CN120314419A - A rapid and non-destructive method for identifying complex bacteria - Google Patents
A rapid and non-destructive method for identifying complex bacteriaInfo
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- CN120314419A CN120314419A CN202510803625.XA CN202510803625A CN120314419A CN 120314419 A CN120314419 A CN 120314419A CN 202510803625 A CN202510803625 A CN 202510803625A CN 120314419 A CN120314419 A CN 120314419A
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Abstract
本发明属于细菌鉴别领域，具体涉及一种快速无损鉴别复合细菌的方法。所述方法包括如下步骤：将细菌单菌落进行培养，完成后分别测定菌液OD600nm吸光度值，然后稀释菌液至OD600nm=0.05，得到稀释菌液；采用固相微萃取对所述稀释菌液进行吸附；然后经气相色谱、质谱检测后，对细菌VOCs代谢谱进行主成分分析、偏最小二乘判别/OPLS‑DA分析，用于确定不同细菌的差异VOCs，从而判别不同的细菌。本发明采用固相微萃取‑气相质谱技术检测细菌挥发性有机化合物，并联合采用多元统计分析方法建立了无损检测、快速鉴别四种不同细菌及耐药铜绿假单胞菌的方法，具有检测时间短、对样本无破坏和高特异性等优势。
The present invention belongs to the field of bacterial identification, and specifically relates to a method for rapid and non-destructive identification of composite bacteria. The method comprises the following steps: culturing a single bacterial colony, determining the absorbance value of the bacterial solution at OD 600nm after completion, and then diluting the bacterial solution to OD 600nm = 0.05 to obtain a diluted bacterial solution; using solid phase microextraction to adsorb the diluted bacterial solution; then after gas chromatography and mass spectrometry detection, the bacterial VOCs metabolic spectrum is subjected to principal component analysis and partial least squares discriminant/OPLS-DA analysis to determine the differential VOCs of different bacteria, thereby discriminating different bacteria. The present invention uses solid phase microextraction-gas mass spectrometry technology to detect bacterial volatile organic compounds, and jointly uses multivariate statistical analysis methods to establish a method for non-destructive detection and rapid identification of four different bacteria and drug-resistant Pseudomonas aeruginosa, which has the advantages of short detection time, no damage to samples, and high specificity.
Description
Technical Field
The invention belongs to the field of bacteria identification, and particularly relates to a method for rapidly and nondestructively identifying composite bacteria.
Background
Volatile organic compounds (Volatile Organic Compounds, VOCs) are a class of carbon-based compounds with small molecular weight (generally <300 Da), low boiling point (50-260 ℃) and volatility at normal temperature, and include aldehydes, ketones, esters, hydrocarbons, sulfides and the like. They result from biological metabolic activities (e.g., bacteria, fungi, host cells) or environmental interactions, which carry a large amount of information on physiological/pathological states as a potential biomarker. Compared with the blood and urine sample acquisition mode, the VOCs are noninvasive, convenient and continuously available, and can dynamically reflect the microbial activity change in real time. Thus, this provides a theoretical basis for the identification of bacteria by VOCs.
Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA), acinetobacter baumannii (Acinetobacter baumannii, AB), klebsiella pneumoniae (Klebsiella pneumoniae, KP) and Staphylococcus aureus (Staphylococcus aureus, SA) are common bacteria, but the prior art still has the following disadvantages:
1. The prior detection technology takes time, and the traditional culture method depends on bacterial amplification and biochemical identification, and takes up to 24-72 hours. The drug resistance detection needs an additional drug sensitivity test, and is further prolonged to a plurality of days.
2. Destructive sample processing limits subsequent applications in which conventional molecular detection (e.g., PCR) and mass spectrometry techniques (e.g., MALDI-TOF) require lysis of bacteria or extraction of nucleic acids/proteins, disrupting sample integrity.
3. Drug resistance detection relies on predicting genotype or phenotype delay, the existing molecular method can only target known drug resistance genes (such as mecA and blaNDM) and cannot identify novel or phenotypic drug resistance mechanisms, and drug sensitivity tests rely on in vitro culture, and sensitivity is influenced by inoculation amount.
Although the existing multiple instrument combination technology has a plurality of advantages, in practical application, the problems in aspects of sample collection, pretreatment, data analysis and the like are required to be solved, so that the accuracy and the reliability of diagnosis are improved. Some existing diagnostic methods based on VOCs have the defects of higher detection limit, weak specificity and the like, and further improvement and perfection are needed.
