msc-alff

Determination of incompatibility group plasmids

Determination of incompatibility group plasmids and copy number of the bla NDM-1 gene in carbapenem-resistant Klebsiella pneumoniae strains recovered from different hospitals in Kerman, Iran

Introduction. New Delhi metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae has become a serious global health concern.

Hypothesis/Gap Statement. Due to the high genetic diversity among NDM-positive K. pneumoniae, we need further surveillance and studies to better understand the relationships between them. In addition, the coexistence of several plasmid replicon types in NDM-positive K. pneumoniae may affect the copy number of bla NDM, the MIC level to antibiotics, as well as increasing the chance of horizontal gene transfer.

Aim. The aim of this study was to determine incompatible plasmid groups and copy numbers of bla NDM, and to investigate the genetic relationship of 37 NDM-positive K. pneumoniae in Kerman, Iran.

Methodology. The bla NDM-1 gene was detected and confirmed by PCR-sequencing. The plasmid replicon types were determined by PCR-based replicon typing (PBRT) and the copy number of bla NDM-1 was determined by quantitaive real time-PCR (qPCR). Random amplified polymorphic DNA (RAPD)-PCR typing was used to detect genetic relationships between the strains.

Results. In this study, 10 different replicon types, including Frep [n=25 (67.5 %)], FIIAs [n=11 (29.7 %)], FIA [n=5 (13.5 %)], FIB [n=3 (8.1 %)], I1-Iγ [n=2 (5.4 %)], L/M [n=7 (18.9 %)], A/C [n=7 (18.9 %)], Y [n=3 (8.1 %)], P [n=1 (2.7 %)] and FIC [n=1 (2.7 %)] were reported. The copy numbers of the bla NDM-1 gene varied from 30.00 to 5.0×106 and no statistically significant correlation was observed between a rise of the MIC to imipenem and the copy numbers of bla NDM-1 (P>0.05). According to RAPD typing results, 35 strains were divided into five clusters, while two strains were non-typeable.Conclusion. The spread of NDM-1-producing K. pneumoniae strains that carry several plasmid replicon types increases the chance of horizontal transfer of antibiotic resistance genes in hospital settings. In this study, 10 different replicon types were identified. We could not find any relationship between the increase of MIC levels to imipenem and the copy numbers of bla NDM-1. Therefore, due to the identification of different replicon types in this study, the type and genetic characteristics of bla NDM-1-carrying plasmids, and other factors such as antibiotic selective pressure, probably affect the copy number of bla NDM-1 and change the MIC level to imipenem.

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msc-alff

pOET-5 Transfer Vector (10ug)

GWB-200106 10 ug Ask for price

pOET3 transfer plasmid

200104 10 µg
EUR 208.32

pOET4 transfer plasmid

200105 10 µg
EUR 208.32

pOET5.1 transfer plasmid

200106 10 µg
EUR 208.32

pOET1.1N_6xHis transfer plasmid

2001011 10 µg
EUR 208.32

pOET1.1C_6xHis transfer plasmid

2001012 10 µg
EUR 208.32

pOET2.1C_6xHis transfer plasmid

2001032 10 µg
EUR 208.32

pOET8.VE2 transfer plasmid

200122 10 µg
EUR 409.2

pOET8.VE3 transfer plasmid

200123 10 µg
EUR 409.2

pOET9 EF1α transfer plasmid

200131 10 µg
EUR 280.24

pOET9 CCAG transfer plasmid

200132 10 µg
EUR 280.24

pOET9 SV40 transfer plasmid

200134 10 µg
EUR 280.24

pOET2.1N/C_6xHis transfer plasmid

2001031 10 µg
EUR 208.32

pOET6 BacMAM transfer plasmid

200107 10 µg
EUR 208.32

pOET 2 N/C_6xHis™ Transfer Vector

GWB-001031 10 ug Ask for price

CCND1 with C-tGFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100009 10 µg Ask for price

LAMP1 with C-tGFP tag for Lysosome marking (10ug transfection-grade plasmid)

RC100016 10 µg Ask for price

LMNB1 with N-tGFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100018 10 µg Ask for price

Rab4 with N-tGFP tag for Endosome marking (10ug transfection-grade plasmid)

RC100025 10 µg Ask for price

Rab5 with N-tGFP tag for Endosome marking (10ug transfection-grade plasmid)

RC100026 10 µg Ask for price

RhoB with N-tGFP tag for Endosome marking (10ug transfection-grade plasmid)

