Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms

Aim: To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm. Methods and Results: Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a period of exposure to 20% sucrose solution (28 days), using Rogosa Agar. The lactobacillus colonies were randomly selected (n = 222) and subcultured. The isolates were identified using pheS or rpoA gene sequence analysis. Lactobacilli identified as Lact. paracasei (n = 75) were subjected to multilocus sequencing typing (MLST) analysis by determining partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG. An increase recovery of lactobacilli after sucrose phase compared with nonsucrose period was observed (31 prior to and 191 following a sucrose exposure period). Seven subjects harboured Lact. paracasei and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. Conclusion: The present data supports the hypothesis that oral lactobacilli may be of exogenous origin. Significance and Impact of the Study: The study allow us to gain insight into the genetic diversity of Lact. paracasei in oral biofilm.     

Profiling the effects of physicochemical indexes on the microbial diversity and its aroma substances in pit mud

Microbial diversity of pit mud (PM) plays a significant role in Baijiu's flavour. Here we explored the microbial community structures and aroma substances of Wenwang Winery with high-throughput sequencing coupling with headspace solid-phase microextraction-gas chromatography-mass spectrometry. We discovered that the odorant was mainly derived from 14 aroma compounds because of their OAVs ≥ 1 (OAV, the ratio of substance concentration to aroma threshold; s, on behalf of the plural), such as ethyl hexanoate (2438), ethyl octanoate (975), caproic acid (52) and etc. Moreover we also revealed that Lactobacillaceae (97·08%) was the mainly bacterial microbial community in 2-year-old PM, companied by the primarily fungi including Aspergillaceae (55·45%), Unclassified Ascomycota (11·13%) and Dipodascaceae (5·72%). Compared with the 2-year-old PM, bacterial floras in 20-year-old PM and 30-year-old PM were more abundant (i.e. Dysgonomonadaceae, Clostridium and Synerggstaceas), while no fungi were detected. Besides, the physicochemical analysis showed that the content of Lactobacillaceae was inversely associated with moisture, pH and ammonia nitrogen. By further Spearman's correlation coefficient analysis, we verified that the content of Lactobacillaceae was positively correlated with ethyl hexanoate, while negatively correlated with ethyl octanoate and caproic acid. Meanwhile, ethyl octanoate and caproic acid were positively correlated with most flora including Ruminococcaceae, Dysgonomonadaceae and Clostridiacea, which were related to physicochemical indexes. This work demonstrates promise for adjusting the physicochemical indexes of PM to affect the micro-organisms and aroma, which may provide a reference for the production of high-quality Baijiu.     

The influence of mutation, recombination, population history, and selection on patterns of genetic diversity in Neisseria meningitidis

Patterns of genetic diversity within populations of human pathogens, shaped by the ecology of host-microbe interactions, contain important information about the epidemiological history of infectious disease. Exploiting this information, however, requires a systematic approach that distinguishes the genetic signal generated by epidemiological processes from the effects of other forces, such as recombination, mutation, and population history. Here, a variety of quantitative techniques were employed to investigate multilocus sequence information from isolate collections of Neisseria meningitidis, a major cause of meningitis and septicemia world wide. This allowed quantitative evaluation of alternative explanations for the observed population structure. A coalescent-based approach was employed to estimate the rate of mutation, the rate of recombination, and the size distribution of recombination fragments from samples from disease-associated and carried meningococci obtained in the Czech Republic in 1993 and a global collection of disease-associated isolates collected globally from 1937 to 1996. The parameter estimates were used to reject a model in which genetic structure arose by chance in small populations, and analysis of molecular variation showed that geographically restricted gene flow was unlikely to be the cause of the genetic structure. The genetic differentiation between disease and carriage isolate collections indicated that, whereas certain genotypes were overrepresented among the disease-isolate collections (the "hyperinvasive" lineages), disease-associated and carried meningococci exhibited remarkably little differentiation at the level of individual nucleotide polymorphisms. In combination, these results indicated the repeated action of natural selection on meningococcal populations, possibly arising from the coevolutionary dynamic of host-pathogen interactions.     

