Evaluation of molecular techniques to biotype Giardia duodenalis collected during an outbreak

Twenty-seven Giardia duodenalis cyst-positive specimens (human, animal, or drinking water) were obtained from a waterborne outbreak in a community in British Columbia, western Canada. Parasite isolates were characterized using molecular techniques at 4 different steps of organism retrieval. None of the drinking water samples (n = 20) infected gerbils and none was successfully amplified using polymerase chain reaction (PCR). We were able to genotype 4 of 7 (human and animal) isolates by amplification of DNA from original specimens at the triosephosphate isomerase (tpi) gene locus using PCR followed by restriction fragment length polymorphism (RFLP) analysis. Five of the original specimens inoculated into Mongolian gerbils (Meriones unguiculatus) were infective and genotyped at the tpi locus using parasite material collected from the gerbil (cysts and trophozoites). Pulsed field gel electrophoresis (PFGE) was used to biotype trophozoites collected from the gerbils as well as trophozoites from the 4 isolates that adapted to culture. Four of these 5 isolates displayed the same (designated outbreak) biotype at all parasite retrieval steps with all molecular techniques including the originally amplified isolates. PCR-RFLP identified an additional biotype group. The 4 isolates that adapted to in vitro culture were also characterized by isoenzyme electrophoresis (IE). Biotype groups identified in these axenized isolates were all the same with each molecular technique (PCR-RFLP, PFGE, IE) tested. Results of this study demonstrate a need for more sensitive molecular methods to detect and characterize Giardia in original host and environmental samples. Results are also consistent with evidence of biotype changes that occur during the presently used process of isolate retrieval.     

Morphological and molecular characterization of <Emphasis Type="Italic">Calothrix</Emphasis> isolates obtained from diverse environments in India

Thirty cyanobacterial strains of Calothrix (family Rivulariaceae) isolated from diverse geographical regions of India were analyzed using morphological and molecular approaches. Most of the isolates were planktonic while some grew benthically. Significant differences were observed with regard to the shape and size of the vegetative cells, heterocysts, and akinetes. Analyses of molecular polymorphisms using Restriction Fragment Length Polymorphisms (RPLP) of 16S rRNA genes with the reference strain led to unambiguous differentiation of the isolates as well as understanding of their genetic relationships.     

A multiple typing approach is recommended for the laboratory-based surveillance of salmonellosis in Romania

In Romania, Salmonella enterica serovar Typhimurium isolates are currently typed by antimicrobial resistance profiles and phage typing, as part of the national laboratory-based surveillance system of human enteric infections. The aim of the present study was to assess the added value of complementing this approach with molecular fingerprinting, namely pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeats analysis (MLVA). Thirty-six S. Typhimurium isolates received by the Reference Center for Human Salmonella Infections for confirmation and typing from the Microbiology Departments of three Public Health Authorities, were selected for this study. Phage typing revealed that 14 isolates (39%) were nontypeable (NT). Twenty-two isolates were assigned to 5 phage types: DT193 (11 isolates), U302 (7 isolates), DT116 (2 isolates), DT41 (1 isolate) and DT86 (1 isolate). Antimicrobial susceptibility testing showed that all the NT and DT116 isolates were multidrug resistant and extended-spectrum betalactamase producers. All the examined isolates were typeable when using the molecular approach. Both methods gave conclusive and comparable results, documenting the genetic relatedness and discriminating the outbreak isolates from sporadic cases. We conclude that in order to improve outbreak investigation and surveillance of salmonellosis in Romania, the current routine typing of Salmonella isolates should be complemented with at least one of these DNA fingerprinting methods.     

Comparative virulence phenotypes and molecular genotypes of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen in China and the United States