RC100027 10 µg Ask for price

CCND1 with C-tRFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100041 10 µg Ask for price

LAMP1 with C-tRFP tag for Lysosome marking (10ug transfection-grade plasmid)

RC100048 10 µg Ask for price

LMNB1 with N-tRFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100050 10 µg Ask for price

Rab4 with N-tRFP tag for Endosome marking (10ug transfection-grade plasmid)

RC100057 10 µg Ask for price

Rab5 with N-tRFP tag for Endosome marking (10ug transfection-grade plasmid)

RC100058 10 µg Ask for price

RhoB with N-tRFP tag for Endosome marking (10ug transfection-grade plasmid)

RC100059 10 µg Ask for price

LAMP1 with C-mGFP tag for Lysosome marking (10ug transfection-grade plasmid)

RC100089 10 µg Ask for price

CCND1 with C-mGFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100094 10 µg Ask for price

LMNB1 with N-mGFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100095 10 µg Ask for price

GBA2 with C-mGFP tag for Microsome marking (10ug transfection-grade plasmid)

RC100099 10 µg Ask for price

LAMP1 with C-mRFP tag for Lysosome marking (10ug transfection-grade plasmid)

RC100123 10 µg Ask for price

CCND1 with C-mRFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100128 10 µg Ask for price

LMNB1 with N-mRFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100129 10 µg Ask for price

GBA2 with C-mRFP tag for Microsome marking (10ug transfection-grade plasmid)

RC100133 10 µg Ask for price

LAMP1 with C-mBFP tag for Lysosome marking (10ug transfection-grade plasmid)

RC100157 10 µg Ask for price

CCND1 with C-mBFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100162 10 µg Ask for price

LMNB1 with N-mBFP tag for Nucleus marking (10ug transfection-grade plasmid)

RC100163 10 µg Ask for price

GBA2 with C-mBFP tag for Microsome marking (10ug transfection-grade plasmid)

RC100167 10 µg Ask for price

LCK with C-tGFP tag for Plasma memberane marking (10ug transfection-grade plasmid)

RC100017 10 µg Ask for price

LCK with C-tRFP tag for Plasma memberane marking (10ug transfection-grade plasmid)

RC100049 10 µg Ask for price

CLTB with N-tGFP tag for Coated pit marking (10ug transfection-grade plasmid)

RC100010 10 µg Ask for price

CLTB with N-tRFP tag for Coated pit marking (10ug transfection-grade plasmid)

RC100042 10 µg Ask for price

CLTB with N-mGFP tag for Coated Pit marking (10ug transfection-grade plasmid)

RC100071 10 µg Ask for price

CLTB with N-mRFP tag for Coated Pit marking (10ug transfection-grade plasmid)

RC100105 10 µg Ask for price

CLTB with N-mBFP tag for Coated Pit marking (10ug transfection-grade plasmid)

RC100139 10 µg Ask for price

BID with C-tGFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100007 10 µg Ask for price

PLK1 with N-tGFP tag for Centrosome marking (10ug transfection-grade plasmid)

RC100023 10 µg Ask for price

PXMP2 with N-tGFP tag for Peroxisome marking (10ug transfection-grade plasmid)

RC100024 10 µg Ask for price

BID with C-tRFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100039 10 µg Ask for price

PLK1 with N-tRFP tag for Centrosome marking (10ug transfection-grade plasmid)

RC100055 10 µg Ask for price

PXMP2 with N-tRFP tag for Peroxisome marking (10ug transfection-grade plasmid)

RC100056 10 µg Ask for price

PLK1 with N-mGFP tag for Centrosome marking (10ug transfection-grade plasmid)

RC100070 10 µg Ask for price

BID with C-mGFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100090 10 µg Ask for price

PLK1 with N-mRFP tag for Centrosome marking (10ug transfection-grade plasmid)

RC100104 10 µg Ask for price

BID with C-mRFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100124 10 µg Ask for price

PLK1 with N-mBFP tag for Centrosome marking (10ug transfection-grade plasmid)

RC100138 10 µg Ask for price

BID with C-mBFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100158 10 µg Ask for price

ACTB with N-tGFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100002 10 µg Ask for price

PDHA1 with C-tGFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100006 10 µg Ask for price

PFN1 with N-tGFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100022 10 µg Ask for price

PDHA1 with C-tRFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100038 10 µg Ask for price

PFN1 with N-tRFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100054 10 µg Ask for price

ATG12 with C-mGFP tag for Auophagasome marking (10ug transfection-grade plasmid)