浓香型白酒窖泥放线菌的群落结构及其多样性

以泸州老窖1、50、100和400年窖泥为研究对象,采用变性梯度凝胶电泳(DGGE)研究浓香型白酒窖泥放线菌的群落结构及其多样性。DGGE图谱显示,除1年样品外,其余窖底泥多样性指数(H)均低于同窖龄窖壁泥,但均匀度指数(EH)较高。不同窖池相同部位窖泥的群落结构变化趋势为:随窖龄的延长,窖壁泥H值逐渐上升,为1.74—2.28;窖底泥下降,为1.73—2.07。EH值均为波动下降,分别在0.986—0.991和0.971—0.994之间。窖底泥相似性系数(SC)逐渐上升,为0.46—0.82;窖壁泥为0.31—0.62。DGGE条带测序结果显示,窖泥放线菌归于Olsenella、Atopobium、Streptomyces和Corynebacterium 4个属。Olsenella和Atopobium属为共有的优势属,且在窖壁泥中的优势度(di)均随窖龄延长而降低,在窖底泥中升高。实验结果表明,浓香型白酒窖泥蕴藏着丰富的放线菌资源,群落结构和多样性存在差异,菌群演替呈现一定规律性。     

浓香型白酒发酵过程中窖内古菌群落分布特征

泥窖池多菌种固态发酵是浓香型白酒的典型特点,其中古菌是重要的酿造功能菌,但目前对发酵过程古菌的群落分布及多样性尚缺乏研究。采用高通量测序技术,分析了浓香型白酒发酵过程酒醅与窖泥中古菌的生物量、群落组成与演替规律,并通过共现性网络分析了古菌与细菌的潜在互作关系。结果表明,窖泥中古菌平均生物量约是酒醅的200倍,两者之间古菌群落的结构差异不显著(r=0.017,P=0.074),但演替规律存在显著相关性(r=0.30,P=0.03)。甲烷杆菌属Methanobacterium是酒醅与窖泥中丰度占比最高的古菌,其他优势群类依次为甲烷八叠球菌属Methanosarcina、甲烷粒菌属Methanocorpusculum、甲烷囊菌属Methanoculleus和甲烷短杆菌属Methanobrevibacter。共现性网络分析显示甲烷杆菌属在酒醅与窖泥中与多数细菌为正相关,特别是与窖泥中主要细菌氢孢菌属Hydrogenispora和产己酸菌属Caproiciproducens。研究结果揭示了浓香型白酒窖池中古菌群落的时空分布特点及潜在功能。     

两株炭疽芽胞杆菌MLST 新序列型(ST)

【目的】2株炭疽芽胞杆菌(Bacillus anthracis)17003-14和17003-32的多位点序列分型(Multilocussequence typing,MLST)研究。【方法】选取B.anthracis基因组7个常见管家基因位点glpF、gmk、ilvD、pta、pur、pycA和tpi进行PCR扩增、测序,与MLST数据库中的等位基因序列进行比对,确定菌株的序列型(sequence type,ST)。【结果】B.anthracis 17003-14和17003-32的等位基因编号分别为113、31、1、43、1、53、7和113、31、1、43、1、53、37,比对结果显示这2株细菌的等位基因编号组合未见报道。【结论】17003-14和17003-32为新ST菌株,已被MLST数据库确认,注册号(pubMLST id)分别为id-1053和id-1054。     

不同时期窖泥理化因子、风味物质和细菌组成的相关性

[背景]窖泥品质是影响浓香型白酒质量的重要因素之一,窖泥的理化特性、微生物群落结构和风味物质等与窖泥的品质有关.[目的]揭示不同时空浓香型白酒窖泥理化因子、细菌群落结构和挥发性风味物质三者之间的关系.[方法]比较演替期(10年)和成熟期(30年)的窖泥理化因子,利用顶空固相微萃取-气相色谱质谱联用分析窖泥中挥发性风味物...     

窖泥群落结构及功能微生物研究进展

窖泥是浓香型白酒的特征发酵载体,包含多种功能微生物,这些功能微生物对浓香型白酒的风格特征和品质有着重要的影响。本文对不同窖龄窖泥、不同浓香型白酒产地的窖泥及不同质量窖泥的窖泥微生物研究进展进行总结分析,并在此基础上对窖泥主体微生物的功能进行阐述。以期为窖泥微生物的研究提供思路,为浓香型白酒的质量和香味成分研究提供思考,促进窖泥微生物的研究,加深对浓香型白酒窖泥的认知。     

Survey of duckweed diversity in Lake Chao and total fatty acid,triacylglycerol, profiles of representative strains