Therefore, the technical scheme of the invention is provided based on the above.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for rapidly and nondestructively identifying composite bacteria, which comprises the following steps:
(1) Culturing single bacterial colonies, respectively measuring the absorbance value of bacterial liquid OD 600nm after the single bacterial colonies are cultured, and then diluting the bacterial liquid until OD 600nm = 0.05 to obtain diluted bacterial liquid;
(2) Absorbing the diluted bacterial liquid by adopting solid-phase microextraction;
(3) And then, after gas chromatography and mass spectrometry detection, performing Principal Component Analysis (PCA) and partial least squares discrimination/OPLS-DA analysis on the bacterial VOCs metabolic spectrum to determine the difference VOCs of different bacteria, thereby discriminating different bacteria.
Preferably, the composite bacteria include pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae and staphylococcus aureus.
Preferably, the pseudomonas aeruginosa includes pseudomonas aeruginosa drug resistant bacteria and pseudomonas aeruginosa sensitive bacteria.
Preferably, in the step (1), the culture condition is that the constant temperature shaking culture is carried out for 10-14 hours under the conditions of 36-38 ℃ and 260-300 r/min.
Preferably, in the step (2), the adsorption mode is that 80 mu m DVB/CWR/PDMS fiber head is adopted for adsorption for 1h under the condition of 36-38 ℃.
Preferably, in the step (3), the temperature programming is carried out at the initial temperature of 40 ℃ for 2min, the temperature is raised to 100 ℃ at the speed of 10 ℃ per min, then raised to 200 ℃ at the speed of 5 ℃ per min, and finally raised to 250 ℃ at the speed of 25 ℃ per min for 2min during gas chromatography detection.
Preferably, in the step (3), during mass spectrum detection, the mass spectrum conditions are ionization mode EI, electron energy 70eV, ion source temperature 230 ℃ and quaternary rod temperature 150 ℃, acquisition of a spectrum after 3min sample injection, and mass scanning range m/z 35-300.
Preferably, in step (3), 9 different VOCs of hexamethylcyclotrisiloxane, 2, 5-dimethylpyrazine, benzaldehyde, octamethyltetrasiloxane, 2-ethylhexanol, 1-undecene, p-trimethylsiloxyphenyl-bis (trimethylsiloxy) ethane, 2, 7-tetramethyl-3-oxa-6-thia-2, 7-disilazane, 2, 4-di-tert-butylphenol are selected in the identification of Pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae and Staphylococcus aureus.
Preferably, in the step (3), 7 different VOCs are selected from 2, 5-dimethylpyrazine, octamethyl cyclotetrasiloxane, 2-ethyl-5-methylpyrazine, 2-ethylhexanol, 1-undecene, dodecyl methyl pentasiloxane and 2, 4-di-tert-butylphenol when the pseudomonas aeruginosa resistant bacteria and the pseudomonas aeruginosa sensitive bacteria are identified.
The beneficial effects of the invention are as follows:
The invention adopts a solid phase microextraction-gas phase mass spectrometry (SPME-GC-MS) technology to detect bacterial Volatile Organic Compounds (VOCs), and combines a multivariate statistical analysis method to establish a method for nondestructive testing and rapid identification of four different bacteria and drug-resistant pseudomonas aeruginosa, which comprises the following steps:
1. Traditional culture methods rely on bacterial amplification and biochemical identification, taking up to 24-72 hours. The drug resistance detection needs an additional drug sensitivity test, and is further prolonged to a plurality of days. The invention realizes the identification of pathogenic bacteria and drug-resistant pseudomonas aeruginosa within 1.5 hours by directly capturing bacterial VOCs, and obviously shortens the clinical diagnosis window period.
2. Conventional molecular detection (e.g., PCR) and mass spectrometry techniques (e.g., MALDI-TOF) require lysis of bacteria or extraction of nucleic acids/proteins, disrupting sample integrity. According to the invention, through optimizing SPME adsorption phase types, extraction modes and adsorption time parameters, the enrichment efficiency of VOCs is improved, the activity of bacterial samples is reserved, and the subsequent drug sensitivity retest or genomics research is supported.
