scholarly journals Genomic Markers for Differentiation of Francisella tularensis subsp. tularensis A.I and A.II Strains

2007 ◽  
Vol 74 (1) ◽  
pp. 336-341 ◽  
Author(s):  
Claudia R. Molins-Schneekloth ◽  
John T. Belisle ◽  
Jeannine M. Petersen
Keyword(s):  
Suppression Subtractive Hybridization ◽  
Francisella Tularensis ◽  
Geographical Location ◽  
Subtractive Hybridization ◽  
Clinical Isolates ◽  
Type B ◽  
Tularensis Subsp ◽  
Genomic Markers ◽  
Pcr Assays ◽  
Genomic Regions

ABSTRACT Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.

scholarly journals Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b

2004 ◽  
Vol 186 (12) ◽  
pp. 3938-3950 ◽  
Author(s):  
David DeShazer
Keyword(s):  
Suppression Subtractive Hybridization ◽  
Burkholderia Pseudomallei ◽  
Genomic Sequence ◽  
Subtractive Hybridization ◽  
Etiologic Agent ◽  
Polyclonal Antiserum ◽  
Genomic Diversity ◽  
Clinical Isolates ◽  
Immunogold Electron Microscopy ◽  
Burkholderia Mallei

ABSTRACT Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated φ1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage φ1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the φ1026b genome was revealed by comparison with bacteriophage φE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The φ1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in φE125. On the other hand, φ1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against φE125 reacted with the tail of φ1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.

scholarly journals Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

2001 ◽  
Vol 67 (10) ◽  
pp. 4781-4788 ◽  
Author(s):  
Maria Dahlenborg ◽  
Elisabeth Borch ◽  
Peter Rådström
Keyword(s):  
Sample Preparation ◽  
Fecal Sample ◽  
Clostridium Botulinum ◽  
Geographical Location ◽  
Type B ◽  
Fecal Samples ◽  
Pcr Assays ◽  
Combined Selection ◽  
Spore Load ◽  
Slaughtered Pigs

ABSTRACT A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymeraserTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 103 spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

Suppression subtractive hybridization identifies bacterial genomic regions that are possibly involved in hBD-2 regulation by enterocytes

2011 ◽  
Vol 55 (10) ◽  
pp. 1533-1542 ◽  
Author(s):  
Darab Ghadimi ◽  
Mohamed Hassan ◽  
Patrisio Njiru Njeru ◽  
Michael de Vrese ◽  
Arnold Geis ◽  
...  
Keyword(s):  
Suppression Subtractive Hybridization ◽  
Subtractive Hybridization ◽  
Genomic Regions

scholarly journals In Vitro Antimicrobial Susceptibilities of Francisella tularensis subsp. holarctica Isolates from Tularemia Outbreaks That Occurred from the End of the 20th Century to the 2020s in Spain

Antibiotics ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 938
Author(s):  
Sonia Martínez-Martínez ◽  
Elías-Fernando Rodríguez-Ferri ◽  
David Rodríguez-Lázaro ◽  
Marta Hernández ◽  
José-Ignacio Gómez-Campillo ◽  
...  
Keyword(s):  
20Th Century ◽  
Francisella Tularensis ◽  
Antimicrobial Agents ◽  
Clinical Isolates ◽  
Tularensis Subsp ◽  
Microdilution Method ◽  
Castilla Y Leon ◽  
Broth Microdilution Method ◽  
Northwestern Spain

A collection of 177 Francisella tularensis subsp. holarctica clinical isolates (29 from humans and 148 from animals, mainly hares and voles) was gathered from diverse tularemia outbreaks in the Castilla y León region (northwestern Spain) that occurred from the end of the 20th century to the 2020s. Along with four F. tularensis subsp. holarctica reference strains, all of these clinical isolates were tested using a broth microdilution method to determine their susceptibility to 22 antimicrobial agents, including β-lactams, aminoglycosides and one member each of the tetracycline, glycylcycline, quinolone and sulphonamide classes. Many multi-resistance profiles were found among the tested isolates, but especially among those of human origin (all but two isolates showed resistance to at least 13 of 18 antimicrobial agents). Even so, all human isolates were susceptible to gentamicin and tobramycin, while more than 96% of animal isolates were susceptible to these two aminoglycosides. Ciprofloxacin showed activity against more than 92% of animal and human isolates. However, almost 21% of human isolates were resistant to tetracycline, and more than 65% were resistant to tigecycline. Finally, a quite similar activity to other F. tularensis subsp. holarctica isolates collected 20 years earlier in Spain was observed.

scholarly journals Presence of Francisella tularensis subsp. holarctica DNA in the Aquatic Environment in France

2021 ◽  
Vol 9 (7) ◽  
pp. 1398
Author(s):  
Camille D. Brunet ◽  
Aurélie Hennebique ◽  
Julien Peyroux ◽  
Isabelle Pelloux ◽  
Yvan Caspar ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Water Samples ◽  
Real Time Pcr ◽  
Aquatic Environment ◽  
Geographic Area ◽  
The West ◽  
Tularensis Subsp ◽  
High Prevalence ◽  
Sources Of Infection ◽  
Pcr Assays

