CN106047898A - Novel integron In1289 containing multiple drug-resistant gene cassettes - Google Patents

Novel integron In1289 containing multiple drug-resistant gene cassettes Download PDF

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CN106047898A
CN106047898A CN201610329147.4A CN201610329147A CN106047898A CN 106047898 A CN106047898 A CN 106047898A CN 201610329147 A CN201610329147 A CN 201610329147A CN 106047898 A CN106047898 A CN 106047898A
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integron
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王冬国
周冬生
陈佳玉
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

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Abstract

The invention discloses a novel integron In1289, wherein the sequence of the integron is shown as SEQ ID NO.1. The integron is discovered from a genome of drug-resistant nontyphoidal salmonella EC7328. Therefore, the integron disclosed by the invention, with discovery thereof, has important theoretical and practical meaning for discussing a molecular mechanism of bacterial drug resistance transfer at a gene level on one side, and on the other side, the integron has a certain guidance function for clinical medication; and the integron is conducive to reduce the application of some antibacterial agents in the clinical field, and the integron is capable of reducing selective pressure of horizontal transfer of the drug-resistant gene cassettes and preventing explosive epidemic of drug-resistant bacteria.

Description

A kind of novel integron In1289 comprising multiple drug resistant gene box
Technical field
The invention belongs to Protocols in Molecular Biology and drug-resistant bacteria monitoring field, relate to a kind of newfound uncommon with large intestine angstrom The closely-related integron of Salmonella drug resistance.
Background technology
Colon bacillus, commonly referred to escherichia coli, be the ingredient of human normal intestinal microbial population, typically at intestinal For non-pathogenic bacteria, but also there are the escherichia coli of some serotypes can cause diarrhoea or the Extra intestinal infection of different symptoms.Pathogenic effects Mainly aggressivity, endotoxin and enterotoxin.According to different biological characteristicses, Escherichia coli is fallen into 5 types: cause a disease Property escherichia coli (EPEC), enterotoxigenic E.Coli (ETEC), enteroinvasive E.Coli (EIEC), enterohemorrhagic large intestine bar Bacterium (EHEC), intestinal adhesion escherichia coli (EAEC).Escherichia coli is caused by pollution drinking-water, food, polluted-water Disease code, the person of being in a bad way, can critical life；It addition, along with broad ectrum antibiotic is widely used, in hospital clinical Occur in that the colon bacillus of Extended-spectrum β-lactamases is popular, cause the biggest puzzlement, serious threat patient to hospital Healthy and safe.The resistance problems of colon bacillus has become the extremely important factor affecting human health.
Integron (integron) is a kind of exogenous gene that can pass through capture, and be translated into functional gene DNA fragmentation, is positioned on chromosome, transposon or plasmid, and can flow betwixt.It is mainly seen in gram-negative bacteria, whole with I class The discovery of zygote is distributed the widest in gram-negative bacteria.The basic structure of I class integron is made up of 3 parts, and two ends are that height is protected Keeping sequence, be called 5 ' conservative ends (5 '-CS) and 3 ' conservative ends (3 '-CS), 5 '-CS are about 1.4kb, 3 '-CS and are about 2kb, Region between 5 '-CS and 3 '-CS is referred to as variable region, and variable region is made up of one or more box genes, and box gene is typically containing anti- Bacterium Drug-resistant gene.Integron is a kind of movably Genetic elements, is positioned on plasmid or the chromosome of antibacterial, and portability is many Individual Drug-resistant genes, and can cause efficient, the fast transfer of drug resistant gene, causes the expansion between of the same race or different strain Dissipate, thus express the multi-drug resistant to different antibiotic.
Further investigation colon bacillus resistance mechanism, especially to the research about integron, can be strengthened infection strain The reasonable employment of EPDML understanding, beneficially antibacterials and the exploitation of antibiotics medicine；Meanwhile, antibacterials are strengthened Reasonable application, the outside induced environment that integron occurs, reduces molecular evolution can be reduced, delay multidrug resistance bacterial strain Raw.
Summary of the invention
