The emergence of plasmid-mediated colistin resistance genes (mcr), which is occurring in numerous countries, is a worldwide concern, primarily because colistin is a last-resort antibiotic. Compared to E. coli, prevalence of mcr genes in Salmonella is unclear in Japan. Here we screened for mcr-1-5 genes in our collection of Salmonella strains isolated from retail meat products collected in Japan from 2012 through 2016.
We found that Salmonella Albany strain 27A-368 encodes mcr-5 and that mcr genes were undetectable among the remaining 202 isolates.
The resistance plasmid p27A-368 was transferred by conjugation to S. Infantis and was stably retained as a transconjugant. Whole-genome sequencing revealed that mcr-5 resided on a 115 kb plasmid (p27A-368).
The plasmid backbone of p27A-368 is more similar to that of pCOV27, an ESBL-encoding plasmid recovered from avian pathogenic E. coli, rather than pSE13-SA01718 of S. Paratyphi B that encodes mcr-5.
Further, mcr-5 is located on a transposon, and its sequence is similar to that of pSE13-SA01718. A phylogenetic tree based on single nucleotide variants implies a relationship between 27A-368 and S. Albany isolated in Southeast Asian countries.
Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale.
Cyanobacteria play a vital role in supplying nitrogen into the soil and aquatic ecosystem. It has an extra chromosomal DNA, whose role is not yet defined well.
Isolation and characterization of extra chromosomal DNA in cyanobacteria might help to understand its survival mechanism.
Cylindrospermum stagnale isolated (and deposited in NRMCF 3001) from soil showed presence of four plasmids namely pCYLM01, pCYLM02, pCYLM03, and pCYLM04.
The following plasmids pCYLM01 and pCYLM02 were subjected to restriction digestion using HindIII restriction enzyme and cloned into pBlueScriptSK(-) vector.
The sequence of pCYLM01 contained 4 potential open reading frames (ORFs) that have amino acids in the range of 59-299. Among them, ORF1 shows high sequence homology to the bacterial replication initiator family protein as evident from BLASTP analysis.
The analysis of 4359 bp plasmid pCYLM02 sequence revealed 7 ORFs which are longer than 50 amino acids in length. The ORF2 of pCYLM02 has 243 amino acids and is represented in the plasmid sequence from 3045 to 3776 bp.
The ORF3 of pCYLM02 corresponds to the plasmid sequence from 2323 to 2976 and codes for a putative protein of 217 amino acids long. A number of small ORFs below 50 bp were also found in the sequence analysis.
Transduction as a Potential Dissemination Mechanism of a Clonal qnrB19-Carrying Plasmid Isolated From Salmonella of Multiple Serotypes and Isolation Sources.
Antimicrobial resistance is an increasing problem worldwide, and Salmonella spp. resistance to quinolone was classified by WHO in the high priority list. Recent studies in Europe and in the US reported the presence of small plasmids carrying quinolone resistance in Enterobacteriaceae isolated from poultry and poultry products.
The aims of this study were to identify and characterize plasmid-mediated quinolone resistance in Salmonella spp. and to investigate transduction as a possible mechanism associated to its dissemination.
First, we assessed resistance to nalidixic acid and/or ciprofloxacin in 64 Salmonella spp. and detected resistance in eight of them. Genomic analyses determined that six isolates of different serotypes and sources carried an identical 2.7-kb plasmid containing the gene qnrB19 which confers quinolone resistance.
The plasmid detected also has high identity with plasmids reported in the US, Europe, and South America.
The presence of similar plasmids was later surveyed by PCR in a local Salmonella collection (n = 113) obtained from diverse sources: food (eggs), wild and domestic animals (pigs, horse, chicken), and human clinical cases. qnrB19-carrying plasmids were found in 8/113 Salmonella tested strains.
A bioinformatics analysis including Chilean and previously described plasmids revealed over 95.0% of nucleotide identity among all the sequences obtained in this study.
Furthermore, we found that a qnrB19-carrying plasmid can be transferred between Salmonella of different serotypes through a P22-mediated transduction.
Altogether our results demonstrate that plasmid-mediated quinolone resistance (PMQR) is widespread in Salmonella enterica of different serotypes isolated from human clinical samples, wild and domestic animals, and food in Chile and suggest that transduction could be a plausible mechanism for its dissemination.
The occurrence of these antimicrobial resistance elements in Salmonella in a widespread area is of public health and food safety concern, and it indicates the need for increased surveillance for the presence of these plasmids in Salmonella strains and to assess their actual impact in the rise and spread of quinolone resistance.
Isolation and Visualization of Plasmids from Gram-Positive Bacteria of Interest in Public Health.
- This chapter describes the methods to extract and characterize plasmids of Gram-positive bacterial species of interest in public health (biomedicine, veterinary, and food safety) as Staphylococcus, Streptococcus, Enterococcus, Listeria, and Clostridium and lactic acid bacteria.
- References for detailed plasmid classification are given in order to provide a comprehensive landscape in the interpretation of their plasmidomes.
Detection, Isolation, and Characterization of Plasmids in the Environment.
Plasmids play a major role in the bacterial adaptation to changing and stressful environmental conditions caused by antibiotics, heavy metals, and disinfectants.
However, the investigation of the ecology and diversity of environmental plasmids is challenging due to their typically low abundance in soil bacterial communities and the low cultivability of their hosts.
Here we discuss the potentials and limitations of cultivation-dependent and cultivation-independent approaches for detecting and quantifying plasmids in total community DNA from environmental samples.
Protocols for PCR-based detection of plasmid-specific sequences in total community DNA are presented. Furthermore, protocols to obtain and characterize plasmids either from isolates (endogenous plasmid isolation) or by capturing into a recipient strain by biparental and triparental mating will be provided.
Plasmid DNA Isolation and Visualization: Isolation and Characterization of Plasmids from Clinical Samples.
Plasmids are important in carrying antibiotic resistance and other genes between bacterial cells, and a number of methods can be employed to characterize plasmids from clinical isolates.
Single colonies typically obtained as part of hospital workflow can undergo S1 nuclease treatment to linearize plasmids followed by pulsed-field gel electrophoresis to enable determination of the number and sizes of plasmids present.
Plasmid DNA Isolation Solution I | |||
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Plasmid DNA Isolation Solution I | |||
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Plasmid DNA Isolation Solution II | |||
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Plasmid DNA Isolation Solution III | |||
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QC Buffer for Plasmid DNA Isolation | |||
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QC Buffer for Plasmid DNA Isolation | |||
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QF Buffer for Plasmid DNA Isolation | |||
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QF Buffer for Plasmid DNA Isolation | |||
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Hybridization of S1/PFGE gels can be used to associate replicon types and passenger genes, such as those conferring antibiotic resistance, with a particular plasmid band. Individual plasmids, obtained by conjugation or transformation, can be compared by gel electrophoresis following restriction digestion of plasmid DNA prepared by alkaline lysis methods, including using specialized kits.