RC100066 10 µg Ask for price

PDHA1 with C-mGFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100091 10 µg Ask for price

ATG12 with C-mRFP tag for Auophagasome marking (10ug transfection-grade plasmid)

RC100100 10 µg Ask for price

PDHA1 with C-mRFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100125 10 µg Ask for price

ATG12 with C-mBFP tag for Auophagasome marking (10ug transfection-grade plasmid)

RC100134 10 µg Ask for price

PDHA1 with C-mBFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100159 10 µg Ask for price

GAP43 with C-tGFP tag for Neuroal axis marking (10ug transfection-grade plasmid)

RC100013 10 µg Ask for price

GAP43 with C-tRFP tag for Neuroal axis marking (10ug transfection-grade plasmid)

RC100045 10 µg Ask for price

RhoB with N-mGFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100075 10 µg Ask for price

Rab4 with N-mGFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100077 10 µg Ask for price

Rab5 with N-mGFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100078 10 µg Ask for price

RhoB with N-mRFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100109 10 µg Ask for price

Rab4 with N-mRFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100111 10 µg Ask for price

Rab5 with N-mRFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100112 10 µg Ask for price

RhoB with N-mBFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100143 10 µg Ask for price

Rab4 with N-mBFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100145 10 µg Ask for price

Rab5 with N-mBFP tag for Early Endosome marking (10ug transfection-grade plasmid)

RC100146 10 µg Ask for price

ATG12 with C-tGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100004 10 µg Ask for price

MAPRE3 with C-tGFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100019 10 µg Ask for price

TUBA1B with N-tGFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100030 10 µg Ask for price

ATG12 with C-tRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100036 10 µg Ask for price

MAPRE3 with C-tRFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100051 10 µg Ask for price

MAPRE3 with C-mGFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100072 10 µg Ask for price

MAPRE3 with C-mRFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100106 10 µg Ask for price

MAPRE3 with C-mBFP tag for Cytoskeleton marking (10ug transfection-grade plasmid)

RC100140 10 µg Ask for price

SYP with N-tGFP tag for Synaptic vesicles marking (10ug transfection-grade plasmid)

RC100028 10 µg Ask for price

TGOLN with C-tGFP tag for Golgi apparatus marking (10ug transfection-grade plasmid)

RC100029 10 µg Ask for price

SYP with N-tRFP tag for Synaptic vesicles marking (10ug transfection-grade plasmid)

RC100060 10 µg Ask for price

TGOLN with C-tRFP tag for Golgi apparatus marking (10ug transfection-grade plasmid)

RC100061 10 µg Ask for price

TGOLN with C-mGFP tag for Golgi Apparatus marking (10ug transfection-grade plasmid)

RC100088 10 µg Ask for price

PXMP2 (PTS) with N-mGFP tag for Peroxisome marking (10ug transfection-grade plasmid)

RC100096 10 µg Ask for price

TGOLN with C-mRFP tag for Golgi Apparatus marking (10ug transfection-grade plasmid)

RC100122 10 µg Ask for price

PXMP2 (PTS) with N-mRFP tag for Peroxisome marking (10ug transfection-grade plasmid)

RC100130 10 µg Ask for price

TGOLN with C-mBFP tag for Golgi Apparatus marking (10ug transfection-grade plasmid)

RC100156 10 µg Ask for price

PXMP2 (PTS) with N-mBFP tag for Peroxisome marking (10ug transfection-grade plasmid)

RC100164 10 µg Ask for price

MAP1LC3A with N-tGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100020 10 µg Ask for price

MAP1LC3B with N-tGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100021 10 µg Ask for price

MAP1LC3A with N-tRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100052 10 µg Ask for price

MAP1LC3B with N-tRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100053 10 µg Ask for price

MAP1LC3A with N-mGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100068 10 µg Ask for price

MAP1LC3B with N-mGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100069 10 µg Ask for price

MAP1LC3A with N-mRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100102 10 µg Ask for price

MAP1LC3B with N-mRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100103 10 µg Ask for price

MAP1LC3A with N-mBFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100136 10 µg Ask for price

MAP1LC3B with N-mBFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100137 10 µg Ask for price

B4GalT1 with C-tGFP tag for Golgi apparatus marking (10ug transfection-grade plasmid)

RC100005 10 µg Ask for price

Di-Ras3 with N-tGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100011 10 µg Ask for price

B4GalT1 with C-tRFP tag for Golgi apparatus marking (10ug transfection-grade plasmid)

RC100037 10 µg Ask for price

Di-Ras3 with N-tRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100043 10 µg Ask for price

