Transformation of Coxiella burnetii to ampicillin resistance

Suhan, Michelle L.; Chen, Shuyin; Thompson, Herbert A.

doi:10.1128/jb.178.9.2701-2708.1996

Cited by 48 publications

(62 citation statements)

References 37 publications

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“…Previous work demonstrated that the efficacies of chloramphenicol and ciprofloxacin against C. burnetii varied (14,17,25,26), depending on the dose, the culture model, and the C. burnetii strain used to test the antimicrobials. The ability of ampicillin to inhibit C. burnetii growth supports prior evidence presented by Suhan et al (21) that C. burnetii Nine Mile, phase I, is susceptible to ampicillin at 50 g/ml. At present, the recommended treatments for C. burnetii infections involve doxycycline for 2 weeks for acute infections and doxycycline plus chloroquine for at least 18 months for chronic infections.…”

Section: Discussionsupporting

confidence: 74%

Evaluation of Coxiella burnetii Antibiotic Susceptibilities by Real-Time PCR Assay

2003

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Coxiella burnetii is an obligate intracellular bacterium. The inability to cultivate this organism on axenic medium has made calculation of infectious units challenging and prevents the use of conventional antibiotic susceptibility assays. A rapid and reliable real-time PCR assay was developed to quantify C. burnetii cells from J774.16 mouse macrophage cells and was applied to antibiotic susceptibility testing of C. burnetii Nine Mile, phase I. For calculation of bacterial replication, real-time PCR performed equally as well as immunofluorescent-antibody (IFA) assay when J774.16 cells were infected with 10-fold serial dilutions of C. burnetii and was significantly (P < 0.05) more repeatable than IFA when 2-fold dilutions were used. Newly infected murine macrophage-like J774.16 cells were treated with 8 g of chloramphenicol per ml, 4 g of tetracycline per ml, 4 g of rifampin per ml, 4 g of ampicillin per ml, or 1 g of ciprofloxacin per ml. After 6 days of treatment, tetracycline, rifampin, and ampicillin significantly (P < 0.01) inhibited the replication of C. burnetii, while chloramphenicol and ciprofloxacin did not. In general, these results are consistent with those from prior reports on the efficacy of these antibiotics against C. burnetii Nine Mile, phase I, and indicate that a real-time PCR-based assay is an appropriate alternative to the present methodology for evaluation of the antibiotic susceptibilities of C. burnetii.

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Section: Discussionsupporting

confidence: 74%

Evaluation of Coxiella burnetii Antibiotic Susceptibilities by Real-Time PCR Assay

2003

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“…Similar conditions were optimal for C. trachomatis L2 (data not shown). Small cells such as chlamydial EBs generally require high electric fields for successful transformation, and similar conditions have been reported to be efficient for Coxiella burnetii transformation (17). Tam et al (6) used 10.0 kV/cm, 400 ⍀, and 25 F in their electroporation protocol but reported poor postelectroporation recovery for C. trachomatis L2.…”

Section: Discussionmentioning

confidence: 99%

Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation

Binet

Maurelli

2009

Proc. Natl. Acad. Sci. U.S.A.

106

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To facilitate genetic investigations in the obligate intracellular pathogens Chlamydia, the ability to construct variants by homologous recombination was investigated in C. psittaci 6BC. The single rRNA operon was targeted with a synthetic 16S rRNA allele, harboring three nucleotide substitutions over 398 bp, which imparts resistance to kasugamycin (Ksm) and spectinomycin (Spc) and causes loss of one HpaI restriction site. A fourth, silent mutation was introduced 654 bp downstream in the beginning of the 23S rRNA gene. C. psittaci 6BC infectious particles were electroporated with various concentrations of circular or linearized plasmids containing different lengths of the rRNA region homologous to the chromosomal copy except for the four nucleotide substitutions. Ksm and Spc were added 18 h after inoculation onto confluent cell monolayers in the plaque assay. Resistant plaques were picked and expanded with selection 10 days later before collecting DNA for analysis by PCR, restriction mapping, sequencing, or Southern. Spontaneous resistance to Ksm and Spc was never observed in mock electroporated bacteria (frequency <6.2 ؋ 10 ؊9 ). Conversely, double resistance and replacement of the 16S rRNA gene were observed when C. psittaci was electroporated with the recombination substrates. Highest efficiency was obtained with 10 g of circular vector prepared in a DNA methylase-deficient Escherichia coli (1.9 ؎ 1.1 ؋ 10 ؊6 , n ‫؍‬ 7). Coinheritance of the silent 23S rRNA mutation was seen in 46 of 67 recombinants analyzed, illustrating DNA exchange of up to 1,052 bp in length. These findings provide the first step toward genetic manipulation of Chlamydia.recombination ͉ selection ͉ plaque assay

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“…This frequency is presumably an underestimate due to the limitations of the rescue cloning procedure and the possible loss over time of transformants harboring deleterious mutations. Earlier C. burnetii transformation experiments utilized resistance to ampicillin as a method of positive selection (36). However, long-term selection resulted in spontaneous mutation to ampicillin resistance by bacteria lacking the introduced ␤-lactamase gene (36).…”

Section: Discussionmentioning

confidence: 99%

“…Transformation of C. burnetii was described by Suhan et al (36) more than 10 years ago. Using a plasmid containing a 5.8-kb C. burnetii autonomous replication sequence, they transformed C. burnetii to ampicillin resistance (4,35,36).…”

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Characterization of a Coxiella burnetii ftsZ Mutant Generated by Himar1 Transposon Mutagenesis

Beare¹,

Howe²,

Cockrell³

et al. 2009

J Bacteriol

102

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Coxiella burnetii is a gram-negative obligate intracellular bacterium and the causative agent of human Q fever. The lack of methods to genetically manipulate C. burnetii significantly impedes the study of this organism. We describe here the cloning and characterization of a C. burnetii ftsZ mutant generated by mariner-based Himar1 transposon (Tn) mutagenesis. C. burnetii was coelectroporated with a plasmid encoding the Himar1 C9 transposase variant and a plasmid containing a Himar1 transposon encoding chloramphenicol acetyltransferase, mCherry fluorescent protein, and a ColE1 origin of replication. Vero cells were infected with electroporated C. burnetii and transformants scored as organisms replicating in the presence of chloramphenicol and expressing mCherry. Southern blot analysis revealed multiple transpositions in the C. burnetii genome and rescue cloning identified 30 and 5 insertions in coding and noncoding regions, respectively. Using micromanipulation, a C. burnetii clone was isolated containing a Tn insertion within the C terminus of the cell division gene ftsZ. The ftsZ mutant had a significantly lower growth rate than wild-type bacteria and frequently appeared as filamentous forms displaying incomplete cell division septa. The latter phenotype correlated with a deficiency in generating infectious foci on a per-genome basis compared to wild-type organisms. The mutant FtsZ protein was also unable to bind the essential cell division protein FtsA. This is the first description of C. burnetii harboring a defined gene mutation generated by genetic transformation.

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Transformation of Coxiella burnetii to ampicillin resistance

Cited by 48 publications

References 37 publications

Evaluation of Coxiella burnetii Antibiotic Susceptibilities by Real-Time PCR Assay

Evaluation of Coxiella burnetii Antibiotic Susceptibilities by Real-Time PCR Assay

Transformation and isolation of allelic exchange mutants of Chlamydia psittaci using recombinant DNA introduced by electroporation

Characterization of a Coxiella burnetii ftsZ Mutant Generated by Himar1 Transposon Mutagenesis

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