Application of Next Generation Sequencing for Rapid Identification of Lactic Acid Bacteria

The rapid identification of lactic acid bacteria,which are essential microorganisms in the food industry,is of great significance for industrial applications.The identification of lactic acid bacteria traditionally relies on the isolation and identification of pure colonies.While this method is well-established and widely used,it is not without limitations.The subjective judgment inherent in the isolation and purification process introduces potential for error,and the incomplete nature of the isolation process can result in the loss of valuable information.The advent of next generation sequencing has provided a novel approach to the rapid identification of lactic acid bacteria.This technology offers several advantages,including rapidity,accuracy,high throughput,and low cost.Next generation sequencing represents a significant advancement in the field of DNA sequencing.Its ability to rapidly and accurately identify lactic acid bacteria strains in samples with insufficient information or in the presence of multiple lactic acid bacteria sets it apart as a valuable tool.The application of this technology not only circumvents the potential errors inherent in the traditional method but also provides a robust foundation for the expeditious identification of lactic acid bacteria strains and the authentication of bacterial powder in industrial applications.This paper commences with an overview of traditional and molecular biology methods for the identification of lactic acid bacteria.While each method has its own advantages,they are not without limitations in practical application.Subsequently,the paper provides an introduction of the principle,process,advantages,and disadvantages of next generation sequencing,and also details its application in strain identification and rapid identification of lactic acid bacteria.The objective of this study is to provide a comprehensive and reliable basis for the rapid identification of industrial lactic acid bacteria strains and the authenticity identification of bacterial powder.

Screening and Identification of Cotton Verticillium Wilt Antagonistic Bacteria Strain 7-30

[Objective]The aim of this study was to screen the antagonistic spore-forming bacteria of Verticillium dahliae and identify its physiological and biochemical characteristics.[Method]Taking the cotton verticillium wilt pathogen Verticillium dahliae V-190 as the test microorganism,the antagonistic spore-forming bacteria were screened.Through the preliminary screening and secondary screening,an antagonistic bacteria strain 7-30 with stronger antibacterial activity was obtained,and its morphological characteristics,physiological and biochemical characteristics were also identified.[Result]84 antagonistic bacteria strains were isolated from soil in various places by the preliminary screening.Especially,18 strains with better antagonistic ability were screened again,so an antagonistic bacteria strain 7-30 with the diameter of inhibition zone 18.9 mm and stronger antibacterial activity was obtained.According to its morphological characteristics,physiological and biochemical characteristics,the antagonistic bacteria strain 7-30 was identified as Bacillus subtilis primarily.[Conclusion]The strain 7-30 was obtained as the antagonistic spore-forming bacteria of Verticillium dahliae.

Screening and Identification of Antagonistic Bacteria against Soil-borne Diseases of Tomato

[Objective] This study was conducted to screen resources for biological control of soil-borne diseases of tomato. [Method] Antagonistic bacteria against Ral- stonia solanacearum and Fusarium oxysporum were isolated and purified from soil samples by dilution plating and confronting incubation on PDA plates, then the antibacterial spectrum and metabolic secretions of the bacterial isolates were measured, and their species were identified by establishing phylogenetic trees with Neighbor-Joining method. [Result] From the 60 soil samples, 10 of 59 antagonistic bacteria isolates against R. solanacearum showed inhibition zone diameter 〉 25 mm, and 4 of 30 strains against F. oxysporum exhibited inhibition rate 〉 30%. The bacteriostatic substances and antibacterial spectrum analysis on above 14 isolates revealed that four strains N23-2, N58-2, NF59-3 and NF61-1 had good antago- nism against pathogenic bacteria and fungi; 12 strains produced cellulose, 11 strains produced proteases, and 6 strains produced siderophores. The molecular identification experiments indicated that four strains were members of Paenibacillus, three strains of Streptomyces, three strains of Pseudomonas strains, and four strains of Bacillus strains.

Isolation and Identification of Antifungal Bacterial Strain KL-1 against Plant Wilt Disease

[Objective] The antifungal bacteria of plant wilt disease was screened and identified to provide foundation for the study on bio-control preparation of plant wilt disease.[Method] Confrontation culture method was adopted to screen the bio-control bacteria with good antifungal effect against plant wilt disease,Biolog bacteria automatic identification system and 16S rDNA sequence analysis method were selected to identify its taxonomic status,the biological safety of the strain towards cotton and mice was also determined.[Result] 12 bacterial strains were isolated from rhizosphere of cotton.Among those strains,5 isolates showed antifungal activity against F.decemcellulare Brick,F.oxysporum f.sp.Diathi,F.oxysporum f.sp.vasinfectum.The antifungal effect of KL-1 strain against three target strains of pathogen reached 69.09%,80.78% and 78.89% respectively.Identification results of Biolog bacteria automatic identification system and 16S rDNA sequence analysis method showed that KL-1strain was Bacillus amyloliquefaciens;primary determination results of biological safety also showed that the strain KL-1 was safe and non-toxic towards cotton and mice.[Conclusion] KL-1strain of B.amyloliquefaciens had antifungal effect against several pathogens of plant wilt diseases,which was safe and non-toxic towards cotton and mice,being the bio-control strain with research and development potential.