In 2018, the incidence of tularemia increased twofold in the west of France, with many pneumonic forms, suggesting environmental sources of infection. We investigated the presence of Francisella tularensis subsp. holarctica and other Francisella species DNA in the natural aquatic environment of this geographic area. Two sampling campaigns, in July 2019 and January 2020, allowed the collection of 87 water samples. Using a combination of real-time PCR assays, we tested the presence of either Francisella sp., F. tularensis/F. novicida, and F. tularensis subsp. holarctica, the latter being the only tularemia agent in Europe. Among 57 water samples of the first campaign, 15 (26.3%) were positive for Francisella sp., nine (15.8%) for F. tularensis and/or F. novicida, and four (7.0%) for F. tularensis subsp. holarctica. Ratios were 25/30 (83.3%), 24/30 (80.0%), and 4/30 (13.3%) for the second campaign. Among the thirty sites sampled during the two campaigns, nine were positive both times for Francisella sp., seven for F. tularensis and/or F. novicida, and one for F. tularensis subsp. holarctica. Altogether, our study reveals a high prevalence of Francisella sp. DNA (including the tularemia agent) in the studied aquatic environment. This aquatic environment could therefore participate in the endemicity of tularemia in the west of France.

scholarly journals Development of PCR Assays for Species- and Type-Specific Identification of Pasteurella multocidaIsolates

1998 ◽  
Vol 36 (4) ◽  
pp. 1096-1100 ◽  
Author(s):  
Kirsty M. Townsend ◽  
Alan J. Frost ◽  
Chiang W. Lee ◽  
John M. Papadimitriou ◽  
Hugh J. S. Dawkins
Keyword(s):  
Pasteurella Multocida ◽  
Pcr Amplification ◽  
Subtractive Hybridization ◽  
Type B ◽  
Amplification Product ◽  
Specific Detection ◽  
Specific Identification ◽  
Dna Fragment ◽  
Pcr Assays ◽  
Sensitive Method

Genomic subtractive hybridization of closely relatedPasteurella multocida isolates has generated clones useful in distinguishing hemorrhagic septicemia-causing type B strains from other P. multocida serotypes. Oligonucleotide primers designed during the sequencing of these clones have proved valuable in the development of PCR assays for rapid species- and type-specific detection of P. multocida and of type B:2 in particular. This study demonstrated that the primer pair designed from the sequence of the clone 6b (KTT72 and KTSP61) specifically amplified a DNA fragment from types B:2, B:5, and B:2,5P. multocida and that the primers KMT1T7 and KMT1SP6 produced an amplification product unique to all P. multocida isolates analyzed. It was also shown that PCR amplification performed directly on bacterial colonies or cultures represents an extremely rapid, sensitive method of P. multocida identification.

scholarly journals Reorganized Genomic Taxonomy of Francisellaceae Enables Design of Robust Environmental PCR Assays for Detection of Francisella tularensis

2021 ◽  
Vol 9 (1) ◽  
pp. 146
Author(s):  
Caroline Öhrman ◽  
Jason W. Sahl ◽  
Andreas Sjödin ◽  
Ingrid Uneklint ◽  
Rebecca Ballard ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Environmental Samples ◽  
Multiple Sources ◽  
Phylogenetic Structure ◽  
Specific Pcr ◽  
Comprehensive Collection ◽  
The Family ◽  
Genomic Taxonomy ◽  
Pcr Assays ◽  
Genomic Regions

In recent years, an increasing diversity of species has been recognized within the family Francisellaceae. Unfortunately, novel isolates are sometimes misnamed in initial publications or multiple sources propose different nomenclature for genetically highly similar isolates. Thus, unstructured and occasionally incorrect information can lead to confusion in the scientific community. Historically, detection of Francisella tularensis in environmental samples has been challenging due to the considerable and unknown genetic diversity within the family, which can result in false positive results. We have assembled a comprehensive collection of genome sequences representing most known Francisellaceae species/strains and restructured them according to a taxonomy that is based on phylogenetic structure. From this structured dataset, we identified a small number of genomic regions unique to F. tularensis that are putatively suitable for specific detection of this pathogen in environmental samples. We designed and validated specific PCR assays based on these genetic regions that can be used for the detection of F. tularensis in environmental samples, such as water and air filters.

scholarly journals Genomic Regions Conserved in Lineage II Escherichia coli O157:H7 Strains

2009 ◽  
Vol 75 (10) ◽  
pp. 3271-3280 ◽  
Author(s):  
Marina Steele ◽  
Kim Ziebell ◽  
Yongxiang Zhang ◽  
Andrew Benson ◽  
Roger Johnson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Suppression Subtractive Hybridization ◽  
Subtractive Hybridization ◽  
Clinical Disease ◽  
Escherichia Coli O157 ◽  
Genomic Features ◽  
Waterborne Pathogen ◽  
E Coli ◽  
Primer Sets ◽  
Genomic Regions

ABSTRACT Populations of the food- and waterborne pathogen Escherichia coli O157:H7 are comprised of two major lineages. Recent studies have shown that specific genotypes within these lineages differ substantially in the frequencies with which they are associated with human clinical disease. While the nucleotide sequences of the genomes of lineage I strains E. coli O157 Sakai and EDL9333 have been determined, much less is known about the genomes of lineage II strains. In this study, suppression subtractive hybridization (SSH) was used to identify genomic features that define lineage II populations. Three SSH experiments were performed, yielding 1,085 genomic fragments consisting of 811 contigs. Bacteriophage sequences were identified in 11.3% of the contigs, 9% showed insertions and 2.3% deletions with respect to E. coli O157:H7 Sakai, and 23.2% did not have significant identity to annotated sequences in GenBank. In order to test for the presence of these novel loci in lineage I and II strains, 27 PCR primer sets were designed based on sequences from these contigs. All but two of these PCR targets were found in the majority (51.9% to 100%) of 27 lineage II strains but in no more than one (<6%) of the 17 lineage I strains. Several of these linage II-related fragments contain insertions/deletions that may play an important role in virulence. These lineage II-related loci were also shown to be useful markers for genotyping of E. coli O157:H7 strains isolated from human and animal sources.

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