Lemnaceae (duckweeds) are widely distributed aquatic flowering plants. Their high growth rate, starch content and suitability for bioremediation make them potential feedstock for biofuels. However, few natural duckweed resources have been investigated in China, and there is no information about total fatty acid (TFA) and triacylglycerol (TAG) composition of duckweeds from China. Here, the genetic diversity of a natural duckweed population collected from Lake Chao, China, was investigated using multilocus sequence typing (MLST). The 54 strains were categorised into four species in four genera, representing 12 distinct sequence types. Strains representing Lemna aequinoctialis and Spirodela polyrhiza were predominant. Interestingly, a surprisingly high degree of genetic diversification within L. aequinoctialis was observed. The four duckweed species revealed a uniform fatty acid composition, with three fatty acids, palmitic acid, linoleic acid and linolenic acid, accounting for more than 80% of the TFA. The TFA in biomass varied among species, ranging from 1.05% (of dry weight, DW) for L. punctata and S. polyrhiza to 1.62% for Wolffia globosa. The four duckweed species contained similar TAG contents, 0.02% mg·DW^?1. The fatty acid profiles of TAG were different from those of TFA, and also varied among the four species. The survey investigated the genetic diversity of duckweeds from Lake Chao, and provides an initial insight into TFA and TAG of four duckweed species, indicating that intraspecific and interspecific variations exist in the content and composition of both TFA and TAG in comparison with other studies.     

泸型酒酒醅与窖泥中梭菌群落结构、演替和功能差异

梭菌对泸型酒风味品质的发酵形成具有重要作用,但目前对酒醅和窖泥中梭菌群落的物种组成、发酵演替规律以及代谢功能的差异尚缺少深入认识。采用分子微生态学技术,在种水平上对比了同一窖池的酒醅和窖泥中梭菌纲细菌群落的结构差异和演替规律,并通过纯培养方法分离和评价了梭菌菌株的主要挥发性脂肪酸组成差异。结果表明,窖泥发酵过程中梭菌纲细菌和总细菌的基因拷贝数比值相对稳定(71.5%–91.2%),而酒醅中梭菌的变化则较大(0.9%–36.5%)。酒醅中优势梭菌纲细菌主要是梭菌属(Clostridium,19.9%)、沉积物菌属(Sedimentibacter,8.8%)和氢孢菌属(Hydrogenispora,7.2%),而窖泥中主要为氢孢菌属(57.2%)、沉积物菌属(5.4%)和产己酸菌属(Caproiciproducens,4.9%)。窖泥及酒醅发酵过程中梭菌群落结构差异显著(P=0.001)。分离获得的20株梭菌菌株具有不同的产挥发性脂肪酸能力。结果表明,窖池中梭菌纲细菌群落存在时空异质性,其结构和功能差异对泸型酒风味形成具有影响。     

厌氧产酸菌群培养技术在窖泥质量快速检测中的应用

【背景】窖泥质量决定了浓香型白酒品质的高低,窖泥中的产酸功能菌群显著影响着窖泥的质量及对应的浓香型白酒品质,但目前对窖泥质量的评价尚缺乏明确和完善的标准。【目的】建立一种快速、准确评价窖泥质量的方法,解析窖泥中参与己酸合成的重要微生物。【方法】通过窖泥产酸菌群培养和产酸代谢特征研究,构建厌氧产酸菌群发酵体系,根据窖泥中厌氧菌的己酸合成能力来评价相应窖泥的质量;利用高通量测序技术分析可培养发酵体系中产己酸功能微生物的群落组成。【结果】葡萄糖碳源相比乳酸碳源可以更有效地富集窖泥中的产己酸菌群;采用毫升级别发酵体系、对窖底泥进行多点取样,产酸菌群发酵代谢产物稳定期的己酸产量、己酸/丁酸值可较准确地评价不同窖泥的质量。16S rRNA基因测序结果表明,利用产酸菌群培养方法可以有效地富集未培养产己酸细菌(Uncultured bacterium Caproiciproducens)。【结论】基于产酸菌群培养的窖泥微生物发酵体系可用于实际生产中快速、准确地评价窖泥质量,并为窖泥微生物己酸合成代谢机制的研究提供参考。     

Genotypic comparison of five isolates of Rickettsia prowazekii by multilocus sequence typing

