Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

Willingham-Lane, Jennifer M.; Berghaus, Londa J.; Giguère, Steeve; Hondalus, Mary K.

doi:10.1128/msphere.00186-16

Cited by 19 publications

(35 citation statements)

References 57 publications

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“…Previous work of our laboratory demonstrated that conjugal transfer of the 33705 pVAPB-type plasmid to the equine-derived pVAPA plasmid-cured 103S P- strain yielded a transconjugant (103S P- /A-p33705) that was able to replicate within murine and equine macrophages [ 20 ]. Those results indicated that the chromosome of an equine R .…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, traditional lysis and plating of infected macrophage monolayers has a tendancy to under represent the total number of bacteria present in the monolayer. Thus, in this cell type, bacterial replication over time is followed by immunostaining infected monolayers and using fluorescent microscopy to enumerate the number of bacteria per 200 macrophages in triplicate at each time point [ 12 , 20 , 24 ]. In parallel, as an additional assessment, the number of macrophages with 10 or greater bacteria is recorded as previously described [ 12 , 20 , 24 ].…”

Section: Resultsmentioning

confidence: 99%

“…Thus, in this cell type, bacterial replication over time is followed by immunostaining infected monolayers and using fluorescent microscopy to enumerate the number of bacteria per 200 macrophages in triplicate at each time point [ 12 , 20 , 24 ]. In parallel, as an additional assessment, the number of macrophages with 10 or greater bacteria is recorded as previously described [ 12 , 20 , 24 ]. As expected [ 20 ], the wild type equine isolate 103S carrying its native pVAPA-type plasmid and the transconjugant 103S P- /A-p33705 in possession of the intact non-native pVAPB-type plasmid increased in number in these macrophages over time ( Fig 6 ).…”

Section: Resultsmentioning

confidence: 99%

“…We recently demonstrated possession of the pVAPB-type plasmid, typical of most swine isolates and commonly found in R . equi strains cultured from people, enables the bacterium to replicate in murine, equine, and swine macrophages [ 20 ]. Since the pVAPB-type plasmid does not contain the vapA or vapN genes, the objectives of this study were to determine the component/s of the pVAPB-type plasmid that allow/s for replication within macrophages and assess whether the identified component/s is/are functionally equivalent to the vapA gene encoded on the equine pVAPA-type plasmid.…”

Section: Introductionmentioning

confidence: 99%

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Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid

2018

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Rhodococcus equi is a facultative intracellular bacterium of macrophages and is an important pathogen of animals and immunocompromised people wherein disease results in abcessation of the lungs and other sites. Prior work has shown that the presence of the major virulence determinant, VapA, encoded on the pVAPA-type plasmid, disrupts normal phagosome development and is essential for bacterial replication within macrophages. pVAPA- type plasmids are typical of R. equi strains derived from foals while strains from pigs carry plasmids of the pVAPB-type, lacking vapA, and those from humans harbor various types of plasmids including pVAPA and pVAPB. Through the creation and analysis of a series of gene deletion mutants, we found that vapK1 or vapK2 is required for optimal intracellular replication of an R. equi isolate carrying a pVAPB plasmid type. Complementation analysis of a ΔvapA R. equi strain with vapK1 or vapK2 showed the VapK proteins of the pVAPB-type plasmid could restore replication capacity to the macrophage growth-attenuated ΔvapA strain. Additionally, in contrast to the intracellular growth capabilities displayed by an equine R. equi transconjugant strain carrying a pVAPB-type plasmid, a transconjugant strain carrying a pVAPB-type plasmid deleted of vapK1 and vapK2 proved incapable of replication in equine macrophages. Cumulatively, these data indicate that VapK1 and K2 are functionally equivalent to VapA.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid

2018

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“…All R . equi strains found to be virulent for mice in laboratory conditions and pathogenic for foals contain the virulence plasmid, and the VapA protein is present in the cell [ 13 – 14 , 22 – 24 ]. Moreover, the absence of the plasmid or the VapA protein has been shown to be associated with the loss of virulence, although the presence of the VapA protein alone, without the other proteins of the family, does not enable the bacteria to multiply in murine macrophages or tissues or to colonize the lungs of experimentally infected foals [ 14 , 23 , 25 – 26 ].…”