Identification of Cloaca Bacteria from Candidate Releasing Chinese Alligators

The Chinese alligator （Alligator sinensis） is a critically endangered species in China. Wild populations of Chinese alligator are on the edge of extinction. Through a release program, some captive-bred alligators will be selected and released into the wild to supplement and renew natural populations. The purpose of this study was to provide data to select healthy individuals for release. Through bacteriological and molecular identification, six different genera, eight species and an unclassified bacterium were identified in 13 bacterial strains, which were isolated from the cloaca of 25 Chinese alligators. One genus and four species were identified in eight bacterial strains, which were isolated from the water where the alligators live. According to the analysis, except for the unclassified bacterium, the other bacteria from the cloaca were not pathogenic and were different from the bacteria isolated from the water. Thus, it was concluded that the 24 Chinese alligators were healthy, and could be selected to be released into the wild. As subject AS 12 was identified carrying an unclassified bacteria, of which the characteristics were unknown, it was suggested that the AS 12 individual not be released.

Rapid and High-throughput Identification of Recombinant Bacteria with Mass Spectrometry Assay

Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） techniques were used to identify 12 recombinant proteins （10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori）. A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-CIinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FIexAnalysis softwares. Results Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities： RC=100%, mean CVA=98.7% （the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively） and PPV=95}. This model can be used to classify the bacteria and their recombinant, which only requires 3.7x103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks （9, 16, 9, and 14 for the four stages, respectively） was found in the various phase of recombinant bacteria. Conclusion MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.

Isolation and Identification of High-Quality Lactic Acid Bacteria in Forage Corn

[ Objective] To screen suitable lactic acid bacterium strains from forage corn which can be used as silage additives. [ Method] The lactic acid bacterium strains were isolated by inoculation on MRS solid medium containing calcium carbonate, and they were preliminarily identified through morphological, physiological and biochemical experiments. The acid production efficiency was determined. Twelve strains having strong acid-pro- duction ability were selected, and their salt tolerance and acid tolerance were detected. The sequences of their 16 S rDNA were also analyzed. [ Result] A total of 44 lactic acid bacterium strains were isolated from the forage com. As evidenced by the physiological and biochemical experi- ments, the twelve strains having strong acid-production ability belonged to Leuconostoc, Lactobacillus and Enterococcus, respectively, and they had strong salt tolerance and acid tolerance. According to the sequences of 16 S rDNA, A4, B9, B11, B12 and B14 were Lactobacillus plantarum; A1, A2., A7, A11 and B8 were Leuconostoc mesenteroides dextran subspecies; and AB and A9 were Enterococcus hirae. [ Conclusion ] The lactic acid bacterium strains with strong acid-production ability isolated from forage corn can be developed into silage additives.

Isolation and Identification of High-Quality Lactic Acid Bacteria from Wheat Haulm

[Objective] The aim of this study was to isolate and identify lactic acid bacteria （LAB） from wheat haulm and to select efficient strains for silage fermentation. [ Method] From 78 LAB strains isolated on the MRS solid medium containing calcium carbonate, we selected 43 strains having better acid-production ability through morphological observation, Gram staining, physiological and biochemical tests, acid production test, acid tolerance test and salt tolerance test. These strains were finally identified by sequencing 16 S rDNA. [ Result] Of the 43 LAB strains having better acid-production ability, 37 belonged to Lactobacillus paracasei subsp., one belonged to Lactobacillus rhamnosus and five belonged to Enterococcus faecium, as shown by the sequences of 16 S rDNA. [ Conclusion ] A total of 43 LAB strains having better acid-production ability were selected, which may be developed as high-quality silage additives.