The invention provides a kind of integron containing drug resistant gene box, by its named In1289, its sequence such as SEQ Shown in ID NO.1.This integron contains dfrA27 box gene, arr-3 box gene, catB3 box gene and aacA4 box gene.On State integron and express multidrug resistant gene, thus cause the strain containing this integron that Multiple Classes of Antibiotics medicine is had drug resistance Property.These antibiotic include: aminoglycoside antibiotics, sulfa antibiotics, chloromycetin series antibiotics and rifampicin class are anti- Raw element.
Common aminoglycoside antibiotics includes: streptomycin, gentamycin, kanamycin, tobramycin, A meter Ka Star.
Common sulfa antibiotics includes: bacteresulf (SIZ), sulfadimidine (SM2), sulfadiazine (SD), sulfamethoxazole (SMZ), sulfamonomethoxine (SMM), sulfasalazine (SASP), mafenide (SML), sulfanilamide Pyrimidine silver, sulfacetamide (SA) and bactrim (trimethoprim+sulfamethoxazole).
Common chloromycetin series antibiotics includes: chloromycetin, CAPS.
Common rifampicin class antibiotic includes: rifampicin, rifapentine.
The integron of the present invention is prepared by the following:
(1) in positive specimen cultivated by Taizhou municipal hospital patient's ICU sputum, 1 strain is isolated to amikacin drug resistance Antibacterial.
(2) then the monoclonal bacterial strain separated (is mended by VITEK 2 and susceptibility flat board agar diffusion method in operation sequence Fill drug sensitive test paper) carry out Bacteria Identification and drug sensitive test, this bacterial strain identified is that colon bacillus is by its named EC7328. Meanwhile, test confirms that this bacterial strain has notable drug resistance to Multiple Classes of Antibiotics.
(3) EC7328 strain culturing is expanded, extract the plasmid of described Resistant strain.
(4) plasmid after step (3) extraction purification is used BamHI enzyme action, agarose gel electrophoresis isolated one section Final length is 4, and this DNA fragmentation is connected by the DNA fragmentation of 324bp in conventional manner with pMD19-T carrier, and then heat shock turns Change method proceeds in competent cell E.coli TOP10, selects positive transformant with blue white macula experiment, order-checking；
(5) utilize ContigExpress Program software to carry out sequence assembly, analyze its structure, identify sequence such as Sequence shown in SEQ ID NO.1.
Present invention also offers a kind of recombiant plasmid containing integron In1289 sequence.Described plasmid comprises integron In1289 sequence.In the specific embodiment of the present invention, described recombiant plasmid is by integron In1289 sequence and pET32a Carrier restructuring forms, and integron insertion point is on pET32a carrier at BamHI restriction enzyme site.The sky that described recombiant plasmid uses Carrier can use any common plasmid vector to replace.The method of described recombiant plasmid is also well known to those skilled in the art Conventional method.
Present invention also offers a kind of joint bacterial strain containing integron In1289.In order to study the drug resistance of integron, The present invention utilizes bacterial plasmid transduction experiment to carry out the function of Testing and appraisal integron In1289, by EC7328 bacterial strain and E.coli J53 AzR(to Hydrazoic acid,sodium salt drug resistance) bacterial strain co-cultures, by containing Hydrazoic acid,sodium salt (300mg/L) and amikacin (0.06mg/L) Plate screening conjugon, does drug sensitive test by conjugon, and recipient bacterium has the drug resistance of donor bacterium.Wherein, amikacin used Available agents replaces: tobramycin, kalamycin, gentamycin, bactrim, SMZ, rifampicin.
Present invention also offers a kind of recombinant bacterial strain containing integron In1289.For being further characterized by above-mentioned joint bacterial strain Resistance derive from the integron in EC7328 bacterial strain, this integron In1289 is cloned in pET32a carrier by the present invention, whole Zygote insertion point is on pET32a carrier at BamHI restriction enzyme site.And be transformed in wild type E.coli JM109, logical Cross the flat board containing Amp and select positive colony (containing the antibacterial of pET32a-integron).Positive colony is done drug sensitive test, result Integron In1289 in display EC7328 bacterial strain can make restructuring E.coli JM109 bacterial strain to following Drug-resistant: A meter Ka Star, tobramycin, butylamine OK a karaoke club, gentamycin, bactrim, chloromycetin, rifampicin.
Advantages of the present invention and beneficial effect:
