Genomic Subtractive Hybridization and Selective Capture of Transcribed Sequences Identify a Novel Salmonella typhimurium Fimbrial Operon and Putative Transcriptional Regulator That Are Absent from the Salmonella typhi Genome

ABSTRACT Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogenSalmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi andSalmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowedS. typhi to evolve as a highly invasive, systemic human pathogen.

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Novel Macrolide Resistance Module Carried by the IncP-1β Resistance Plasmid pRSB111, Isolated from a Wastewater Treatment Plant

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00802-06 ◽

2006 ◽

Vol 51 (2) ◽

pp. 673-678 ◽

Cited By ~ 27

Author(s):

Rafael Szczepanowski ◽

Irene Krahn ◽

Nadine Bohn ◽

Alfred Pühler ◽

Andreas Schlüter

Keyword(s):

Wastewater Treatment ◽

Wastewater Treatment Plant ◽

Treatment Plant ◽

Transcriptional Regulator ◽

Macrolide Resistance ◽

Nucleotide Sequence Analysis ◽

Inverted Repeats ◽

Resistance Plasmid ◽

The Individual ◽

High Level

ABSTRACT The macrolide resistance plasmid pRSB111 was isolated from bacteria residing in the final effluents of a wastewater treatment plant. The 47-kb plasmid confers resistance to azithromycin, clarithromycin, erythromycin, roxithromycin, and tylosin when it is carried by Pseudomonas sp. strain B13 and is very similar to prototype IncP-1β plasmid pB3, which was previously isolated from an activated-sludge bacterial community of a wastewater treatment plant. The two plasmids differ in their accessory regions, located downstream of the conjugative transfer module gene traC. Nucleotide sequence analysis of the pRSB111 accessory region revealed that it contains a new macrolide resistance module composed of the genes mphR(E), mph(E), and mrx(E), which putatively encode a transcriptional regulator, a macrolide phosphotransferase, and a transmembrane transport protein, respectively. Analysis of the contributions of the individual genes of the macrolide resistance module revealed that mph(E) and mrx(E) are required for high-level macrolide resistance. The resistance genes are flanked by two insertion sequences, namely, ISPa15 and ISRSB111. Two truncated transposable elements, IS6100 and remnants of a Tn3-like transposon, were identified in the vicinity of ISRSB111. The accessory element of pRSB111 apparently replaced the Tn402-like element present on the sister plasmid, pB3, as suggested by the conservation of Tn402-specific terminal inverted repeats on pRSB111.

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Genomic Subtraction Identifies Salmonella typhimurium Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon, stf, Which Are Absent inSalmonella typhi

Journal of Bacteriology ◽

10.1128/jb.181.18.5652-5661.1999 ◽

1999 ◽

Vol 181 (18) ◽

pp. 5652-5661 ◽

Cited By ~ 36

Author(s):

Melanie Emmerth ◽

Werner Goebel ◽

Samuel I. Miller ◽

Christoph J. Hueck

Keyword(s):

Salmonella Typhimurium ◽

Virulent Strain ◽

Inbred Mice ◽

Subtractive Hybridization ◽

Salmonella Typhi ◽

Virulence Plasmid ◽

Sugar Transporters ◽

Subtraction Procedure ◽

Hybridization Procedure ◽

K 12

ABSTRACT Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon,stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi,Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region betweenfhuB and hemL in Escherichia coliK-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57.

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Suppression of Salmonella Growth by Wild-Type and Large-Plaque Variants of Bacteriophage Felix O1 in Liquid Culture and on Chicken Frankfurters

Journal of Food Protection ◽

10.4315/0362-028x-66.2.220 ◽

2003 ◽

Vol 66 (2) ◽

pp. 220-225 ◽

Cited By ~ 89

Author(s):

JEAN M. WHICHARD ◽

NAMALWAR SRIRANGANATHAN ◽

F. WILLIAM PIERSON

Keyword(s):