Stripe rust (yellow rust) of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases in both China and the United States. The Chinese and US populations of the stripe rust fungus were compared for their virulence phenotypes on wheat cultivars used to differentiate races of the pathogen in China and the US and molecular genotypes using simple sequence repeat (SSR) markers. From 86 Chinese isolates, 54 races were identified based on reactions on the 17 Chinese differentials and 52 races were identified based on the 20 US differentials. The selected 51 US isolates, representing 50 races based on the US differentials, were identified as 41 races using the Chinese differentials. A total of 132 virulence phenotypes were identified from the 137 isolates based on reactions on both Chinese and US differentials. None of the isolates from the two countries had identical virulence phenotypes on both sets of differentials. From the 137 isolates, SSR markers identified 102 genotypes, of which 71 from China and 31 from the US. The virulence data clustered the 137 isolates into 20 virulence groups (VGs) and the marker data clustered the isolates into seven molecular groups (MGs). Virulence and SSR data had a low (r = 0.34), but significant (P = 0.01) correlation. Principal component analyses using either the virulence data or the SSR data separated the isolates into three groups: group a consisting of only Chinese isolates, group b consisting of both Chinese and US isolates and group c consisting of mostly US isolates. A neighbour-joining tree generated using the molecular data suggested that the P. striiformis f. sp. tritici populations of China and the US in general evolved independently.     

Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas

Cryptococcus gattii has recently emerged as a pathogen of humans and animals in the temperate climate of Vancouver Island, British Columbia (B.C.). The majority (approximately 95%) of the isolates from the island belong to the VGII molecular type, and the remainder belong to the VGI molecular type. The goals of this study were to compare patterns of molecular variation among C. gattii isolates from B.C. with those from different areas of the world and to investigate the population structure using a comparative gene genealogy approach. Our results indicate that the C. gattii population in B.C. comprises at least two divergent lineages, corresponding to previously identified VGI and VGII molecular types. The genealogical analysis of strains suggested a predominantly clonal population structure among B.C. isolates, while there was evidence for sexual recombination between different molecular types on a global scale. We found no geographic pattern of strain relationships, and nucleotide sequence comparisons revealed that genotypes among isolates from B.C. were also present among isolates from other areas of the world, indicating extensive strain dispersal. The nucleotide sequence diversity among isolates from B.C. was similar to that among isolates from other areas of the world.     

Molecular characterization and distribution of virulence-associated genes amongst Aeromonas isolates from Libya

AIMS: The aim of the study was to type 52 Aeromonas spp. isolates from chicken carcasses, children with diarrhoea and a hospital environment in Libya, and to determine the distribution of putative virulence genes amongst them. METHODS AND RESULTS: Macrorestriction analysis using pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of 16S rRNA and aroA genes were used to type the isolates. Whereas 30 of 32 chicken isolates were identified as Aeromonas veronii, eight of 12 environmental isolates were Aer. caviae. Three species were identified amongst the eight isolates from children. Aeromonas veronii and Aer. caviae isolates could be divided into eight and five PFGE types, respectively. All species could be further subtyped into one of 21 aroA PCR-RFLP groups. Aerolysin-like haemolysin or enterotoxin gene sequences were detected in all the isolates. Overall carriage rates for hlyA and alt were 77 and 75%, respectively. CONCLUSIONS: Seven of eight isolates from children were of different subtypes, indicating a lack of any common source of acquisition. Isolates of common molecular type did not share identical distributions of putative virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the effectiveness of using molecular typing to identify and study genetic variation amongst Aeromonas isolates.     

Comparison of molecular and antibiotic resistance profile methods for the population analysis of Bradyrhizobium spp. (TGx) isolates that nodulate the new TGx soybean cultivars in Africa

AIMS: Comparison of molecular and antibiotic resistance profile methods to identify an easy method that can differentiate between strains of introduced Bradyrhizobium japonicum and the indigenous Bradyrhizobium spp. (TGx) isolates which nodulate the newly developed TGx soybean cultivars in Africa. METHODS AND RESULTS: Restriction fragment length polymorphism (RFLP) of 16S rDNA generated by five restriction enzymes, banding patterns in Southern hybridization using nod and nif genes as probes, and resistance patterns of the isolates to nine antibiotics, were used to group 26 Bradyrhizobium spp. (TGx) isolates and four other Bradyrhizobium strains. The clusters of isolates obtained from the four grouping methods were all different, although all methods revealed large genetic diversity among the isolates. CONCLUSIONS: Results indicate that the antibiotic resistance profile method is as good as the three molecular methods used in this study for phylogenetic grouping of the Bradyrhizobium spp. (TGx) isolates, which may serve as a basis for further characterization of selected isolates from each group. SIGNIFICANCE AND IMPACT OF THE STUDY: The antibiotic resistance profile method can be used as a simple means of assessing genetic variability and grouping of a large number of Bradyrhizobium spp. (TGx) isolates. Representative isolates from each group can then be selected for further characterization.     