(1) present invention firstly discovers that the integron including multidrug resistant gene box that sequence is SEQ ID NO.1 In1289, the nucleotide sequence of this integron has no report in the prior art.
(2) discovery of integron sequence of the present invention, provides certain directive function for clinical application.Be conducive to clinically Reduce the use of some antibacterials, reduce the selection pressure of such drug resistant gene box horizontal transfer, prevent the quick-fried of drug-resistant bacteria The property sent out is popular.
Accompanying drawing explanation
Fig. 1 is the structural representation of the integron In1289 of the present invention.
Specific embodiment
Further illustrating the present invention below in conjunction with specific embodiment, embodiments of the invention are only used for explaining the present invention, It is not intended to limit protection scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition such as sambrook et al., Molecular cloning: retouched in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) The condition stated, or according to the condition proposed by manufacturer.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The qualification of embodiment 1 integron In1289
1, the separation of EC7328 bacterial strain and qualification
1.1 material
Bacterial susceptibility card: the AST-GN13 of France bioMerieux.AST-GN13 susceptibility kind has: amikacin, ammonia benzyl XiLin, ampicillin/sulbactam, aztreonam, cefazolin sodium, cefepime, cefotetan, ceftazidime, ceftriaxone, ring third Sha Xing, ertapenem, gentamycin, imipenum, levofloxacin, nitrofurantoin, piperacillin/Tazobactam Sodium, appropriate cloth are mould Element, SMZ.
Supplement drug sensitive test paper (experiment of susceptibility flat board agar diffusion): the scraps of paper derive from Oxoid company of Britain, have the new promise of compound recipe Bright (30 μ g), chloromycetin (30 μ g) and rifampicin (5 μ g).
1.2 method
Instrumental: Taizhou municipal hospital patient's ICU sputum is cultivated positive bacterial strain transferred species and separates training to blood plate Support (at 35 DEG C containing 5%CO2Incubator is cultivated 16-18h), then by the pure antibacterial of separation in operation sequence on VITEK 2 Machine makees Bacteria Identification and drug sensitive test.
Supplement drug sensitive test: spread upon on MH flat board by the bacterium solution of 0.5 Maxwell unit, stick the new promise of compound recipe in operation sequence Bright, chloromycetin and the rifampicin scraps of paper, at 35 DEG C containing 5%CO2After 16-18h cultivated by incubator, identify by CLSI 2015 standard Drug sensitivity tests.
1.3 result
1.3.1 instrumental result
It is accredited as colon bacillus through VITEK 2 microbiological analysis instrument, identifies that probability is 99%, it is named EC7328。
1.3.2 drug sensitive experiment result
Drug sensitive experiment result shows, EC7328 bacterial strain produces resistance for following medicine: amikacin, gentamycin, appropriate Obramycin, bactrim, SMZ, chloromycetin, rifampicin.
2, the separation of integron In1289 and qualification
2.1 method
2.1.1EC7328 the plasmid extraction of bacterial strain
The plasmid using TaKaRa extracts test kit on a small quantity, and operating procedure extracts bacteria plasmid DNA to specifications, as Pcr template ,-20 DEG C of Refrigerator stores are standby.
2.1.2 the acquisition of integron
(1) cleaning is dried and sterilized 0.5mL eppendorf pipe is numbered, be separately added into micropipette rifle EC7328 plasmid DNA 1 μ g and 10 × restriction enzyme reaction buffer 2 μ L (Fermentas), add redistilled water and make always Volume 19 μ L, by adding 1 μ L enzyme liquid (Fermentas) after solution mixing in pipe, gets rid of with microcentrifuge, makes solution concentrate At the bottom of pipe.
(2), after mixing reaction system, eppendorf pipe is placed on suitable holder and (is inserted on plastic foamboard), 37 DEG C of water bath heat preservation 2-3h, make endonuclease reaction complete.
(3) often pipe adds 2 μ L 0.1mol/L EDTA (pH8.0), and mixing, with stopped reaction.
(4) product after enzyme action is separated through agarose gel electrophoresis.
2.1.3 the qualification of integron
(I) DNA fragmentation that final length is 4,324bp through agarose gel electrophoresis isolated is reclaimed.
(2) use conventional method, the DNA fragmentation that step (1) reclaims is connected with pMD19-T carrier, the most heat-shock transformed Method proceeds in competent cell E.coli TOP10, selects positive transformant with blue white macula experiment, order-checking.
