Cuscuta odorata) connections.

First Report on the Association of a 16SrII-A Phytoplasma with Sesame (Sesamum indicum) Exhibiting Abnormal Stem Curling and Phyllody in Taiwan.

Sesame (Sesamum indicum L.), an annual plant, is grown as an oilseed crop and the seeds are used in bakery products in Taiwan. In June 2013, plants exhibiting symptoms including phyllody and abnormal stem curling were observed in sesame fields in Pitou Township, Changhua County, Taiwan. Incidence of infected plants was estimated to be greater than 90% within a single field. Phytoplasmas associated with sesame exhibiting phyllody, witches'-broom, or virescence have been classified as strains of 16SrI-B in Myanmar (GenBank Accession No. AB558132), 16SrII-A in Thailand (JN006075), 16SrII-D in Oman (EU072505) and India (KF429486), 16SrIV-C in Iran (JF508515), and 16SrVI-A (KF156894) and 16SrIX (KC139791) in Turkey (1). Three symptomatic and four asymptomatic plants were uprooted and transplanted in a greenhouse for further study. Transmission electron microscopy (TEM) revealed clusters of phytoplasma cells ranging from 300 to 800 nm in diameter only in phloem sieve elements of stems of three symptomatic and two asymptomatic plants. Comparable tissues from two other symptomless plants were devoid of phytoplasma cells. Total DNA was extracted with a modified CTAB method (2) from plant tissues (100 mg each) including stem, leaf, petiole, and root from the same plants used for TEM work. Analyses by a nested PCR using universal primer pairs P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT/5'-CGTCCTTCATCGGCTCTT) followed by R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG/5'-TGACGGGCGGTGTGTACAAACCCCG) were performed to detect putative phytoplasma DNA (3). Each primer pair amplified a single PCR product of either 1.8 or 1.2 kb, respectively, only from the three symptomatic and two asymptomatic plant tissues that had phytoplasma cells in their sieve elements. It is likely that these two asymptomatic plants were in the early stage of infection before symptoms became noticeable. The nested PCR products (1.2 kb) amplified from the symptomatic plants were cloned separately and sequenced (GenBank Accession Nos. KF923391, KF923392, and KF923393). BLAST analysis of the sequences revealed that they shared 99.2% sequence identity with strains reported from India and Thailand (KF429486 and JN006075), which were classified to the 16SrII-D and 16SrII-A subgroups, respectively. Moreover, iPhyClassifier software (4) was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile. The 16S rDNA sequences shared 99.4% identity with that of the 'Candidatus Phytoplasma australasiae' (Y10097) and the RFLP patterns were identical to that of the 16SrII-A subgroup, indicating the Taiwanese strain is a 'Ca. P. australasiae'-related strain. To our knowledge, this is the first report of a 16SrII-A subgroup phytoplasma causing phyllody and abnormal stem curling on sesame in Taiwan. The occurrence of phytoplasma on sesame could have direct implications for the cultivation of this economically important oilseed plant and the bakery industry in Taiwan.