3. The existing molecular method can only target known drug resistance genes (such as mecA and blaNDM) and cannot identify novel or phenotypic drug resistance mechanisms, and the drug sensitivity test depends on in vitro culture, and the sensitivity is influenced by the inoculation amount. According to the invention, through analyzing the drug-resistant pseudomonas aeruginosa specific VOCs metabolic spectrum, phenotype drug resistance characteristics are directly related, and no genetic marker is required to be predicted.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph comparing the effect of different fiber heads on extraction. Wherein A is a PDMS (100 μm) fiber head, B is a CWR/PDMS (95 μm) fiber head, and C is a DVB/CWR/PDMS (80 μm) fiber head.
FIG. 2 is a graph of adsorption time versus peak area.
Fig. 3 is a graph of VOCs stability data.
FIG. 4 is a metabolic profile of different bacteria VOSs. Wherein LB is LB culture medium, PA is Pseudomonas aeruginosa, SA is Staphylococcus aureus, AB is Acinetobacter baumannii, KP is Klebsiella pneumoniae.
FIG. 5 is a graph showing the VOCs profile of various bacteria, pseudomonas aeruginosa and susceptible bacteria. Wherein LB is LB culture medium, PA is Pseudomonas aeruginosa, SA is Staphylococcus aureus, AB is Acinetobacter baumannii, KP is Klebsiella pneumoniae, and 150, 160, 170, D3, D4 and D5 are drug-resistant Pseudomonas aeruginosa.
FIG. 6 is a thermogram of differential composition analysis of drug-resistant bacteria and susceptible bacteria of four bacteria and Pseudomonas aeruginosa. Wherein the components correspond to the peak numbers of hexamethyl-cyclotrisiloxane (peak number 1), 2, 5-dimethylpyrazine (peak number 10), benzaldehyde (peak number 13), octamethyl-cyclotrisiloxane (peak number 15), 2-ethyl-5-methylpyrazine (peak number 17), 2-ethylhexanol (peak number 20), 1-undecene (peak number 26), p-trimethylsiloxyphenyl-bis (trimethylsiloxy) ethane (peak number 30), 2, 7-tetramethyl-3-oxa-6-thia-2, 7-disilazane (peak number 35), dodecylpentasiloxane (peak number 36), 2, 4-di-tert-butylphenol (peak number 37).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Examples
The embodiment provides a method for rapidly and nondestructively identifying composite bacteria, which comprises the following steps:
(I) Method optimization of Solid Phase Microextraction (SPME) for adsorbing VOCs
(I-1) examining comparison of adsorption effect of SPME species on VOCs
According to the characteristics of VOCs, three coatings SPME of PDMS (100 μm), CWR/PDMS (95 μm) and DVB/CWR/PDMS (80 μm) are selected for the embodiment, and the VOCs are adsorbed for about 1h. As shown in FIG. 1, by comparing the areas of the chromatographic peaks of the VOCs, the adsorption effect of DVB/CWR/PDMS (80 μm) on the VOCs is determined to be optimal, and the obtained VOCs are more abundant.
(I-2) Effect of adsorption time on VOCs adsorption Effect
As shown in FIG. 2, the adsorption effect of the VOCs is better by adopting DVB/CWR/PDMS (80 μm) to adsorb samples (such as respiratory gas) for 10min and 60min, and the adsorption effect of the VOCs on the VOCs components is better by determining that the adsorption time is increased after the adsorption time reaches 1h and the VOCs enter an equilibrium state. Therefore, the selective adsorption time was 1h.
(I-3) stability study of VOCs gas
As shown in fig. 3, the stability of VOCs gas plays a key role in the stability of analysis results, and by examining the respiratory gas placement for 0, 3, 4, 5, 6 hours, it was finally determined that VOCs remained stable for less than 4 hours.
(II) analysis of different bacterial compositions
(II-1) cultivation of bacteria Single colonies of bacteria were picked and placed in sterile EP tubes containing LB medium and cultured at 37℃under constant temperature and 280r/min for 12 hours with shaking. After the completion, the absorbance value of the bacterial liquid OD 600nm was measured, and then the bacterial liquid was diluted with LB to OD 600nm =0.05.
(II-2) VOCs adsorption, namely accurately sucking 10mL of the diluted bacterial liquid, placing the bacterial liquid in a 20mL headspace sample injection bottle, and adsorbing by adopting 80 mu m DVB/CWR/PDMS fiber head for 1h (37 ℃). After the end, the sample was analyzed at 250℃for 3min at the gas inlet. Analysis was performed using a gas chromatography-mass spectrometry (GC-MS) combination.