Salmonella Typhimurium ◽

Host Range ◽

Liquid Culture ◽

Brain Heart Infusion ◽

Salmonella Typhi ◽

Broad Host Range ◽

Wild Type ◽

Large Plaque ◽

Phage T4 ◽

Plaque Phenotype

The bacteriophage Felix O1, a member of Myoviridae, is specific for, and possesses a broad host range within, the genus Salmonella. This work explores a Felix O1 phage-based intervention for Salmonella enterica serotype Typhimurium DT104 that is potentially applicable at several stages of animal production and processing. A variant of Felix O1 was obtained that produces a larger, clearer plaque phenotype (LP) on Salmonella Typhi than wild-type Felix O1 (WT) does, not unlike r mutants of phage T4. LP exhibited slightly more extensive overall suppression of Salmonella Typhi in brain heart infusion (BHI) broth, as ascertained on the basis of culture turbidity (optical density at 600 nm). Both phage variants suppressed log phase BHI broth cultures containing 8.2 × 106 CFU of Salmonella Typhimurium DT104 per ml. A PFU/CFU ratio of 1.0 was effective for WT and LP, whereas increasing the PFU/CFU ratio to 5.0 did not increase suppression. Untreated Salmonella-contaminated frankfurters were compared with treated samples (PFU/CFU ratio, 1.9 × 104) to test WT and LP for their ability to suppress Salmonella growth on chicken frankfurters contaminated with 300 CFU of Salmonella Typhimurium DT104. Suppression levels of 1.8 and 2.1 log units were achieved with WT and LP, respectively (P = 0.0001), but no difference was found between the performances of the two variants (P = 0.5088).

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Pengaruh ekstrak buah naga daging merah (Hylocereus polyrhizus) terhadap berat badan, indeks fagositosis makrofag dan produksi nitrit oksida makrofag (Studi pada mencit BALB/c yang diinfeksi Salmonella typhimurium)

JURNAL GIZI INDONESIA ◽

10.14710/jgi.2.2.73-77 ◽

2014 ◽

Vol 2 (2) ◽

pp. 72-76

Author(s):

Kusdalinah Kusdalinah ◽

Andrew Johan ◽

Noor Wijayahadi

Keyword(s):

Treatment Group ◽

Salmonella Typhimurium ◽

Control Group ◽

No Production ◽

Macrophage Phagocytosis ◽

Post Test ◽

Phagocytosis Index ◽

High Level ◽

And Control ◽

Hylocereus Polyrhizus

Background: Salmonella typhimurium has ability to avoid the phagosome and escape from the trap-free in cytoplasm.Macrophage was activated to phagocyte microbe and produced microbicidal agent nitric oxide (NO).Polyphenolic compounds are proved as an immunomodulatory agent.Hylocereus polyrhizus contains high level of polyphenols and thus increases appetite.Objective: Proving the effects various doses (6 mg, 12 mg, and 24 mg/20 g mice’s weight) of Hylocereus polyrhizus extract to increase weight, increase macrophage phagocytosis and NO production of macrophage.Methods:True experiment research for fourteen day on males BALB/c mice, post-test only controlled group design.The treatment groups were a dose of 6 mg/day (X1), 12 mg/day (X2), and 24 mg/day (X3), whereas the control group (K) was not given the extract of Hylocereus polyrhizus.All treatment and control groups were infected by Salmonella typhimurium intraperitoneally 105 CFU in tenth day.Results: Weight in the treatment group was higher than the control group (p=0.037). Index of macrophage phagocytosis was higher in the treatment group compared to the control group but it was not significant (p=0.154). Macrophage NO production was higher in the treatment group compared to the control group but was not significant (p=0.332).Conclusion: Weight increased significantly.Increasing of macrophage phagocytosis index and macrophage NO production were not significant.

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

Journal of Bacteriology ◽

10.1128/jb.186.12.3938-3950.2004 ◽

2004 ◽

Vol 186 (12) ◽

pp. 3938-3950 ◽

Cited By ~ 49

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.

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ArmA Methyltransferase in a Monophasic Salmonella enterica Isolate from Food

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00308-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5262-5266 ◽

Cited By ~ 17

Author(s):

Sophie A. Granier ◽

Laura Hidalgo ◽

Alvaro San Millan ◽

Jose Antonio Escudero ◽

Belen Gutierrez ◽

...

Keyword(s):

16S Rrna ◽

Salmonella Enterica ◽

Multidrug Resistant ◽

Broad Host Range ◽

En Bloc ◽

Content Type ◽

Route Of Transmission ◽

Amoxicillin Clavulanate ◽

High Level ◽

Level Resistance

ABSTRACTThe 16S rRNA methyltransferase ArmA is a worldwide emerging determinant that confers high-level resistance to most clinically relevant aminoglycosides. We report here the identification and characterization of a multidrug-resistantSalmonella entericasubspecies I.4,12:i:− isolate recovered from chicken meat sampled in a supermarket on February 2009 in La Reunion, a French island in the Indian Ocean. Susceptibility testing showed an unusually high-level resistance to gentamicin, as well as to ampicillin, expanded-spectrum cephalosporins and amoxicillin-clavulanate. Molecular analysis of the 16S rRNA methyltransferases revealed presence of thearmAgene, together withblaTEM-1,blaCMY-2, andblaCTX-M-3. All of these genes could be transferreden blocthrough conjugation intoEscherichia coliat a frequency of 10−5CFU/donor. Replicon typing and S1 pulsed-field gel electrophoresis revealed that thearmAgene was borne on an ∼150-kb broad-host-range IncP plasmid, pB1010. To elucidate howarmAhad integrated in pB1010, a PCR mapping strategy was developed for Tn1548, the genetic platform forarmA.The gene was embedded in a Tn1548-like structure, albeit with a deletion of the macrolide resistance genes, and an IS26was inserted within themelgene. To our knowledge, this is the first report of ArmA methyltransferase in food, showing a novel route of transmission for this resistance determinant. Further surveillance in food-borne bacteria will be crucial to determine the role of food in the spread of 16S rRNA methyltransferase genes worldwide.

