CN108823124B - Staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and screening method and application thereof - Google Patents

Staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and screening method and application thereof Download PDF

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CN108823124B
CN108823124B CN201810603967.7A CN201810603967A CN108823124B CN 108823124 B CN108823124 B CN 108823124B CN 201810603967 A CN201810603967 A CN 201810603967A CN 108823124 B CN108823124 B CN 108823124B
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staphylococcus epidermidis
resistant
gram
drug
bacteria
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CN108823124A (en
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李敏
刘倩
孟红委
刘瑶
刘俊兰
汪骅
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Renji Hospital Shanghai Jiaotong University School of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/44—Staphylococcus
    • C12R2001/45—Staphylococcus epidermidis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A61K2035/115—Probiotics

Abstract

The invention discloses staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and a screening method thereof, wherein the staphylococcus epidermidis is separated from a staphylococcus epidermidis strain planted in a nasal cavity of a healthy person, is named as N173-2, has obvious bacteriostatic action on clinically common gram-positive drug-resistant pathogenic bacteria, and has no bacteriostatic action on gram-negative drug-resistant bacteria. The genome size of the staphylococcus epidermidis strain N173-2 is 2459364bp, the staphylococcus epidermidis strain N173-2 contains 2 plasmids, the sizes of the plasmids are 65046bp and 55156bp respectively, the staphylococcus epidermidis strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018 at 03.04, and the preservation number of the staphylococcus epidermidis strain is CGMCC No. 15550. The invention also relates to application of the staphylococcus epidermidis N173-2 in preparing medicaments for resisting gram-positive drug-resistant bacteria. The invention screens the strains with broad-spectrum antibacterial activity to the clinical common drug-resistant pathogenic bacteria from the human symbiotic/colonization flora, and lays a foundation for the development of novel antibacterial molecules which are low-toxic and harmless to human bodies, have brand-new structures, are not easy to generate drug resistance and have stronger pertinence.