Di-Ras3 with N-mGFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100067 10 µg Ask for price

COX8A (MTS) with C-mGFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100092 10 µg Ask for price

Di-Ras3 with N-mRFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100101 10 µg Ask for price

COX8A (MTS) with C-mRFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100126 10 µg Ask for price

Di-Ras3 with N-mBFP tag for Autophagosome marking (10ug transfection-grade plasmid)

RC100135 10 µg Ask for price

COX8A (MTS) with C-mBFP tag for Mitochondria marking (10ug transfection-grade plasmid)

RC100160 10 µg Ask for price

FAK with N-tGFP tag for Focal adherin fiber marking (10ug transfection-grade plasmid)

RC100012 10 µg Ask for price

VCL with N-tGFP tag for Focal adherin fiber marking (10ug transfection-grade plasmid)

RC100031 10 µg Ask for price

ZYX with C-tGFP tag for Focal adherin fiber marking (10ug transfection-grade plasmid)

RC100032 10 µg Ask for price

FAK with N-tRFP tag for Focal adherin fiber marking (10ug transfection-grade plasmid)

RC100044 10 µg Ask for price

VCL with N-tRFP tag for Focal adherin fiber marking (10ug transfection-grade plasmid)

RC100063 10 µg Ask for price

ZYX with C-tRFP tag for Focal adherin fiber marking (10ug transfection-grade plasmid)

RC100064 10 µg Ask for price

FAK with N-mGFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100084 10 µg Ask for price

VCL with N-mGFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100085 10 µg Ask for price

ZYX with C-mGFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100086 10 µg Ask for price

FAK with N-mRFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100118 10 µg Ask for price

VCL with N-mRFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100119 10 µg Ask for price

ZYX with C-mRFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100120 10 µg Ask for price

FAK with N-mBFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100152 10 µg Ask for price

VCL with N-mBFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100153 10 µg Ask for price

ZYX with C-mBFP tag for Focal Adherin Fiber marking (10ug transfection-grade plasmid)

RC100154 10 µg Ask for price

Rab11a with N-mGFP tag for Recycling Endosome marking (10ug transfection-grade plasmid)

RC100082 10 µg Ask for price

Rab11a with N-mRFP tag for Recycling Endosome marking (10ug transfection-grade plasmid)

RC100116 10 µg Ask for price

Rab11a with N-mBFP tag for Recycling Endosome marking (10ug transfection-grade plasmid)

RC100150 10 µg Ask for price

CALR (ETS) with mRFP tag for Endoplasmic Reticulum marking (10ug transfection-grade plasmid)

RC100108 10 µg Ask for price

CALR (ETS) with mBFP tag for Endoplasmic Reticulum marking (10ug transfection-grade plasmid)

RC100142 10 µg Ask for price

B4GalT1 (GTS) with C-mGFP tag for Golgi Apparatus marking (10ug transfection-grade plasmid)

RC100087 10 µg Ask for price

B4GalT1 (GTS) with C-mRFP tag for Golgi Apparatus marking (10ug transfection-grade plasmid)

RC100121 10 µg Ask for price

B4GalT1 (GTS) with C-mBFP tag for Golgi Apparatus marking (10ug transfection-grade plasmid)

RC100155 10 µg Ask for price

pOET Sequencing Primers

GWB-200100 2 x 100 ul Ask for price

Gltp (untagged) - Mouse glycolipid transfer protein (Gltp), (10ug)

MC203852 10 µg Ask for price

Pltp (untagged) - Mouse phospholipid transfer protein (Pltp), (10ug)

MC216773 10 µg Ask for price

Pctp (untagged) - Mouse phosphatidylcholine transfer protein (Pctp), (10ug)

MC209154 10 µg Ask for price

Ttpa (untagged) - Mouse tocopherol (alpha) transfer protein (Ttpa), (10ug)

MC209849 10 µg Ask for price

Gltpd2 (untagged) - Mouse glycolipid transfer protein domain containing 2 (Gltpd2), (10ug)

MC201992 10 µg Ask for price

Mttp (untagged) - Mouse microsomal triglyceride transfer protein (Mttp), transcript variant 2, (10ug)

MC204263 10 µg Ask for price

Pctp (GFP-tagged) - Mouse phosphatidylcholine transfer protein (Pctp), (10ug)

MG219494 10 µg Ask for price

Ttpa (GFP-tagged) - Mouse tocopherol (alpha) transfer protein (Ttpa), (10ug)