Isolation and Identification of Cellulose- decomposing Bacteria from Deep-litter Systems

In this study, 43 cellulose-decomposing strains were isolated from deep-litter systems. After preliminary screening with Congo red identification medium and filter paper strip medium, five strains with large transparent circles that disintegrated filter paper strips were obtained. After further liquid fermentation, CMC activity, FPA activity and natural eellulase activity of these five strains were determined, and two cellulose-decomposing strains with higher enzyme activity were screened, which were named F7 and F21. Based on molecular biological identification and phylogenetic analysis of 16S rRNA gene sequences, these two cellulose- decomposing strains were identified as Bacillus subtilis and Streptomyces sp. , respectively.

Sanger Sequencing Implementation in Clinically Ill Patients for Bacterial and Fungal Pathogens Identification

The diagnosis of bacterial or fungal infections requires the identification of the pathogen etiology in the shortest time possible. Although some biomarkers are used as indicators of bacterial infections, their specificity and sensitivity are highly variable, and there is no direct relationship between the level increase of these biomarkers for mycosis. It is common to obtain negative microbiological cultures in patients infected by non-culturable, intracellular bacteria or mycosis, even though there is a high clinical suspicion of infection. This study identifies the pathogen present in critically infected patients through 16S and 18S/eEF1 genes detection by polymerase chain reaction (PCR) coupled with Sanger sequencing. Thirty clinical samples were evaluated by PCR, of which 40% were positive for fungi, 23.33% for bacteria, 26.7% for fungi and bacteria, and 10% for no pathogen. The PCRs outcomes period for bacteria or fungi was one day compared to seven and up to 14 days (on average) of microbiological culture for bacteria and fungi. Then, we assessed the relationship with the most used biomarkers (procalcitonin, C-reactive protein, globular sedimentation velocity, and the neutrophil-lymphocyte index). This combination of molecular techniques has been shown as helpful in identifying intracellular bacteria and fungi that are difficult to culture by conventional methods. Screening with genomic markers 16S and 18S/eEF1 by PCR allowed us to optimize the time to obtain the result of the infection caused by bacteria or fungi. Also, identifying the specific etiological microorganism by Sanger sequencing was very helpful in avoiding the progression of the disease and setting targeted treatment with better clinical outcomes.

Identification and Mutagenesis of Lactic Acid Bacteria from Chinese Sauerkraut

In order to analyze the fermentation properties of lactic acid bacteria in Chinese sauerkraut and to improve acid production, 21 samples of Chinese sauerkraut from Inner Mongolia and Northeast China were collected and isolated with a Man-Rogosa-Sharpe （MRS） culture. Sixteen strains of lactic acid bacteria were identified by combining both phenotype and genotype methods. After activation, the 16 strains were inoculated into the MRS medium with a concentration of 4% and then incubated at 37 ~C. The pH and the absorbance of the culture were mea- sured. The activated strains were then mutagenized in a field of 4 KV/cm mutation, with dosages administered within 20 minutes and 30 minutes, respectively. The variation curves of the pH and the absorbance of the culture were determined. The experimental results showed that the lactic acid bacteria isolated from the soup were identified as Lactobacillus and the acid production of the bacteria was signifi- cantly improved by the mutagenesis of the corona electric field.

Identification and Susceptibility Test of Ralstonia solanacearum Isolated from White Burley

[ Objective ] The paper was to isolate and identify Ralstonia solanacearum from white burley, and determine its susceptibility to 6 fungicides. [ Mcth- od] Using the combination method of semiselective medium （PCCG） and apolymerase chain reaction （PCR） technique, R. solanacearum in stalk of white burley from Dazhou City in Sichnan Province was isolated, and its biochemical type was identified. Through susceptibility test, the susceptibility of R. solanacearum to bismerthiazol, ethylicin, streptomycin, lime sulfur, 47% polylysine, 99% kojic acid was studied in laboratory. [Result] A total of 23 strains OfR. solanacearum were isolated, all belonging to biochemical type Ill. R. solanacearum obtained in the test were more susceptible to ethylicin, streptomycin and bismerthiazol, and ethylicin had good control effect against R. solanacearum with ECso of 0.086 ml/L. [ Conclusion ] The study provide theoretical basis for control of R. solanaceanon in white burley.

Model identification with BPNN on restrictive ecological factors of SRB for sulfate-reduction

The model of back-propagation neural network (BPNN) was presented to demonstrate the effect of restrictive ecological factors, COD/SO 4 2- ratio, pH value, alkalinity (ALK) and SO 4 2- loading rate (Ns), on sulfate reduction of Sulfate Reducing Bacteria (SRB) in an acidogenic sulfate reducing reactor supplied with molasses as sole organic carbon source and sodium sulfate as electron acceptor. The compare of experimental results and computer simulation was also discussed. It was shown that the method of BPNN had a powerful ability to analyze the ecological characteristic of acidogenic sulfate reducing ecosystem quantitatively.