Description

Staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and screening method and application thereof
Technical Field
The invention relates to the field of microbial animal cell lines, in particular to staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria and a screening method thereof.
Background
Multiple drug resistant bacterial infections have become a global and intractable public health issue. In 2017, the WHO issued a global warning of 12 clinical drug-resistant bacteria, carbapenem antibiotics drug-resistant enterobacteriaceae (CRE), vancomycin drug-resistant enterococci (VRE), methicillin drug-resistant staphylococcus aureus (MRSA) and the like were the most troublesome pathogenic types for clinical anti-infective therapy. Most of the existing antibiotics are derived from metabolites of environmental (soil, etc.) microorganisms, but the limited resource of environmental microorganisms has been exploited to the greatest extent in the sixties of the last century. With the progress of human microbiology research in recent years, researchers are concerned about human symbiotic bacteria/colonized flora adapted to and co-evolved with human beings for millions of years, and the human symbiotic bacteria consisting of about 100 trillion bacteria affect the health of human bodies. The symbiotic flora at different parts of the human body forms a first defense line for resisting pathogen invasion, can compete with the pathogen for nutrition, prevent the pathogen from adhering and planting, decompose and absorb carbohydrates which can not be digested by the host, synthesize and secrete vitamins necessary for the host, and have dynamic and lasting influence on intestinal mucosa immunity and organism immune system. More importantly, the symbiotic flora of the human body can generate special metabolites acting on human body and environmental cells, wherein one of the metabolites is an antibacterial molecule which can inhibit the colonization and invasion of other bacteria at the part and provide necessary competitive advantages for producing bacteria.
The skin and upper respiratory tract of human body are the first defense line for resisting the invasion of external pathogenic microorganism, and the flora colonized on the skin and upper respiratory tract mainly comprises Coagulase Negative Staphylococcus (CNS), including Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Staphylococcus wowensis, Staphylococcus ledendri, etc. The presence of the CNS is of great importance in maintaining the normal physiological functions of the skin and upper respiratory tract and in protecting against the invasion of other pathogenic microorganisms.
At present, no relevant literature is reported on the relevance between staphylococcus epidermidis and gram-positive resistant bacteria.
Disclosure of Invention
The invention aims to screen a strain with broad-spectrum antibacterial activity on clinical common drug-resistant pathogenic bacteria from human symbiotic/colonization flora, and particularly relates to a staphylococcus epidermidis strain isolated from nasal cavity of a healthy person, wherein antibacterial molecules generated by the strain have broad-spectrum antibacterial activity on clinical common gram-positive drug-resistant bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first purpose of the invention is to provide a staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria, wherein the size of the staphylococcus epidermidis is 2459364bp, the content of G + C is 32%, the MLST is classified as ST73, and the staphylococcus epidermidis contains 4 prophages, 3 genomic islands and 2 plasmids, wherein the sizes of the plasmids are 65046bp and 55156bp respectively.
Furthermore, the Staphylococcus epidermidis is named as N173-2, the Staphylococcus epidermidis is classified and named as Staphylococcus epidermidis (CGMCC), the preservation number is CGMCC No.15550, the preservation date is 2018, 03 days 04 and 2018, the preservation unit is China general microbiological culture Collection center (CGMCC), and the preservation unit address is Beijing university Hodgkin district North Chen West Lu No.1 institute, China academy of sciences microbial research institute.
Further, the gram-positive resistant bacteria include: methicillin-resistant staphylococcus aureus, macrolide-resistant streptococcus pneumoniae, and micrococcus luteus.
Further, the methicillin-resistant staphylococcus aureus includes community-acquired methicillin-resistant staphylococcus aureus (CA-MRSA), hospital-acquired methicillin-resistant staphylococcus aureus (HA-MRSA), and livestock-derived methicillin-resistant staphylococcus aureus (LA-MRSA).
It is a second object of the present invention to provide a method for screening staphylococcus epidermidis having broad-spectrum antibacterial activity against gram-positive resistant bacteria, as described above, which comprises the steps of:
step 1) carrying out conventional flora culture on nasal swab samples of healthy subjects, and carrying out gene sequencing identification on each sample, wherein the analysis result shows that the nasal culturable flora of the healthy subjects contains a certain proportion of staphylococcus epidermidis on the level;
step 2) analyzing the inhibition effect of staphylococcus epidermidis fixed on the nasal cavity of a healthy subject on the growth of tested gram-positive drug-resistant pathogenic bacteria through a flat plate bacteriostasis test;
and 3) screening staphylococcus epidermidis which has obvious bacteriostatic action on all tested gram-positive drug-resistant pathogenic bacteria, and naming the staphylococcus epidermidis as N173-2.
In order to further optimize the screening method, the technical measures adopted by the invention also comprise the following steps:
further, the staphylococcus epidermidis in the step 1) has a certain proportion that: the proportion of staphylococcus in the nasal cavity culturable flora of healthy people is 82.0 percent; among the genus Staphylococcus, the proportion of Staphylococcus epidermidis is 79.8%.
Further, the healthy subjects in the step 1) and the step 2) are subjects 18-25 years old with normal physical examination, no past disease history and no hospital exposure history in the last half year.