Natural Populations of Chickpea Rhizobia Evaluated by Antibiotic Resistance Profiles and Molecular Methods

The aims of this study were to investigate the hypothesis that intrinsic antibiotic resistance (IAR) profiles of chickpea rhizobia are correlated with the isolates site of origin, and to compare the discriminating power of IAR profiles with molecular approaches in rhizobial strain identification and differentiation. Rhizobial diversity from five Portuguese soils was assessed by IAR profiles and molecular methods [16S rDNA restriction fragment length polymorphism (RFLP) analysis, direct amplified polymorphic DNA (DAPD) fingerprinting, and SDS–PAGE analysis of protein profiles]. For each analysis, a dendrogram was generated using the software BioNumerics. All three molecular methods generated analogous clustering of the isolates, supporting previous results on 16S rDNA sequence-based phylogeny. Clusters obtained with IAR profile are similar to the species groups generated with the molecular methods used. IAR groups do not correlate significantly with the geographic origin of the isolates. These results may indicate a chromosomal location of antibiotic resistance genes, and suggest that IAR is species related. DAPD and IAR profiles proved to be the most discriminating approaches in strain differentiation and can be used as fast methods to screen diversity in new isolates.     

Characterization and correlation of pathogenicity of Botryodiplodia theobromae isolates,the causal agent of black root rot of mulberry (Morus spp.)

Abstract

The study reports the characterization of 10 isolates of mulberry black root rot causing fungus, Botryodiplodia theobromae obtained from the infected gardens of Karnataka, Andhra Pradesh and Tamil Nadu. The analysis based on cultural, morphological, pathogenicity and molecular markers (RAPD and SSRs) revealed significant variations among the isolates. Based on the disease reaction on susceptible V-1 variety, isolates were grouped as pathogenic (60%), moderate pathogenic (20%) and non-pathogenic (20%). Among all isolates, RAPDs revealed higher marker polymorphism however, based on Shannon’s Information Index (I) SSRs were more informative (0.781) compared to the former (0.444). Stepwise Multiple Regression Analysis (MRA) indicated a total of 5, 5 and 3 molecular markers were found to correlate with disease symptoms. Screening of germplasm using multiple strains of virulent isolates will enhance possibilities of locating diverse resistant genes. Pyramiding of these genes will aid in development of mulberry variety with durable resistance and sustainable sericulture.     

Molecular serotype identification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.     

Comparative Analysis of Different Typing Methods for Helicobacter pylori Clinical Isolates

The goal of the present work was to compare different techniques of molecular typing using as an example clinical isolates of Helicobacter pylori obtained from patients in different regions of Russia. DNA-macroarray genome scanning using individual genes was employed to set up our basic classification of isolates that did or did not contain pathogenicity islands. In parallel, DNA of the same isolates was used in the conventional random amplified polymorphic DNA (RAPD) PCR analysis, and the isolates were also genotyped (cagA, vacA, iceA, and babA status) and their proteomic maps were obtained by means of unidimensional SDS polyacrylamide gel electrophoresis (1D-SDS-PAGE) coupled with identification using peptide mass fingerprinting by MALDI-TOF mass spectrometry. A statistically significant correlation (coefficient of correlation r = 0.25, p = 0.005) was observed between the results of genome scanning and 1D-SDS-PAGE. No correlation was found between RAPD-PCR typing and genome scanning.     

Cryphonectria naterciae: a new species in the Cryphonectria-Endothia complex and diagnostic molecular markers based on microsatellite-primed PCR

In a recent study intended to assess the distribution of Cryphonectria parasitica in Portugal, 22 morphologically atypical orange isolates were collected in the Midwestern regions. Eleven isolates were recovered from Castanea sativa, in areas severely affected by chestnut blight and eleven isolates from Quercus suber in areas with cork oak decline. These isolates were compared with known C. parasitica and Cryphonectria radicalis isolates using an integrated approach comprising morphological and molecular methods. Morphologically the atypical isolates were more similar to C. radicalis than to C. parasitica. Phylogenetic analyses based on internal transcribed spacer (ITS) and β-tubulin sequence data grouped the isolates in a well-supported clade separate from C. radicalis. Combining morphological, cultural, and molecular data Cryphonectria naterciae is newly described in the Cryphonectria-Endothia complex. Microsatellite-primed PCR fingerprinting with (GACA)(4) primer discriminated between C. naterciae, C. radicalis, and C. parasitica.     