Isolation and Identification of an Endophytic Strain with Antibiosis Ability from Davidia involucrate Brail.

[Objective]The aim was to isolate and identify an endophytic bacteria strain with antimicrobial activity from Davidia involucrate Brail.[Method]Endophytic strain with antibiosis ability was isolated from D.involucrate Brail by using cylinder-plate method.Then,it was identified through physiological and biochemical tests,16S rDNA homology analysis as well as some gene-specific sequence analysis.[Result]B221 stain had antimicrobial activity against a variety of rice plant pathogens,and it was identified as Bacillus subtilis.[Conclusion]This study enriches the research on endophyte within D.involucrate Brail,application of Bacillus bio-control,and therefore has laid a good foundation for the development of fungus used in biological control of crop pathogens.

林麝肠道中乳酸菌的分离筛选及益生特性

本研究旨在从健康林麝粪便中分离筛选用于改善林麝健康状况的潜在益生菌株,并对其进行体外益生性评价。共分离获得12株乳酸菌,结合细胞个体形态观察及细菌16S rDNA序列鉴定,体外评价其耐酸和耐胆盐能力,测定其对5种目标病原菌(大肠埃希氏菌Escherichia coli ATCC 25922、金黄色葡萄球菌Staphylococcus aureus ATCC 25923、铜绿假单胞菌Pseudomonas aeruginosaPAO1、肠炎沙门氏菌Salmonella enteritidisATCC 13076和化脓隐秘杆菌Arcanobacterium pyogenes LSN3)的拮抗活性、自聚集和疏水性、抗生素敏感性、抗药性基因型和生长曲线。结果显示,5株植物乳植杆菌(Lactiplantibacillus plantarum)和1株乳酸片球菌(Pediococcus acidilactici)在pH 4酸性条件或质量分数0.5%胆盐环境中,存活率分别达88.5%和87%以上;能够抑制前4种目标病原菌(抑菌圈直径17.20~20.19 mm)和化脓隐秘杆菌LSN3(抑菌圈直径14.23~15.53 mm);与对照菌株副干酪乳酪杆菌(L. paracasei)L22具有相近的自聚集(53.1%~56.2%)和表面疏水率(52.9%~56.0%);对链霉素、环丙沙星和万古霉素耐药;携带万古霉素抗性基因vanX;具有快速生长的特点(培养24 h后OD600分别达9.0和7.1左右)。本研究筛选出的6株乳酸菌可作为后续开发林麝用益生菌制剂的一种可靠的菌株来源。

大豆镰刀菌根腐病拮抗菌的筛选及生防效果

【目的】镰刀菌根腐病是世界范围内大豆生产上最具破坏性的土传病害之一,为获得对禾谷镰刀菌Fusarium graminearum具有较好拮抗效果的生防菌株。【方法】从健康大豆根际土壤分离细菌,平板对峙法筛选拮抗菌株,通过形态观察、生理生化特性、胞外酶活性及促生特性对菌株进行鉴定分析,采用盆栽试验进一步测定菌株生防及促生效果。【结果】筛选出的4株芽孢杆菌和1株假单胞杆菌对F.graminearum的抑菌率均达到60%以上,对F.oxysporum,F.solani,F.longifundum以及F.equiseti也均有一定的抑制作用,其发酵液及挥发代谢物均会影响F.graminearum的生长。5株拮抗菌具有产蛋白酶、纤维素酶以及葡聚糖酶的活性,解磷、解钾、固氮以及产铁载体的能力。综合以上测试结果,菌株20-8具有较强的抑菌及大豆促生效果。根据形态特征及16S rRNA序列分析,将菌株20-8鉴定为暹罗芽孢杆菌(Bacillus siamensis)。该菌株的发酵上清液可以破坏F.graminearum菌丝体结构,无细胞发酵上清液可以显著抑制孢子萌发。室内盆栽防效测定结果表明,20-8的稀释发酵液对F.graminearum引起大豆根腐病的防效可达46.08%,并且促进大豆植株生长。【结论】筛选鉴定的暹罗芽孢杆菌20-8具有解磷、解钾、固氮以及产铁载体及多种胞外酶功能,其稀释发酵液具有较强的抗真菌活性及大豆促生能力,菌株20-8可以用于防治F.graminearum引起的大豆根腐病。