Further, the gene sequencing in the step 1) is to perform gene sequencing of 20 single clones of MALDI-TOF MS and 16S rRNA for each sample.
The third purpose of the invention is to provide the application of the staphylococcus epidermidis in preparing the medicine for resisting gram-positive drug-resistant bacteria.
Furthermore, the gram-positive drug-resistant bacteria resisting drugs comprise methicillin-resistant staphylococcus aureus resisting drugs, macrolide antibiotic resisting streptococcus pneumoniae drugs and micrococcus tenhuangensis resisting drugs.
Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:
the invention screens a strain with broad-spectrum antibacterial activity to clinical common drug-resistant pathogenic bacteria from human symbiotic/colonization flora, in particular to a staphylococcus epidermidis strain N173-2 which is isolated from nasal cavity colonization of healthy people and is a staphylococcus epidermidis with relatively less genetic background. The antibacterial molecules generated by the strain have broad-spectrum antibacterial activity on common clinical gram-positive drug-resistant bacteria, and the foundation is laid for the development of novel antibacterial molecules which are low-toxic and harmless to human bodies, have brand-new structures, are not easy to generate drug resistance and have stronger pertinence.
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The Staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria is named as N173-2, is classified and named as Staphylococcus epidermidis (Staphylococcus epidermidis), has the preservation number of CGMCC No.15550, the preservation date of 2018, 03 months 04, the preservation unit is China general microbiological culture Collection center (CGMCC), and the preservation unit address is Beijing university Hodgkin district North Chen West Lu No.1 institute, China academy of sciences microbial research institute.
FIG. 1 is a schematic representation showing the broad spectrum bacteriostatic effect of Staphylococcus epidermidis N173-2/supernatant against pathogenic methicillin-resistant Staphylococcus aureus from different sources; note: the plates are coated with clinical common methicillin-resistant staphylococcus aureus from different national sources, staphylococcus epidermidis N173-2 bacteria/supernatant are inoculated on the plates coated with the drug-resistant bacteria, and the bacteriostatic result is observed after 24 hours.
FIG. 2 is a schematic diagram showing the results of whole genome sequencing analysis and phylogenetic analysis of Staphylococcus epidermidis N173-2 strain; wherein A: sequencing by adopting a third-generation PacBio RS II platform, and analyzing sequencing data by adopting mGenomeSubtractor software; b: the genome size of the staphylococcus epidermidis N173-2 strain is 2459364bp, the G + C content is 32%, the MLST is classified into ST73, the MLST contains 4 Prophage (Prophage), 3 GI-like region (genome island) and 2 plasmids, and the sizes of the plasmids are 65046bp and 55156bp respectively; c: phylogenetic analysis (evolution tree based on whole Genome core SNP) is carried out by adopting kSNP 3.0 software, and the evolution distance of the staphylococcus epidermidis N173-2 strain is far away from the staphylococcus epidermidis which is subjected to whole sequencing and included by the NCBI _ Genome at present.
Detailed Description
The invention discloses staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria, which is separated from a staphylococcus epidermidis strain planted in the nasal cavity of a healthy person, is named as N173-2, is preserved in 2018 at 03.04.7, and has the preservation number of CGMCC No. 15550. The invention also relates to a screening method of the staphylococcus epidermidis.
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
This example is a screening procedure for staphylococcus epidermidis having broad-spectrum antibacterial activity against gram-positive resistant bacteria, which comprises:
step 1) performing conventional flora culture on nasal cavity swab samples of 200 male and female patients of 18-25 years old who have normal physical examination in Shanghai regions, no past disease history and no hospital contact history in the last half year, and performing 20 monoclonal MALDI-TOF MS and 16S rRNA gene sequencing identification on each sample. The analysis result shows that the staphylococcus accounts for 82.0% of the nasal cavity culturable bacterial flora of healthy people on the genus level; further, the species of Staphylococcus bacteria were identified, and it was found that Staphylococcus epidermidis accounts for 79.8%.
And 2) analyzing the inhibition effect of the staphylococcus epidermidis fixed on the nasal cavity of a healthy person on the growth of pathogenic methicillin-resistant staphylococcus aureus (MRSA), macrolide antibiotic-resistant streptococcus pneumoniae (MRSP), micrococcus luteus (M.luteus), carbapenem antibiotic-resistant enterobacteriaceae (CRE) and the like separated in different countries, communities and hospitals through a plate bacteriostasis test (specifically tested methicillin-resistant staphylococcus aureus is shown in Table 1).
And 3) the staphylococcus epidermidis in the nasal cavity can generate antibacterial molecules with different effects, so that the colonization and invasion of clinical drug-resistant pathogenic bacteria can be effectively resisted. The N173-2 strain has obvious bacteriostatic action on all tested gram-positive drug-resistant pathogenic bacteria, and staphylococcus epidermidis planted in other nasal cavities cannot inhibit all tested gram-positive drug-resistant pathogenic bacteria (the bacteriostatic result is shown in figure 1).
Table 1: pathogenic methicillin-resistant staphylococcus aureus types for screening
Numbering Name of Strain MLST typing Source Source of
1 SF8300 ST8 USA300 CinAf-eMctiRoSnA
2 SF8300agr mutant ST8 USA300 CA-MRSA
3 MW2 ST1 USA400 CA-MRSA
4 MW2agr mutant ST1 USA400 CA-MRSA
5 BD02-25 ST8 USA500 HA-MRSA
6 BD-02-25agr mutant ST8 USA500 HA-MRSA
7 07-02662 ST80 Germany CA-MRSA
8 SF-1497 ST30 USA1100 CA-MRSA
9 CN1 ST72 South Korea CA-MRSA
10 SF 1208 ST36 USA200 HA-MRSA