Characterization of a Single-Spore Isolate Population of Plasmodiophora brassicae Resulting from a Single Club

The single‐spore isolates ‘e₃’ and ‘e₆’ of Plasmodiophora brassicae with different virulence patterns were distinguished by restriction fragment length polymorphisms in fingerprint‐like patterns and by electrophoretic karyotypes using repetitive fragments as hybridization probes. These molecular tools were used to characterize a set of isolates originated from an infected root, which was inoculated with a mixture of the two single‐spore isolates (e₃ and e₆). Spores harvested from mixed‐infected roots were used to establish 53 new single‐spore isolates. All these single‐spore isolates revealed parental patterns according to their molecular fingerprints and their virulence pattern. No sexual recombination could be detected with repetitive molecular probes. However, one isolate (M₃₆ES49) showed the same fingerprint pattern and virulence pattern but different sizes of small chromosomes than the parental type ‘e₆’, which is taken as an indication of chromosome rearrangement during the infection cycle.     

Acute diarrhea outbreak caused by Shigella flexneri at a school in Madrid,Cundinamarca: phenotypic and genotypic characterization of the isolates

Shigellosis is an acute diarrhoeal disease that is the main cause of morbidity and mortality in developing countries. In 1997, the Colombian Instituto Nacional de Salud Microbiology Group organized a network surveillance program with the country's Public Health Laboratories (PHLs) to monitor the principal etiological agents responsible for acute diarrhoeal disease. In May, 2001, the PHL of the state of Cundinamarca reported a food poisoning outbreak involving an elementary school community. The main goal of the Microbiology Group involvement was to establish the molecular relationships among the isolates from the outbreak by phenotypic and genotypic methods of characterization. Stool cultures were obtained from 22 of 195 affected individuals. The Microbiology Group confirmed the identification of the isolates by biochemical and serological probes. The antimicrobial susceptibilities were tested against the following battery of antibiotics: chloramphenicol, trimehoprim-sulfamethozazole, cefotaxime, gentamicin, ampicillin and ciprofloxacin. The isolates were subjected to pulsed field gel electrophoresis (PFGE) using the following CDC (U.S. Centers for Disease Control) protocols: Xbal restriction enzyme, Shigella sonnei CDC F2353 as the reference standard, and lambda phage as a molecular weight marker. In 15 of 22 (68%) stool cultures Shigella was recovered, all isolates were identified as Shigella flexneri serotype 6 biotype Newcastle with the same antimicrobial susceptibility profile. PFGE showed that 3 (20%) isolates were identical (100% genetic similarity) and the other 12 (80%) were very closely related (genetic similarity between 86-98%). The network system permitted the INS ready access to the isolates and the implementation of the PFGE permitted a quantitative characterization of the clonal relationship among the isolates from the outbreak.     

Genotyping of Mycoplasma bovis isolates using multiple-locus variable-number tandem-repeat analysis

Mycoplasma bovis has been considered an important cause of mastitis, pneumonia, and arthritis in bovines with a highly detrimental economic impact in the livestock industry. Previous epidemiological studies, using different typing techniques showed that the isolates were usually heterogeneous and results were difficult to compare between different laboratories. Reliable and comparable molecular typing techniques using geographically and temporal distinct isolates have never been used. The objective of this study was to use multiple-locus variable-number tandem-repeat analysis (MLVA) for the differentiation of M. bovis isolates. This typing scheme was developed using the sequenced genome of the M. bovis PG45 type strain. Nine tandem-repeat sequences were selected and the genetic diversity of 37 isolates of wide geographic and temporal origins was analyzed. The results were compared to those obtained with random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) for the same isolates. Cluster concordance between techniques was evaluated using Adjusted Rand and Wallace coefficients, and different cutoff levels of similarity were tested. Acceptable values of ≥0.5 for all techniques for the Wallace coefficient were only observed at the most relaxed cutoff level, i.e., 52% for MLVA, 29% for PFGE and 18% for RAPD. The Simpson's index of diversity was 0.91 for MLVA, 0.99 for RAPD analysis and 0.99 for PFGE. This MLVA assay is compatible with simple PCR and agarose gel-based electrophoresis steps as well as with high-throughput automated methods. The molecular typing scheme presented in this study provides a fast, reliable, and cost-effective typing method for surveillance of M. bovis epidemiology.     