(II-3) GC-MS analysis the column was a DB-5MS column (30 m. Times.0.25 mm,0.25 μm). The temperature programming is that the initial temperature is 40 ℃ and kept for 2min, the temperature is increased to 100 ℃ at the speed of 10 ℃ per min, then is increased to 200 ℃ at the speed of 5 ℃ per min, and finally is increased to 250 ℃ at the speed of 25 ℃ per min and kept for 2min. The temperature of the sample inlet is 250 ℃, sample injection is not split, and the carrier gas is helium. Constant flow mode 1mL/min, auxiliary temperature 250 ℃. The mass spectrum conditions are ionization mode EI, electron energy 70eV, ion source temperature 230 ℃ and quaternary rod temperature 150 ℃, the mass spectrum is acquired after 3min of sample injection, and the mass scanning range m/z is 35-300. And searching the corresponding mass spectrograms in the chromatograms by adopting a workstation spectrum library.
(II-4) by performing Principal Component Analysis (PCA) and partial least squares discrimination (PLS-DA)/OPLS-DA analysis on the metabolic spectra of VOCs of Pseudomonas aeruginosa (Pseudomonas aeruginosa, PA), acinetobacter baumannii (Acinetobacter baumannii, AB), klebsiella pneumoniae (Klebsiella pneumoniae, KP) and Staphylococcus aureus (Staphylococcus aureus, SA), and finally determining the differential VOCs of different bacteria by examination. Unlike single VOCs differential marker compounds, this example determines multi-component VOCs for discriminating different bacteria by multivariate statistical algorithm, and the characteristic patterns of VOCs of different bacteria are shown in fig. 4.
The results of PCA analysis of the different bacteria VOCs showed that the different bacteria were each approximately grouped into classes, the VOCs were significantly different from LB, indicating that the different bacteria had respective specific VOCs, and the PCA analysis of the different bacteria showed R 2X=0.662,Q2 = 0.380, as shown by A in FIG. 5, and the OPLS-DA analysis showed R 2X=0.805,R2Y=0.835,Q2 = 0.78. VIP was screened for more than 1, p <0.05 as the difference VOCs, and hexamethyl cyclotrisiloxane, 2, 5-dimethylpyrazine, benzaldehyde, octamethyl cyclotrisiloxane, 2-ethylhexanol, 1-undecene, p-trimethylsiloxyphenyl-bis (trimethylsiloxy) ethane, 2, 7-tetramethyl-3-oxa-6-thia-2, 7-disilazane, 2, 4-di-tert-butylphenol, and a total of 9 difference VOCs were screened as shown in B in fig. 5. Comparison of the relative amounts of the different components as shown in A in FIG. 6, the above-mentioned relative amounts of the different components were significantly different among different bacteria. Wherein, the content of 2, 5-dimethyl pyrazine (peak number 10), p-trimethylsiloxyphenyl-bis (trimethylsiloxy) ethane (peak number 30) and 2, 4-di-tert-butylphenol (37) in the PA is relatively less, the content of 2, 5-dimethyl pyrazine (peak number 10), benzaldehyde (peak number 13) and 2, 4-di-tert-butylphenol (peak number 37) in the AB is relatively higher, the content of 2, 4-di-tert-butylphenol (peak number 37) in the KP is the highest, the content of other components is relatively lower, and the content of 2, 5-dimethyl pyrazine (peak number 10), benzaldehyde (peak number 13) and octamethyl cyclotetrasiloxane (peak number 15) and 2, 4-di-tert-butylphenol (peak number 37) in the SA are higher.