Section: Introductionmentioning

confidence: 99%

Molecular analysis of the chromosomal 16S rRNA gene and vapA plasmid gene of Polish field strains of R. equi

et al. 2018

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Rhodococcus equi (R. hoagii) is an opportunistic pathogen commonly found in foals up to 6 months old and animal environment. The R. equi genome contains genetically stable chromosomal DNA and an 80–90 kb plasmid containing vapA gene, responsible for virulence. Most reports from around the world focus on the determination of R. equi plasmid profiles. Few studies have attempted to determine differences in nucleotide sequences between virulent strains of R. equi isolated from foals and breeding environment. The aim of the study was to perform a molecular analysis of a fragment of the chromosomal gene encoding the 16S rRNA subunit and the vapA plasmid gene of virulent R. equi strains isolated on Polish studs from foals and from the breeding environment of horses. The sequencing method was used to compare the primary structure of fragments of the chromosomal and plasmid DNA of the virulent R. equi strains. The sequences of 22 clinical and 18 environmental R. equi isolates were compared with the sequences of the gene fragments of reference strains available in the NCBI GenBank database. All sequenced 16S rRNA amplicons of Polish field strains were identical and showed 99.5% similarity to the four randomly selected sequences of this gene fragment in the GenBank database. The results confirm that fragments of the 16S rRNA gene of R. equi strains are highly conserved and do not undergo variation in field conditions. Analysis of the sequencing results for the vapA gene fragment of the strains used in our study revealed two polymorphic variants and clear differences between the sequences of strains isolated from foals and from soil samples. Presumably, R. equi strains present in the breeding environment are more exposed than clinical strains to adverse external factors. This may result in changes in the DNA sequence due to natural selection.

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Antimicrobial susceptibility of Rhodococcus equi strains isolated from foals in Chile

Zúñiga

Badillo

Abalos

et al. 2023

World J Microbiol Biotechnol

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Rhodococcus equi is responsible for foal pneumonia worldwide, with a significant economic impact on the production and breeding of horses. In Chile, the first case was reported in 2000, and since then, its incidence has been increasing. Distinctive characteristics of R. equi as an intracellular pathogen in macrophages, emergence of virulence plasmids encoding surface lipoprotein antigens, and appearance of antibiotic resistance against macrolides and rifampicin have significantly complicated the treatment of R. equi pneumonia in foals. Therefore, in vitro susceptibility studies of first-line and newer antibiotics against R. equi are the first step to establishing effective treatments and optimizing new therapeutic options. The aim of the present study is to determine the susceptibility profile of fourteen strains of R. equi isolated from foals in Chile to several antibiotics of the macrolide group including azithromycin, amikacin, tildipirosin and gamithromycin as well as others such as rifampicin, doxycycline and ceftiofur. Identification of R. equi in collected isolates from foals in Chile has been performed by CAMP test and PCR based on detecting of the gene encoding the 16 S rRNA. The presence of genes encoding virulence plasmids was also determined using PCR. Results obtained have demonstrated presence of virulent R. equi strains in Chile. In vitro susceptibility pattern to different antibiotics has shown better results for doxycycline and rifampicin similar to previous studies performed. Current macrolides have been evaluated in order to consider alternative treatment options in a context of emerging resistance to classic macrolides and rifampicin, obtaining better results with gamithromycin (MIC range of 0.125 to 128 mg/ml) than with tildipirosin (MIC range of 16 to 128 mg/ml). An adequate diagnosis of bacterial susceptibility based on antibiograms is necessary to treat the Rhodococcus equi infection in foals.

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Influence of Plasmid Type on the Replication of Rhodococcus equi in Host Macrophages

Cited by 19 publications

References 57 publications

Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid

Identification of a VapA virulence factor functional homolog in Rhodococcus equi isolates housing the pVAPB plasmid

Molecular analysis of the chromosomal 16S rRNA gene and vapA plasmid gene of Polish field strains of R. equi

Antimicrobial susceptibility of Rhodococcus equi strains isolated from foals in Chile

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