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RamB, a Novel Transcriptional Regulator of Genes Involved in Acetate Metabolism of Corynebacterium glutamicum

Journal of Bacteriology ◽

10.1128/jb.186.9.2798-2809.2004 ◽

2004 ◽

Vol 186 (9) ◽

pp. 2798-2809 ◽

Cited By ~ 90

Author(s):

Robert Gerstmeir ◽

Annette Cramer ◽

Petra Dangel ◽

Steffen Schaffer ◽

Bernhard J. Eikmanns

Keyword(s):

Corynebacterium Glutamicum ◽

Isocitrate Lyase ◽

Transcriptional Regulator ◽

Regulatory Elements ◽

Malate Synthase ◽

Acetate Kinase ◽

Acetate Metabolism ◽

Peptide Mass ◽

Specific Activities ◽

High Level

ABSTRACT The adaptation of Corynebacterium glutamicum to acetate as a carbon and energy source involves transcriptional regulation of the pta-ack operon coding for the acetate-activating enzymes phosphotransacetylase and acetate kinase and of the aceA and aceB genes coding for the glyoxylate cycle enzymes isocitrate lyase and malate synthase, respectively. Deletion and mutation analysis of the respective promoter regions led to the identification of highly conserved 13-bp motifs (AA/GAACTTTGCAAA) as cis-regulatory elements for expression of the pta-ack operon and the aceA and aceB genes. By use of DNA affinity chromatography, a 53-kDa protein specifically binding to the promoter/operator region of the pta-ack operon was purified. Mass spectrometry and peptide mass fingerprinting identified the protein as a putative transcriptional regulator (which was designated RamB). Purified His-tagged RamB protein was shown to bind specifically to both the pta-ack and the aceA/aceB promoter/operator regions. Directed deletion of the ramB gene in the genome of C. glutamicum resulted in mutant strain RG1. Whereas the wild type of C. glutamicum showed high-level specific activities of acetate kinase, phosphotransacetylase, isocitrate lyase, and malate synthase when grown on acetate and low-level specific activities when grown on glucose as sole carbon and energy sources, mutant RG1 showed high-level specific activities with all four enzymes irrespective of the substrate. Comparative transcriptional cat fusion experiments revealed that this deregulation takes place at the level of transcription. The results indicate that RamB is a negative transcriptional regulator of genes involved in acetate metabolism of C. glutamicum.

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Correlation between Detection of a Plasmid and High-Level Virulence of Vibrio nigripulchritudo, a Pathogen of the Shrimp Litopenaeus stylirostris

Applied and Environmental Microbiology ◽

10.1128/aem.02680-07 ◽

2008 ◽

Vol 74 (10) ◽

pp. 3038-3047 ◽

Cited By ~ 14

Author(s):

Yann Reynaud ◽

Denis Saulnier ◽

Didier Mazel ◽

Cyrille Goarant ◽

Frédérique Le Roux

Keyword(s):

Genetic Markers ◽

New Caledonia ◽

Subtractive Hybridization ◽

Epidemiological Studies ◽

Etiological Agent ◽

Subtraction Library ◽

High Virulence ◽

High Level ◽

Mass Mortalities ◽

Coral Pathogen

ABSTRACT Vibrio nigripulchritudo, the etiological agent of Litopenaeus stylirostris summer syndrome, is responsible for mass mortalities of shrimp in New Caledonia. Epidemiological studies led to the suggestion that this disease is caused by an emergent group of pathogenic strains. Genomic subtractive hybridization was carried out between two isolates exhibiting low and high virulence. Our subtraction library was constituted of 521 specific fragments; 55 of these were detected in all virulent isolates from our collection (n = 32), and 13 were detected only in the isolates demonstrating the highest pathogenicity (n = 19), suggesting that they could be used as genetic markers for high virulence capacity. Interestingly, 10 of these markers are carried by a replicon of 11.2 kbp that contains sequences highly similar to those of a plasmid detected in Vibrio shilonii, a coral pathogen. The detection of this plasmid was correlated with the highest pathogenicity status of the isolates from our collection. The origin and consequence of this plasmid acquisition are discussed.

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