Antibiotic Resistance Markers for Genetic Manipulations of
            <i>Leptospira</i>
            spp

D, Poggi; Giuseppe, P.O.; Picardeau, Mathieu

doi:10.1128/aem.00775-10

Cited by 28 publications

(18 citation statements)

References 22 publications

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“…Analysis of the nucleotide sequence of the 16S rRNA gene of the small ribosomal subunit, however, permitted us to identify a C1273T transition that may be associated with spectinomycin resistance in this C. coli strain. Indeed, this mutation corresponds to the C1192T substitution (E. coli numbering) of the upper region of helix 34 identified in many bacterial species, which confers a high level of resistance to spectinomycin (51)(52)(53). Further investigations will be necessary to confirm the association of the C1273T transition in the 16S rRNA gene with spectinomycin resistance in C. coli, which has not previously been described.…”

Section: Resultsmentioning

confidence: 83%

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants

et al. 2018

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A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant and isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [(″)- and (″)- ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in : a lincosamide nucleotidyltransferase gene [(G)], likely associated with lincomycin resistance, and two resistance enzyme genes ( and ) similar to those found in, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

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Section: Resultsmentioning

confidence: 83%

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants

et al. 2018

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“…The cells (5 ϫ 10 9 ) were collected every 4 generations, plated on semisolid TYGVS plates containing a MIC of erythromycin (40 g/ml) (8) or gentamicin (20 g/ml) (31), and incubated in an anaerobic chamber for 2 weeks. The frequencies of spontaneous resistance to the antibiotics were expressed as the number of colonies divided by the number of cells plated (17,23). The development of gentamicin resistance in T. denticola is extremely rare (spontaneous mutation frequency of Ͻ1.5 ϫ 10 Ϫ10 ) and is substantially lower than that of erythromycin (approximate frequency of 5 ϫ 10 Ϫ8 ) during the period of 28-generation cultivations, indicating that aacC m can serve as a reliable antibiotic selectable marker for constructing T. denticola mutants.…”

Section: Previous Studies Report That the Insertion Of Either Erm(f)-mentioning

confidence: 99%

Development of a Modified Gentamicin Resistance Cassette for Genetic Manipulation of the Oral Spirochete Treponema denticola

Bian

Fenno

2012

Appl Environ Microbiol

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b Herein, we report that a modified gentamicin cassette and a PCR-based method can be used for targeted mutagenesis of the oral spirochete Treponema denticola. This approach minimizes polar effects and spontaneous antibiotic resistance. Therefore, it can serve as a reliable tool for genetic manipulation of T. denticola. The oral spirochete Treponema denticola is a member of the "red complex" bacteria. It is an important pathogen that is associated with human periodontal disease (7, 14), a chronic infection that occurs in 80% of the adult population worldwide (5,22,26). However, due to the paucity of genetic tools and its fastidious growth requirements, the biology and pathogenicity of T. denticola are poorly understood (7,10,15). Although significant progress has been achieved during the last 2 decades, tools for genetic manipulation of T. denticola are still very limited (2,18,19,25,31). The first tool applied to genetic studies in T. denticola is an erythromycin resistance cassette [erm(F)-erm(B)] developed by Li et al. (19). It has since become a standard method for genetic manipulations of T. denticola. However, the spirochete has a high spontaneous mutation rate to the presence of erythromycin, and the insertion of the erm(F)-erm(B) cassette in a targeted gene often has a polar effect on downstream genes (13,20). These two drawbacks have substantially hampered the use of this cassette. Two modified erm(F)-erm(B) cassettes have been developed (13,20), but the aforementioned problems still hold. Chloramphenicol and coumermycin antibiotic resistance cassettes have been used for trans-complementation of certain T. denticola mutants (4, 25). However, these two cassettes have not yet been successfully used for the targeted mutagenesis of T. denticola, as the resistance to chloramphenicol is not stable and the coumermycin resistance is not reliable due to the pleiotropic effects of gyrase mutation (24,27).Constructing a mutated gentamicin resistance cassette. A gentamicin cassette (aacC1) (6, 28-30) has been recently used as a selectable marker for transposon mutagenesis in the T. denticola ATCC 35405 (Td35405) strain (31). We reasoned that this cassette could also be used as a selectable marker for targeted mutagenesis in the Td35405 strain. Unexpectedly, all of the attempts to use it for targeted mutagenesis failed. Our recent work revealed that Td35405 carries genes encoding three type II DNA restriction endonucleases. One of these enzymes (TDE0911) recognizes and cleaves targeted DNAs containing the sequence GGNCC (2). Sequence analysis shows that the aacC1 cassette contains a cleavage site recognized by TDE0911 (G 97 GCCC). We hypothesized that the failure of the aacC1 cassette in Td35405 could be due to the restriction cleavage mediated by TDE0911. To test this hypothesis, the cleavage site (G 97 GCCC) within aacC1 was mutated to A 97 GCCC by site-directed mutagenesis. The wild-type and mutated (designated aacC m ) cassettes were treated with the crude cell extract of Td35405. As expected, the aacC1 cassett...

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“…Transformed Leptospira can be visualized on solid medium as subsurface colonies after 1 week for saprophytes and up to 4 weeks for pathogens. Markers for the selection of transformants include kanamycin, spectinomycin, and gentamicin resistance cassettes (3,5,6). The replication origins of L. biflexa phage LE1 (3), L. biflexa plasmid p74 (7), and a phage-related genomic island from L. interrogans (8) have previously been used to construct L. biflexa-E. coli plasmid shuttle vectors.…”

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confidence: 99%

A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

Pappas

Benaroudj

Picardeau

2015

Appl Environ Microbiol

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bLeptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells.In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp. L eptospirosis, which is caused by one of the 10 pathogenic Leptospira spp. described to date, is a neglected zoonotic disease that has a worldwide distribution with a high incidence in tropical countries. The virulence mechanisms and, more generally, the biology of pathogenic Leptospira spp. remain largely unknown. This hindrance is partly due to a lack of efficient genetic tools available for use in pathogenic Leptospira spp. (1, 2). While genetic modification tools allow flexible manipulation of the genome of the saprophyte Leptospira biflexa, including targeted mutagenesis and cis/trans complementation (2), genetic modification of the pathogen is limited primarily to random transposon mutagenesis.Previously, genetic analysis of Leptospira was impeded by the absence of methods for the introduction of DNA into leptospiral cells. Currently, DNA can be introduced into Leptospira spp. by electroporation (3) or conjugation between Escherichia coli and Leptospira spp. by using RP4 derivative conjugative plasmids (4). Transformed Leptospira can be visualized on solid medium as subsurface colonies after 1 week for saprophytes and up to 4 weeks for pathogens. Markers for the selection of transformants include kanamycin, spectinomycin, and gentamicin resistance cassettes (3, 5, 6). The r...

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Antibiotic Resistance Markers for Genetic Manipulations of Leptospira spp

Cited by 28 publications

References 22 publications

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants

Development of a Modified Gentamicin Resistance Cassette for Genetic Manipulation of the Oral Spirochete Treponema denticola

A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

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