Application of different molecular techniques for deciphering genetic diversity among yeast isolates of traditional fermented food products of Western Himalayas

Forty-three yeast isolates derived from various fermented foods, alcoholic beverages and traditional inocula of Western Himalayas were characterized by using traditional and molecular techniques. Traditional characterization identified these isolates as belonging to seven genera and eight species. Twenty-three yeast isolates were identified as Saccharomyces cerevisiae, six as Debaromyces hansenii, five as Issatchenkia orientalis, four as Saccharomyces fermentati, two as Schizosaccharomyces pombe and one each as Endomyces fibuliger, Brettanomyces bruxellensis and Candida tropicalis. The molecular characterization using four marker systems i.e. universal rice primers (URP), randomly amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and delta typing was carried out, which revealed strainal level differences along with geographical origin clustering of various yeast isolates which otherwise could not be revealed through conventional characterization. URP markers were found to be best for revealing the genetic polymorphism hidden among forty-three yeast isolates followed by delta typing, RAPD and ISSR. In the above study, URP 6R and URP 9F were found to be species specific thereby producing specific banding pattern for a specific species.     

Application of RAPD technique to study polymorphism among Bacillus thuringiensis isolates from Jordan

The soil-borne bacterium Bacillus thuringiensis (Bt) is an important biological agent used against human and plant pests and diseases. Seven Jordanian Bt isolates, which have been analysed for toxicity against important pests, were also differentiated through serotyping. In this study, they were analysed at the molecular level using random amplified polymorphic DNA markers. Five more international strains were incorporated in the analysis. The DNA markers used showed high polymorphism among the isolates tested. However, the data did not align completely with earlier serotyping for most isolates. Therefore, it is recommended to engage several analyses (e.g. biochemical and molecular) when classifying newly surveyed Bt isolates in the world.     

Polypeptide analysis of genotypic variants of occluded Heliothis spp. baculoviruses

The structural polypeptides of 12 baculovirus isolates which included nuclear polyhedrosis viruses (NPVs) and granulosis viruses (GVs) obtained from four different species of the insect genus Heliothis collected in different geographical regions of the world were characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The matrix proteins were compared according to their molecular weights and peptide profiles produced after limited proteolysis. Examination of the matrix and virion polypeptide profiles revealed three major polypeptide phenotypes which corresponded to the three baculovirus morphological groups; singly embedded nuclear polyhedrosis viruses (SNPVs), multiply embedded nuclear polyhedrosis viruses (MNPVs), and granulosis viruses (GVs). Enveloped nucleocapsid polypeptide profiles of isolates within each NPV phenotype differed in only one polypeptide whereas the two GV isolates differed by as many as five polypeptides. Nucleocapsid polypeptide profiles of isolates within each of the NPV subgroups were identical while those profiles from the GV nucleocapsids differed slightly in molecular weight of one polypeptide.     

Molecular characterization of Mycobacterium tuberculosis isolates from Izmir, Turkey

In recent years, molecular typing methods have been used in epidemiologic studies of Mycobacterium tuberculosis isolates in various areas of the world. However, there have been few data on this issue in Turkey. We describe the molecular characterization of 56 Mycobacterium tuberculosis isolates recovered from individual patients in Izmir and the surrounding area by three different molecular methods. Isolated M. tuberculosis strains were characterized by IS6110 RFLP, spoligotyping and major genetic group designation. In total, 51 RFLP and 35 spoligopatterns were identified. Fourteen (25%) isolates were indicated as low copy number. Based on three genotypic characterization methods together, five clusters with two isolates each were identified. Most of the isolates (98.2%) were assigned as genetic groups 2 or 3. Only one isolate was identified as Beijing family strain (principal genetic group 1). The shared international clades were found to be Beijing-family, var T1 (ST 37), LAM (Latin-American-Mediterranean) 7 (ST 41), LAM 9 (ST 42), Haarlem 1 (ST 47), Haarlem 3 (ST 50) and T1 (ST 53). In this study, IS6110 RFLP, spoligotyping and major genetic group designation were found to be useful methods for molecular epidemiologic studies.