(3) utilize ContigExpress Program software to carry out sequence assembly, analyze its structure, identify sequence such as Shown in SEQ IDNO.1.
2.2 result
Sequencing result is analyzed and shows after splicing, and the DNA fragmentation of a length of 4,324bp belongs to I class integron, this integron Including basic structure and multiple box gene of I type integron, sequence as shown in SEQ ID NO.1, structural representation such as Fig. 1 institute Show.
The function of embodiment 2 plasmid transduction experimentation integron In1289
1, method
(1) donor bacterium is EC7328 bacterial strain, and recipient bacterium is E.coli J53 AzR(to Hydrazoic acid,sodium salt drug resistance).By donor Bacterium, recipient bacterium are inoculated on LB flat board respectively, 35 DEG C of incubated overnight.The single bacterium colony of picking is inoculated in the LB meat soup of 4mL respectively, 37 DEG C of 220r/min shake bacterium and cultivate to exponential phase.Respectively take 0.5mL for, recipient bacterium in the LB meat soup of 4mL, 37 DEG C of static mistakes Night cultivates.Conjugon screens with pancreas soy agar (TSA) plate, containing Hydrazoic acid,sodium salt (300mg/L) and A meter Sha star in flat board (0.06mg/L).Put 35 DEG C and hatch 18-24h.Extract the plasmid (step is with embodiment 1) of Resistant strain, integrate with PCR method The detection of sub-In1289 sequence.
The primer of amplification integron In1289 sequence is as shown in table 1:
Table 1 primer sequence
The reaction system (50 μ L) of PCR:
Mg2+2.5mmol/L, primer 30pmol/L, dNTP 0.2mmol/L, 2.5U Tap enzyme and 2 μ L DNA profilings.
Amplification condition:
94 DEG C of denaturations 3min, then 94 DEG C of degeneration 1min, anneal under 50-59 DEG C of suitable temperature 1min, 72 DEG C of extensions 1min, 30 circulations, last 72 DEG C extend to 10min.
Above-mentioned PCR primer is carried out agarose gel electrophoresis, reclaims the DNA fragmentation of all amplifications, will expand in conventional manner Increasing fragment to be connected with pMD19-T carrier respectively, the most heat-shock transformed method proceeds in competent cell E.coli TOP10, white with indigo plant Positive transformant is selected in speckle experiment, order-checking.
(2) positive strain being carried out drug sensitive experiment, step is with embodiment 1.
1.2 result
Drug sensitive experiment result shows, integron In1289 sequence expresses the drug sensitive reaction of positive strain with EC7328 bacterial strain phase With, represent integron In1289 sequence joint and shift successfully, and this integron result in conjugative bacteria and creates agents Drug resistance: amikacin, gentamycin, tobramycin, SMZ, bactrim, chloromycetin, rifampicin.
The function of embodiment 3 gene recombinaton experimentation integron In1289
1, method
(1) PCR primer (nucleotides sequence is classified as SEQ ID NO.1) in embodiment 2 is connected with pMD19-T carrier, even Thing of practicing midwifery adds in the competence of 100 μ L E.coli JM109 and converts, and cultivates on the flat board containing IPTG, x-gal, Amp, IPTG, x-gal, Amp Resistant strain is extracted plasmid order-checking (step is with embodiment 1), and whether detection integron In1289 inserts In genome.Then integron In1289 sequence being cloned in pET32a carrier, the insertion point of integron carries at pET32a On body at BamHI restriction enzyme site, conventionally prepare, obtain the pET32a recombiant plasmid containing integron In1289 sequence. By in recombinant plasmid transformed to competence E.coli JM109 cell.
(2) the clone bacterium successfully constructed is carried out Resistance detection
Use France's VITEK 2 Bacteria Identification of bioMerieux and Analysis of Drug Susceptibility system and susceptibility flat board agar diffusion side Method (supplementing drug sensitive test paper) carries out Resistance detection to recombinant bacterium E.coli JM109 (pET32a-integron In1289).
2, result
Drug sensitive experiment result shows, the drug sensitive reaction of recombinant bacterium E.coli JM109 (pET32a-integron In1289) is same EC7328 bacterial strain is identical, represents integron In1289 sequence and is the most successfully incorporated in the genome of E.coli JM109, and should Integron result in recombinant bacterium E.coli JM109 and creates the drug resistance of agents: amikacin, gentamycin, appropriate cloth are mould Element, SMZ, bactrim, chloromycetin, rifampicin.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for this For the those of ordinary skill in field, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement And modification, these improve and modify also by the protection domain falling into the claims in the present invention.