MG221845 10 µg Ask for price

Pitpna (untagged) - Mouse phosphatidylinositol transfer protein, alpha (Pitpna), (10ug)

MC206047 10 µg Ask for price

Pitpnb (untagged) - Mouse phosphatidylinositol transfer protein, beta (Pitpnb), (10ug)

MC210037 10 µg Ask for price

Pitpnm2 (untagged) - Mouse phosphatidylinositol transfer protein, membrane-associated 2 (Pitpnm2), (10ug)

MC224144 10 µg Ask for price

Ttpal (untagged) - Mouse tocopherol (alpha) transfer protein-like (Ttpal), transcript variant 2, (10ug)

MC202535 10 µg Ask for price

CETP (untagged) - Human cholesteryl ester transfer protein, plasma (CETP), transcript variant 2

SC336099 10 µg Ask for price

Pitpnm2 (GFP-tagged) - Mouse phosphatidylinositol transfer protein membrane-associated 2 (Pitpnm2), (10ug)

MG216492 10 µg Ask for price

Pitpnb (GFP-tagged) - Mouse phosphatidylinositol transfer protein beta (Pitpnb), (10ug)

MG224376 10 µg Ask for price

CETP (untagged)-Human cholesteryl ester transfer protein, plasma (CETP)

SC124106 10 µg Ask for price

Pitpnc1 (untagged) - Mouse phosphatidylinositol transfer protein, cytoplasmic 1 (Pitpnc1), (10ug)

MC211221 10 µg Ask for price

Gltpd1 (untagged) - Mouse glycolipid transfer protein domain containing 1 (Gltpd1), (10ug)

MC200085 10 µg Ask for price

Plasmids shape the diverse accessory resistomes of Escherichia coli ST131

The human gut microbiome includes beneficial, commensal and pathogenic bacteria that possess antimicrobial resistance (AMR) genes and exchange these predominantly through conjugative plasmids. Escherichia coli is a significant component of the gastrointestinal microbiome and is typically non-pathogenic in this niche. In contrast, extra-intestinal pathogenic E. coli (ExPEC) including ST131 may occupy other environments like the urinary tract or bloodstream where they express genes enabling AMR and host cell adhesion like type 1 fimbriae.

The extent to which commensal E. coli and uropathogenic ExPEC ST131 share AMR genes remains understudied at a genomic level, and we examined this here using a preterm infant resistome. We found that individual ST131 had small differences in AMR gene content relative to a larger shared resistome. Comparisons with a range of plasmids common in ST131 showed that AMR gene composition was driven by conjugation, recombination and mobile genetic elements.

Plasmid pEK499 had extended regions in most ST131 Clade C isolates, and it had evidence of a co-evolutionary signal based on protein-level interactions with chromosomal gene products, as did pEK204 that had a type IV fimbrial pil operon. ST131 possessed extensive diversity of selective type 1, type IV, P and F17-like fimbriae genes that was highest in subclade C2. The structure and composition of AMR genes, plasmids and fimbriae vary widely in ST131 Clade C and this may mediate pathogenicity and infection outcomes.

Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs)

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A.

To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps.

Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

Emergence of plasmid-mediated tigecycline resistance tet(X4) gene in Escherichia coli isolated from poultry, food and the environment in South Asia

The recent emergence of mobile-tigecycline resistance tet(X) genes in human and animals in China seriously threats the clinical utility of tigecycline. Here we focused on the isolation and molecular characterization of plasmid-mediated tigecycline resistance tet(X4)-positive E. coli from different sources in Pakistan using MinION and Illumina sequencing. The tet(X4) gene was detected in four E. coli isolates from poultry, chicken meat, wild bird and the slaughterhouse wastewater in Pakistan. Co-existence of colistin resistance mcr-1 gene was also detected in three isolates.

The four isolates belonged to different sequence types and the tet(X4) gene was located on plasmids ranging from 12,331 bp to 113,610 bp belonging to IncFII and IncQ replicon types with two genetic contexts ISCR2-tet(X4)-abh-ISCR2-lysR-floR-virD2 and ΔISCR2-abh-tet(X4)-ISCR2-virD2-floR, respectively. In all the four E. coli strains, tet(X4) was transferable by conjugation to E. coli J53 host strain. In addition, three of four strains transferred tet(X4) to a wild-type carbapenem resistant E. coli strain. To our knowledge, this is the first report of the emergence of plasmid-mediated tet(X4) gene from Pakistan. The convergence of tigecycline and colistin resistance in South Asia is a serious threat to human health.

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