11 BAA-40 ST239 Canada HA-MRSA
12 BAA-43 ST239 Canada HA-MRSA
13 SF-2561 ST5 USA100 HA-MRSA
14 S0385 ST398 Holland LA-MRSA
15 2014-58 ST9 China LA-MRSA
16 2012-RJ2 ST59 China CA-MRSA
17 2012-3 ST398 China CA-MRSA
18 09-770 ST239 China HA-MRSA
19 05-72 ST5 China HA-MRSA
In the embodiment, the staphylococcus epidermidis is found to be a main strain by identifying the nasal cavity symbiotic/colonized flora of the 18-25-year-old healthy people with normal physical examination, no past disease history and no hospital contact history in the last half year. Through carrying out plate bacteriostasis test screening on all staphylococcus epidermidis strains fixedly planted in the nasal cavities of healthy people, a staphylococcus epidermidis strain separated from the nasal cavities of the healthy people is found, the strain is named as N173-2, and the staphylococcus epidermidis strain has broad-spectrum bacteriostasis effect on all tested clinical gram-positive drug-resistant pathogenic bacteria (figure 1), but has no obvious bacteriostasis effect on clinical drug-resistant gram-negative pathogenic bacteria. The invention filters and concentrates the culture supernatant of staphylococcus epidermidis strain N173-2 planted in the nasal cavity, and the antibacterial effect still exists, which shows that the antibacterial molecules generated by staphylococcus epidermidis N173-2 are secreted into the supernatant outside the bacteria.
Example 2
This example is the gene sequencing of Staphylococcus epidermidis N173-2, which is a molecule with broad-spectrum antibacterial activity against the clinically gram-positive drug-resistant pathogenic bacteria selected in example 1. The results are as follows:
in this example, whole genome sequencing analysis was performed on staphylococcus epidermidis strain N173-2, which produces molecules with broad-spectrum antibacterial activity against clinically gram-positive drug-resistant pathogenic bacteria (part a of fig. 2), and it was found that the size was 2459364bp, the G + C content was 32%, the MLST type was ST73, which contained 4 prophages (prophages), 3 GI-like regions (genomic islands), and 2 plasmids, and the sizes of the plasmids were 65046bp and 55156bp, respectively (part B of fig. 2); and the phylogenetic analysis of the phylogenetic tree based on the whole Genome core SNP is carried out on the strain of the bacteria through kSNP 3.0 software, and the staphylococcus epidermidis N173-2 strain is found to have a longer evolution distance (part C in figure 2) than the whole sequenced staphylococcus epidermidis recorded by the current NCBI _ Genome, which shows that the staphylococcus epidermidis N173-2 is a strain of staphylococcus epidermidis with a less common genetic background.
The Staphylococcus epidermidis strain N173-2 in the embodiment is deposited in 03.04.2018, is classified and named as Staphylococcus epidermidis (Staphylococcus epidermidis), and has a preservation number of CGMCC No.15550, a preservation unit of China general microbiological culture Collection center (CGMCC), a preservation unit address of Beijing university Hokko No.1 Hopkins, Naja district, China academy of sciences and microbiology institute.
Example 3
This example is a bacteriostatic experiment verification of staphylococcus epidermidis strain N173-2, taking staphylococcus aureus USA300 as an example, the verification steps are as follows:
(1) staphylococcus aureus USA300 was picked and inoculated with 10ml of fresh TSB, incubated overnight at 37 ℃ and 220 rpm.
(2) Detecting the bacteria OD600 by a microplate reader, adjusting the bacteria OD600 to 0.0001, inoculating into 50ml of solid TSB culture medium, mixing well, and plating.
(3) A single clone of the N173-2 strain isolated from the nasal cavity of a healthy person was picked and inoculated into 10ml of fresh TSB, incubated at 37 ℃ and shaken at 220rpm overnight.
(4) Respectively taking the bacterial suspension, the supernatant obtained after the centrifugation of the bacterial culture and 5 mul of precipitate, dibbling the mixture on a solid plate containing USA300 bacteria, drying the solid plate, putting the dried solid plate in an incubator at 37 ℃ for overnight culture, and observing the inhibition zone the next day.
The verification experiment shows that the N173-2 strain has an obvious bacteriostatic action on staphylococcus aureus USA 300.
According to the embodiment, the staphylococcus epidermidis strain N173-2 separated from the nasal cavity of a healthy person for field planting is provided, and antibacterial molecules generated by the strain have broad-spectrum antibacterial activity on common clinical gram-positive drug-resistant bacteria, so that a foundation is laid for the development of novel antibacterial molecules which are low-toxic and harmless to human bodies, have brand-new structures, are not easy to generate drug resistance and have stronger pertinence.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (4)

1. The Staphylococcus epidermidis with broad-spectrum antibacterial activity on gram-positive drug-resistant bacteria is characterized in that the Staphylococcus epidermidis (Staphylococcus epidermidis) is named as N173-2, the preservation number of the Staphylococcus epidermidis is CGMCC No.15550, and the preservation unit is the China general microbiological culture Collection center; the size of the staphylococcus epidermidis is 2459364bp, the G + C content is 32%, the MLST type is ST73, the staphylococcus epidermidis contains 4 prophages, 3 genome islands and 2 plasmids, and the sizes of the plasmids are 65046bp and 55156bp respectively.
2. The staphylococcus epidermidis according to claim 1, wherein the gram-positive resistant bacteria comprise: methicillin-resistant staphylococcus aureus, macrolide-resistant streptococcus pneumoniae, and micrococcus luteus.
3. The application of staphylococcus epidermidis as set forth in any one of claims 1-2 in preparation of medicines for resisting gram-positive resistant bacteria.
4. The use of claim 3, wherein the gram-positive resistant bacteria drug comprises methicillin-resistant Staphylococcus aureus drug, macrolide-resistant Streptococcus pneumoniae drug, and Micrococcus tenuis drug.
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