Claims (9)

1. an integron, it is characterised in that described integron sequence is as shown in SEQ ID NO.1, and described integron is named In1289。
2. a recombiant plasmid, it is characterised in that comprise the integron described in claim 1 in described recombiant plasmid.
Recombiant plasmid the most according to claim 2, it is characterised in that the load used in the preparation process of described recombiant plasmid Body constitution grain is pET32a carrier.
4. a conjugative bacteria, it is characterised in that containing the integron described in claim 1 in described conjugative bacteria；Described connect Closing antibacterial is by EC7328 bacterial strain and E.coliJ53 AzRPrepared by bacterial strain.
5. a recombinant bacteria, it is characterised in that containing the integron described in claim 1 in described recombinant bacteria；Described heavy Group antibacterial is to proceed to the recombiant plasmid described in Claims 2 or 3 be prepared from E.coli JM109 bacterial strain.
6. the acquisition methods of the integron described in claim 1, it is characterised in that said method comprising the steps of:
(1) in positive specimen cultivated by Taizhou municipal hospital patient's ICU sputum, isolate thin to amikacin drug resistance of 1 strain Bacterium；
(2) monoclonal bacterial strain step (1) separated is in operation sequence by VITEK 2 and susceptibility flat board agar diffusion method, same Time supplement drug sensitive test paper and carry out Bacteria Identification and drug sensitive test, this bacterial strain identified is escherichia coli, named EC7328；
(3) EC7328 strain culturing is expanded, extract the plasmid of described EC7328 bacterial strain；
(4) plasmid after step (3) extraction purification being used BamHI enzyme action, agarose gel electrophoresis isolated one section is last The DNA fragmentation of a length of 4,324bp, is connected with pMD19-T carrier in conventional manner by this DNA fragmentation, the most heat-shock transformed method Proceed in competent cell E.coli TOP10, select positive transformant with blue white macula experiment, order-checking；
(5) utilize ContigExpress Program software to carry out sequence assembly, analyze its structure, identify sequence such as SEQ Sequence shown in ID NO.1.
7. the preparation method of the conjugative bacteria described in a claim 4, it is characterised in that the concrete operations of described preparation method Step is as follows: by EC7328 bacterial strain and E.coli J53 AzRBacterial strain co-cultures, by containing Hydrazoic acid,sodium salt and EC7328 bacterial strain The plate screening conjugon of tolerance medicine.
Preparation method the most according to claim 7, it is characterised in that described EC7328 bacterial strain tolerance medicine is A meter Ka Star.
9. the preparation method of the recombinant bacteria described in a claim 5, it is characterised in that the concrete operations of described preparation method Step is as follows: be cloned in pET32a carrier by integron In1289, and then recombinant plasmid transformed enters wild type E.coli In JM109, select positive colony by the flat board containing amikacin.
CN201610329147.4A 2016-05-10 2016-05-10 Novel integron In1289 containing multiple drug-resistant gene cassettes Pending CN106047898A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913399A (en) * 2019-04-12 2019-06-21 贵州省水产研究所 A kind of Aeromonas media integron containing multiple drug resistant gene boxes and its acquisition methods and application

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450765A (en) * 2014-11-13 2015-03-25 王冬国 Integron In1069

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104450765A (en) * 2014-11-13 2015-03-25 王冬国 Integron In1069

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GENBANK: "JF731030.1", 《GENBANK》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109913399A (en) * 2019-04-12 2019-06-21 贵州省水产研究所 A kind of Aeromonas media integron containing multiple drug resistant gene boxes and its acquisition methods and application
CN109913399B (en) * 2019-04-12 2021-08-20 贵州省水产研究所 Aeromonas intermedia integron containing multiple drug-resistant gene cassettes and obtaining method and application thereof

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