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- JANEWANGJK55
- http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037152#pone.0037152-Zhang1
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- Materials and methods
Section of the bacterial isolate for study
The bacterial isolate was identified using the VITEK 2 compact platform (bioMérieux Vitek, Hazelwood, MO, USA) and 16S rDNA gene-based sequencing. The plasmid-borne carbapenemase- and ESBL-encoding genes were assessed by PCR.
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- Plasmid conjugal transfer
The K. oxytoca KOX3 isolate was analyzed for its ability to transfer the carbapenem resistance phenotype to a plasmid-free E. coli recipient.
Detection of carbapenemase activity
Antimicrobial susceptibility testing
The results were interpreted according to the 2016 CLSI guidelines (9).
Plasmid sequencing and annotation
To assess the genomic background of the resistance genes,
In addition to blaNDM-1, blaIMP-4 and blaKPC-2, other β-lactamase-encoding genes were also identified in K. oxytoca KOX3 including blaOXA-10 (encoding a non-ESBL β-lactamase), blaSHV-12 (encoding ESBL) and blaTEM-1 variant (encoding β-lactamase) (Table 2). Of note, this was the first report of a CRE isolate containing three different carbapenemase-resistance encoding genotypes.
Following annotation, 300 predicted coding sequences (CDSs) and 14 subsystems were identified. These included, a mercury resistance-encoding operon, a conjugation system, a plasmid stabilization system and a toxin/antitoxin resistance system among others. The protein products of nine of these CDSs were predicted to be associated with resistance to a variety of antimicrobial classes. These antimicrobial resistance genes were identified as: aadA5 and aadA2 (streptomycin/spectinomycin), sul1 and sul2 (sulfonamides), strA and strB (streptomycin), catA2 (chloramphenicol), dfrA1 and dfrA12 (trimethoprim) (Table 2 and Figure 1).
and were reported previously in clinical isolates worldwide. Integron In27, was also detected earlier in the two NDM-1-harbouring plasmids described above. Notably, most integron In27 structures reported to date were associated with IncP-1 plasmids (11).
Situated proximally to the intl1 gene, was a recently recognized Tn3-like transposon, Tn1696, (Figure 1) (14). This arrangement was similar in physical organization to other complex plasmids, but varying in the composition of their gene cassettes (data not shown) (14, 15). A Tn3-type transposase gene was located upstream of the integron-containing region, and this feature suggested that the ISCR1-associated class 1 integron may have inserted into this complex transposable element. Similar features, such as Tn1696-intl1, were reported in IncA/C2, IncHI1, IncL/M and IncHI2 plasmids from Enterobacter cloacae, Klebsiella oxytoca, Salmonella enterica and Cronobacter sakazakii isolates (14-16). The flanking regions of the integrating element are highly conserved, whilst the internal gene content varies, suggesting this type of mobile element can integrate into plasmids with differing backbone structures, thereby contributing to their dissemination whilst expanding their repertoires of resistance (15).
, including plasmid pNDM-SX04 (GenBank no. KC876051) in K. pneumonia, plasmid pNDM-HF727 in Enterobacter cloacae (GenBank no. KF976405) and in plasmid pFR90 in Providencia rettgeri (GenBank no. JQ362415)
The backbone structures of these plasmids, including the loci responsible for replication, partitioning, maintenance and conjugal transfer, were highly conserved, suggesting that they may have originally evolved from the same ancestral plasmid. A study of a worldwide collection of NDM-1-producing enterobacterial isolates showed that the current spread of the blaNDM-1 gene is not related to the spread of specific clones, specific plasmids, or single genetic structure (21).
From the annotated data, 95 CDSs were predicted, of which 45 genes encoded replication, partitioning and DNA restriction/modification functions. Seventeen genes were predicted to have putative roles in transposition and recombination.
The backbone regions shared by plasmid pKOX3-P4-IMP and the IncN-type plasmids included the repA-encoding gene, a stbABC operon (for plasmid stability), the ard genes (with antirestriction function), the two transfer gene clusters (encoding the conjugative apparatus) and genes for plasmid maintenance (Figure 2b). The two resistance genes (qnrS1 and blaIMP-4) were found in the variable region of these plasmids.