一株降解黄曲霉毒素B1、拮抗黄曲霉的菌株鉴定及应用研究

目的筛选、鉴定一株降解黄曲霉毒素B1(aflatoxin B1,AFB1)及拮抗黄曲霉的菌株,并进行应用研究。方法以青岛市海洋、陆地、河流等不同地区的土壤、水质微生物为研究对象,利用平板划线法、AFB1降解率和黄曲霉对峙试验法筛选得到有效菌;通过形态学观察和同源性分析,鉴定有效菌的种类;分离该菌的上清液、悬菌液、胞内液并对各组分的AFB1降解率进行测定;提取有效菌的活性物质,对其提取物进行抑菌试验;主动侵染花生籽仁,验证有效菌对降解AFB1及抑制黄曲霉生长的效果。结果通过筛选,得到有效菌株LE160。综合形态学特征和16S rRNA基因分析结果,鉴定菌株LE160为毕节假单胞菌(Pseudomonas bijieensis),命名为毕节假单胞菌LE160。该菌株胞外上清液、菌悬液和胞内液对AFB1降解率分别为80.4%、16.3%、11.9%。该菌降解AFB1不是依赖细菌胞体的吸附作用,而是细菌代谢产生并分泌至胞外的活性物质主导的生物降解作用。菌株提取液对黄曲霉菌有明显抑制作用。花生应用试验结果表明该菌株的发酵液对降解AFB1及抑制黄曲霉生长有一定效果。结论菌株LE160对AFB1具有良好的降解效果,能有效拮抗黄曲霉的生长,具有应用的潜力。本研究结果为深入研究该菌的降解机制奠定了基础,也为花生等农产品防控黄曲霉侵染提供了新思路。

云木香内生细菌的分离鉴定及其对大丽轮枝菌的抑制作用

从药用植物云木香中分离得到41株内生细菌,采用细菌16S rRNA基因测序的方法对这些菌株进行鉴定,结果显示,这些云木香内生细菌分别属于6个属,其中假单胞菌属Pseudomonas和拉恩氏菌属Rahnella为优势菌属。采用对峙培养方法对这些菌株进行筛选,发现其中12株内生细菌对大丽轮枝菌Verticillium dahliae具有抑制作用,其中有2株属于拉恩氏菌属Rahnella,7株属于假单胞菌属Pseudomonas,3株属于沙雷氏菌属Serratia。12株内生细菌对棉花黄萎病的温室防效评估结果表明,假单胞菌属菌株R1-6、R1-13和S1-2对棉花黄萎病的温室防效达到55%以上,具有潜在的生产利用价值。

基质辅助激光解吸电离飞行时间质谱快速检测无菌体液分离病原菌的研究

目的探讨基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization-time of flight mass spectrometry,MALDI-TOF MS)快速检测增菌报阳后无菌液体病原菌的临床应用价值。方法收集河北省人民医院肉汤增菌培养阳性的无菌体液标本412份。报阳后的标本进行涂片革兰染色镜检,同时接种固体培养基进行培养,35℃培养4~6 h长出菌膜,35℃培养18~24 h长出单个菌落,应用质谱仪VITEK MS分别对菌膜和菌落进行鉴定,以质谱鉴定菌落的结果作为参考标准,分析菌膜的鉴定符合率。结果VITEK MS(菌落)共鉴定出412株病原菌,包括革兰阳性球菌212株(51.45%)、革兰阴性杆菌172株(41.75%)和真菌28株(6.80%)。与VITEK MS(菌落)鉴定结果相比,VITEK MS(菌膜)总体鉴定符合率为91.50%(377/412),革兰阳性球菌鉴定符合率为89.15%(189/212),革兰阴性杆菌鉴定符合率为94.19%(162/172),差异均有统计学意义(χ^(2)=36.553、24.319、10.299,均P<0.05);真菌鉴定,VITEK MS(菌膜)鉴定符合率为92.86%(26/28),差异无统计学意义(χ^(2)=2.074,P=0.150)。结论应用基质辅助激光解吸电离飞行时间质谱技术对报阳后的无菌体液标本进行4~6 h菌膜鉴定,可进一步缩短病原菌鉴定时间,快速准确提供鉴定结果,为临床侵袭性感染疾病的诊疗提供早期依据。

清香型白酒大曲中产酯化酶微生物分离筛选及鉴定

从清香型白酒大曲中分离纯化高产酯化酶的微生物菌株,经过初筛、复筛,最终得到1株细菌HXX7,其麸曲酯化力可达20.6 mg/g,具有较强催化合成乙酸乙酯的能力。对HXX7菌株及微观细胞形态进行观察,发现HXX7为革兰氏阳性细菌;应用16S rDNA测序方法对其进行分子生物学鉴定,结果显示HXX7为枯草芽孢杆菌(Bacillus subtilis)。