Unlike VOCs distribution of different bacteria, pseudomonas aeruginosa resistant bacteria (resistant bacteria numbers 150, 160, 170, D3, D4, D5) are distributed around their susceptible bacteria (PA), and PCA analysis R 2X=0.519,Q2 =0.161, as shown in C in fig. 5, indicates that the differences in VOCs of resistant bacteria and susceptible bacteria are small. The PLS-DA analysis results are shown as D in FIG. 5, R 2X=0.642,R2Y=0.732,Q2 = 0.600 for PLS-DA, VIP >1, p <0.05 were chosen as the difference VOCs, 7 difference VOCs for 2, 5-dimethylpyrazine, octamethylcyclotetrasiloxane, 2-ethyl-5-methylpyrazine, 2-ethylhexanol, 1-undecene, dodecylpentasiloxane, 2, 4-di-tert-butylphenol. As shown in B in fig. 6, the difference composition thermal diagram shows that the relative contents of 2, 5-dimethylpyrazine (peak No. 10), octamethylcyclotetrasiloxane (peak No. 15), dodecamethylpentasiloxane (peak No. 36) and 2, 4-di-tert-butylphenol (peak No. 37) were significantly increased and the relative contents of 2-ethyl-5-methylpyrazine (peak No. 17), 2-ethylhexanol (peak No. 20) and 1-undecene (peak No. 26) were decreased as compared with PA.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. A method for rapid, non-destructive identification of a composite bacterium, said method comprising the steps of:
(1) Culturing single bacterial colonies, respectively measuring the absorbance value of bacterial liquid OD 600nm after the single bacterial colonies are cultured, and then diluting the bacterial liquid until OD 600nm = 0.05 to obtain diluted bacterial liquid;
(2) Absorbing the diluted bacterial liquid by adopting solid-phase microextraction;
(3) And then, after gas chromatography and mass spectrometry detection, carrying out principal component analysis and partial least squares discrimination/OPLS-DA analysis on the bacterial VOCs metabolic spectrum, and determining the difference VOCs of different bacteria so as to discriminate different bacteria.
2. The method of rapid and non-destructive identification of a composite bacterium according to claim 1, wherein the composite bacterium comprises pseudomonas aeruginosa, acinetobacter baumannii, klebsiella pneumoniae, and staphylococcus aureus.
3. The method for rapid and non-destructive identification of complex bacteria according to claim 2, wherein the pseudomonas aeruginosa comprises pseudomonas aeruginosa drug resistant bacteria and pseudomonas aeruginosa sensitive bacteria.
4. The method for rapid nondestructive identification of composite bacteria according to claim 1, wherein in the step (1), the culture condition is that the culture is carried out under constant temperature shaking at 36-38 ℃ and 260-300 r/min for 10-14 h.
5. The method for rapidly and nondestructively identifying composite bacteria according to claim 1, wherein in the step (2), the adsorption mode is that 80 μm DVB/CWR/PDMS fiber heads are adopted for adsorption for 1h under the condition of 36-38 ℃.
6. The method for rapid nondestructive identification of complex bacteria according to claim 1, wherein in step (3), the temperature programming is performed at an initial temperature of 40 ℃ for 2min, at a rate of 10 ℃ to 100 ℃ and at a rate of 5 ℃ to 200 ℃, and at a rate of 25 ℃ to 250 ℃ for 2min for a total analysis time of 32min.
7. The method for rapidly and nondestructively identifying composite bacteria according to claim 1, wherein in the step (3), during mass spectrum detection, mass spectrum conditions are ionization mode EI, electron energy 70eV, ion source temperature 230 ℃ and quaternary rod temperature 150 ℃, acquisition of a spectrum after 3min sample injection, and mass scanning range m/z 35-300.
8. The method for rapid and nondestructive identification of complex bacteria according to claim 2, wherein 9 kinds of different VOCs, namely hexamethyl cyclotrisiloxane, 2, 5-dimethylpyrazine, benzaldehyde, octamethyl cyclotrisiloxane, 2-ethylhexanol, 1-undecene, p-trimethylsiloxyphenyl-bis (trimethylsiloxy) ethane, 2, 7-tetramethyl-3-oxa-6-thia-2, 7-disilazane and 2, 4-di-tert-butylphenol, are selected in step (3) when the pseudomonas aeruginosa, the acinetobacter baumannii, the klebsiella pneumoniae and the staphylococcus aureus are identified.
9. The method for rapid and nondestructive identification of composite bacteria according to claim 3, wherein in the step (3), 7 different VOCs of 2, 5-dimethylpyrazine, octamethyl cyclotetrasiloxane, 2-ethyl-5-methylpyrazine, 2-ethylhexanol, 1-undecene, dodecyl pentasiloxane and 2, 4-di-tert-butylphenol are screened out during identification of pseudomonas aeruginosa resistant bacteria and pseudomonas aeruginosa sensitive bacteria.
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