No conjugation-related genes were detected in plasmid pKOX3-P5-KPC, suggesting that it is not mobile. This latter finding was consistent with these data obtained from conjugation experiments (data not shown). Although no conjugative genes were detected, several features of this plasmid, including the repA gene (the IncP-6-type replication), the typical parABC locus of IncP-6-type plasmids (promoting the plasmid mobilization), the mob gene cluster (enhancing the mobilization frequency), suggest that it may be able to replicate and adapt in a variety of hosts (26).
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- Growth curve assays to assess plasmid-related fitness costs-
To assess any potential fitness costs associated with transmissible plasmids identified in the original isolates, growth curve profiles were determined for each transconjugant and compared with the plasmid-free recipient. Growth assays were initiated with 1:10 mixtures of independent overnight cultures of test isolate grown in fresh LB broth. Growth was measured at 37 °C for 24 h, using the Multiskan TM FC Microplate Photometer (Thermo Fisher Scientific). The OD610 nm was measured at 15-min intervals over a period of 24 h. The experiment was repeated three times.
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- Interestingly, the mcr-1 positive IncI2 plasmid was co-occurrence with the other plasmids with diverse replicon types such as IncHI2 and IncFII. 2016-Sci rep-fengyoujun-clonal. ISApl1 was present upstream of the mcr-1 gene, a ISApl1-mcr-1-orf-ISApl1 structure has been found in IncHI2-type plasmids and bacterial chromosomes with the ability to generate a circular intermediate harbouring ISApl1 and mcr-1 [8-11]2016-JAC-li.
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- The transconjugants showed drastically increased minimal inhibitory concentrations (MICs) for colistin (Table 1). However,. S1-PFGE confirmed that ***, suggesting the mcr-1
Conjugation assay showed that mcr-1 was successfully transferred. All the transconjugants showed 2- or 4-fold increases in the MICs of colistin, when compared with the recipients.
Following conjugation the MIC values of colistin recorded for S455 was 2 () or 1 () determined by broth dilution. Interestingly,
When these MIC values were measured for the transconjugants, all were recorded at ** and there was between a 2- to 4-fold increase, in this value the MICs of transconjugants correspondingly were higher.
and hybridization performed to determine the location of mcr-1 showed that four plasmids (~224, ***, *** and ** kb) existed in S. Indiana S455, and that the mcr-1 gene was located in a plasmid of ~60 kb.
Isolates with colistin MICs ranging from 4 to 8 mg/L (Table S1). Conjugation experiments and S1-PFGE demonstrated the
Suggesting the gene has been circulating in human-restricted pathogens for some time but carries a selective fitness cost.
and the minimal inhibitory concentration (MIC) values for colistin was determined as 2 μg/mL using broth dilution tests.
These genomic regions code for several virulence- or fitness-associated functions such as a type 6 secretion system, a type 3 secretion system. Interestinly, *** It should be note that Transferability
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- Can be transferred between different species of Enterobacteriaceae.
Diversified plasmids act as major vectors for the dissemination of the mcr-1 gene in Enterobacteriaceae. So far, the mcr-1-harbouring plasmids can be grouped into eight types…… As a prevalent type, IncI2 plasmid is widespread in various species of diversified origins.
Broth microdilution is considered the reference standard for polymyxin susceptibility testing (Al-Tawfiq et al., 2017). In this study, the MICs of colistin in each sample were tested in triplicate.
This gene has mainly been found in E. coli, and to a lesser extent in Klebsiella spp. and Salmonella spp. The spread of mcr-1 has already occurred worldwide, probably due to its location on conjugative plasmids.
Could be transferred by conjugation among enterobacterial species.
. Regardless of their incompatibility types, plasmids carrying the mcr-1 gene could be transferred to several enterobacterial species (at least to E. coli, K. pneumonia, K. oxytoca, and E. aerogenes) at a conjugation rate varying from 10-4 to 10-6.
A high-level colistin-resistant clinical strain of Enterobacter cloacae (MIC, 128 μg/mL) (Lin et al., 2017).