Genetic traits of five Rickettsia prowazekii isolates, including the first from Africa and North America, and representatives from human and flying squirrels were compared using multilocus sequence typing. Four rickettsial genes encoding 17 kDa genus-common antigen (17 kDa gene), citrate synthase (gltA), OmpB immunodominant antigen (ompB) and 120 kDa cytoplasmic antigen (sca4) were examined. Sequence identities of 17 kDa gene and gltA were 100% among the isolates. Limited sequence diversity of ompB (0.02-0.11%) and sca4 (0.03-0.20%) was enough to distinguish the isolates, and evaluation of the combined four genes provided a method to easily differentiate R. prowazekii from other rickettsiae.     

流溪河生态公益林下土壤细菌多样性

【背景】森林土壤细菌多样性在一定程度上是衡量森林质量的重要指标之一,土壤细菌群落结构组成及变化能够反映森林生态系统的结构和功能,在森林生态系统物质养分循环中发挥着重要的作用。【目的】以广州市流溪河国家森林公园3种不同密度林分生态公益林的土壤为研究对象,分析不同密度林分类型对土壤细菌群落结构的影响,探讨不同林分密度土壤养分和土壤细菌的分布规律,为退化生态系统的恢复、合理利用公益林土壤资源、维护地力及提高公益林生态系统生产力和服务功能提供参考。【方法】选择高密度林分(HD)、中密度林分(MD)、低密度林分(LD)3种林分类型,采用"S"形取样法采集土壤样品,提取土壤微生物总DNA,采用Illumina MiSeq高通量测序技术对16S rRNA基因进行序列测定,利用R语言和SPSS21.0等软件分析林分密度对细菌多样性及群落结构的影响。【结果】高林分密度土壤肥力状况较中低林分密度高;不同林分密度下土壤细菌多样性指数、丰富度指数略有差异,中密度林分指数最高;流溪河区域土壤细菌多样性较高,变形菌门和酸杆菌门是主要类群。【结论】流溪河生态公益林土壤细菌具有丰富的多样性,以变形菌门和酸杆菌门为主;林分密度对土壤细菌的多样性、丰富度及其群落结构产生明显影响;流溪河区域中密度林分(1 800-2 200株/hm2)更适合土壤细菌的繁衍生息;土壤的肥力状况受林分密度、灌木杂草的影响;16SrRNA基因的基因组异质性会导致对细菌多样性的过高估计。     

江苏水产养殖区拟态弧菌种群结构及遗传多样性

【背景】拟态弧菌(Vibrio mimicus)是一种常见的革兰氏阴性病原菌,广泛分布于水环境和水生动物体内,可导致多种水产动物和人类感染。多位点序列分型(multilocus sequence typing, MLST)已被应用于多种病原菌的分子分型,其通过分析不同菌株之间的遗传关系,监测细菌传播的时间和地理分布,确定感染和传播途径,但目前未见有关拟态弧菌MLST的报道。【目的】开发一种基于MLST的拟态弧菌分型方法,并用于江苏水产养殖区拟态弧菌的种群结构和遗传进化分析,为拟态弧菌感染所引起的疾病防治提供理论基础。【方法】选择拟态弧菌的7个管家基因dnaE、gyrB、mdh、recA、rpoD、pntA和pyrH作为靶点,对江苏水产养殖区分离的155株拟态弧菌进行PCR扩增和测序。将测序结果分配等位基因,制作等位基因谱,分配不同的序列类型(sequence type, ST),利用软件goeBURST-1.2.1和MEGA-X对分配的ST型进行克隆复合体和遗传进化树聚类分析;此外,利用Kirby-Bauer圆盘扩散法测试155株拟态弧菌的药敏特性。【结果】155株拟态弧菌被分为56个STs,其中ST11占比最高;在双位点变异(double locus variants, DLV)水平分析发现56个STs分为3个克隆复合体和3个单体;系统发育树显示,56个STs被分为3个集群(cluster I、cluster II、cluster III)。药敏结果显示,155株拟态弧菌对红霉素类抗生素的耐药性最高(88.39%, 137/155),对氯霉素类抗生素敏感性最高(91.61%, 142/155)。【结论】本研究建立的MLST方法具有良好的分辨率,可作为拟态弧菌系统发育和未来流行病学调查有用的分子分型工具。根据抗生素耐药谱结果,提示在养殖过程中可选用氟苯尼考等国家批准使用的专用抗菌药对拟态弧菌进行防治。     

Niche segregation and genetic structure of Campylobacter jejuni populations from wild and agricultural host species

Bacterial populations can display high levels of genetic structuring but the forces that influence this are incompletely understood. Here, by combining modelling approaches with multilocus sequence data for the zoonotic pathogen Campylobacter, we investigated how ecological factors such as niche (host) separation relate to population structure. We analysed seven housekeeping genes from published C. jejuni and C. coli isolate collections from a range of food and wild animal sources as well as abiotic environments. By reconstructing genetic structure and the patterns of ancestry, we quantified C. jejuni host association, inferred ancestral populations, investigated genetic admixture in different hosts and determined the host origin of recombinant C. jejuni alleles found in hybrid C. coli lineages. Phylogenetically distinct C. jejuni lineages were associated with phylogenetically distinct wild birds. However, in the farm environment, phylogenetically distant host animals shared several C. jejuni lineages that could not be segregated according to host origin using these analyses. Furthermore, of the introgressed C. jejuni alleles found in C. coli lineages, 73% were attributed to genotypes associated with food animals. Our results are consistent with an evolutionary scenario where distinct Campylobacter lineages are associated with different host species but the ecological factors that maintain this are different in domestic animals such that phylogenetically distant animals can harbour closely related strains.     

A multilocus sequence analysis of Xanthomonas campestris reveals a complex structure within crucifer-attacking pathovars of this species

Previous classification of Xanthomonas campestris has defined six pathovars (aberrans, armoraciae, barbareae, campestris, incanae, and raphani) that cause diseases on cruciferous plants. However, pathogenicity assays with a range of strains and different hosts identifies only three types of symptom: black rot, leaf spot and bacterial blight. These findings raise the question of the genetic relatedness between strains assigned to different pathovars or symptom phenotypes. Here we have addressed this issue by multilocus sequence analysis of 42 strains. The X. campestris species was polymorphic at the 8 loci analysed and had a high genetic diversity; 23 sequence types were identified of which 16 were unique. All strains that induce black rot (pathovars aberrans and campestris) were genetically close but split in two groups. Only three clonal complexes were found, all within pathovar campestris. The assignment of the genome-sequenced strain 756C to pathovar raphani suggested from disease symptoms was confirmed, although this group of strains was particularly polymorphic. Strains belonging to pathovars barbareae and incanae were closely related, but distinct from pathovar campestris. There is evidence of genetic exchanges of housekeeping genes within this species as deduced from a clear incongruence between individual gene phylogenies and from network structures from SplitsTree analysis. Overall this study showed that the high genetic diversity derived equally from recombination and point mutation accumulation. However, X. campestris remains a species with a clonal evolution driven by a differential adaptation to cruciferous hosts.     

Multilocus sequence typing of Campylobacter jejuni isolates from New South Wales, Australia

AIMS: Multilocus sequence typing (MLST) was used to examine the diversity and population structure of Campylobacter jejuni isolates associated with sporadic cases of gastroenteritis in Australia, and to compare these isolates with those from elsewhere. METHODS AND RESULTS: A total of 153 Camp. jejuni isolates were genotyped. Forty sequence types (STs) were found, 19 of which were previously undescribed and 21 identified in other countries. The 19 newly described STs accounted for 43% of isolates, 16 of which were assigned to known clonal complexes. Eighty-eight percent of isolates were assigned to a total of 15 clonal complexes. Of these, four clonal complexes accounted for 60% of isolates. Three STs accounted for nearly 40% of all isolates and appeared to be endemic, while 21 STs were represented by more than one isolate. Seven infections were acquired during international travel, and the associated isolates all had different STs, three of which were exclusive to the travel-acquired cases. Comparison of serotypes among isolates from clonal complexes revealed further diversity. Eight serotypes were identified among isolates from more than one clonal complex, while isolates from six clonal complexes displayed serotypes not previously associated with those clonal complexes. CONCLUSIONS: Multilocus sequence typing is a useful tool for the discrimination of subtypes and examination of the population structure of Camp. jejuni associated with sporadic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the genotypic diversity of Camp. jejuni in Australia, demonstrating that STs causing disease have both a global and a local distribution evident from the typing of domestically and internationally acquired Camp. jejuni isolates.