Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

白僵菌不同菌株DNA扩增图谱与其来源的相关性分析

采用RAPD技术对28个不同来源的球孢白僵菌菌株的DNA指纹图谱进行了测定。菌株间显示了DNA图谱的多态性,但系统聚类分析结果表明,其多态性与菌株的原寄主和原采集地之间未表现出相关性。     

Rapid genomic fingerprinting of Lactococcus lactis strains by arbitrarily primed polymerase chain reaction with 32P and fluorescent labels.

Arbitrarily primed polymerase chain reaction, with incorporation of either radioactive or fluorescent labels, was used as a rapid and sensitive method for obtaining genomic fingerprints of strains of Lactococcus lactis. Closely related strains produced almost identical fingerprints. Fingerprints of other strains showed only some similarities.     

双孢蘑菇遗传多样性分析

应用AFLP指纹技术对双孢蘑菇的20个野生菌株和5个栽培品种的遗传多样性进行了研究。AFLP指纹揭示出20个野生异核体菌株所固的基因型。5个商业品种表现出比较一致的AFLP指纹,但也显示出它们之间的一些差别。由单孢分离获得的同核体菌株携带着部分异核体菌株的AFLP指纹;由同一子实体分离得到的大部分单孢菌株是异核体菌株,它们具有与其亲本一致的AFLP指纹。UPGMA分析揭示出2个与地理分布（美洲、欧洲）和相对应的组。研究结果表明：（1）在野生菌株之间存在着明显的遗传差异;（2）大多数单孢分离的菌株具有与母本一致的遗传物质;（3）野生菌株间的遗传变异大于栽培品种间的变异;（4）在双孢蘑菇的遗传多样性分析中,AFLP技术是非常有效的工具。     

Genetic diversity of a natural population of Sinorhizobium meliloti, detected during analysis of a cryptic plasmid and ISRm2011-2 fingerprints]

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Genetic Diversity of a Natural Population of Sinorhizobium melilotiRevealed in Analysis of Cryptic Plasmids and ISRm2011-2 Fingerprints

Fifty-six natural strains of alfalfa nodule bacteria were isolated from samples of the soil under wild legume and alfalfa in two different field sites of Irkutsk oblast. Based on the results of analysis of plasmid profile, 11 different types of strains were detected, and 43 types were identified based on the results of hybridization with the insertion sequence element ISRm2011-2. Significant differences were found in the plasmid profile and IS fingerprints between strains isolated from the soil under alfalfa and the soil under legume. In contrast, strains growing at some distance from each other differed only in the IS fingerprints. From a comparison of results obtained in the assessment of plasmid profile and in analysis of IS fingerprints with results of RFLP analysis in strains, the conclusion about the transference of cryptic plasmids between strains and genetic rearrangements in strains of this population was drawn.     

Repetitive elements sequence (REP/ERIC)-PCR based genotyping of clinical and environmental strains of Yersinia enterocolitica biotype 1A reveal existence of limited number of clonal groups

REP- and ERIC-PCR genotyping were used to assess genetic heterogeneity among 81 strains of Yersinia enterocolitica biotype 1A isolated from India, Germany, France and the USA. Although both gave comparable results, ERIC fingerprints discriminated the strains better. The rep- (REP and ERIC) PCR genotyping showed that strains having different serotypes produced identical rep-profiles indicating their limited genetic diversity. The concatenated dendrogram of REP- and ERIC-PCR fingerprints clustered the biotype 1A strains into two major groups. In each group, majority of the Indian, European and American strains exhibited similarities ranging from 85% to >95%. Similarity of rep-PCR fingerprints amongst strains isolated from widely separated geographical regions revealed existence of a limited number of clonal groups of Y. enterocolitica biotype 1A. The present study failed to reveal unequivocal relationships between rep-PCR genotypes and the source of isolation. However, the clinical serotype O:6,30-6,31 strains formed a tight cluster and the aquatic O:6,30-6,31 strains formed a yet another tight cluster.     

Discrepancy between Penner serotyping and polymerase chain reaction fingerprinting of Campylobacter isolated from poultry and other animal sources

Thirty-four Campylobacter jejuni or coli strains, isolated from various livestock and darkling beetles from two Dutch poultry farms during different broiler production cycles, were subjected to Penner serotyping and polymerase chain reaction (PCR) fingerprint analysis. Ten different Penner serotypes were determined in the isolates. Visual scoring of the PCR fingerprints resulted in 14 clearly different profiles. Some strains with identical Penner serotypes exhibited different PCR fingerprints and conversely strains with different serotypes produced identical PCR fingerprints. Discrepancies between Penner serotyping and PCR fingerprinting were most obvious between isolates from different animal sources. Indications for the occurrence of genomic rearrangements were found. The inconsistency between serotyping and fingerprinting of Campylobacter strains suggests that conventional typing methods should be used in combination with fingerprinting if the epidemiological factors that contribute to Campylobacter colonization of live chickens are to be assessed reliably.     

An inhibitor typing scheme applicable to Lancefield group E streptococci

A previously described inhibitor typing scheme for hemolytic streptococci has been utilized to test 12 well-characterized strains of group E streptococci. These strains were differentiated into five inhibitor production (P) types and four inhibitor sensitivity (S) types: seven different strain "fingerprints" (combinations of P-type and S-type) being demonstrated. Inhibitor production by group E streptococci was increased under conditions of anaerobic incubation. The inhibitor fingerprints were stable on repeated testing of strains and it is suggested that the scheme is of value both for the labelling of serologically untypable strains and for subdividing strains belonging to existing serotypes.     

Characteristics of Ralstonia solanacearum Biovar N2 Strains in Asia

Ralstonia solanacearum biovar N2 strains isolated in Asia were compared by biochemical tests with biovar N2 strains from South America and biovar 2 (race 3) strains from Africa, America, Asia and Europe. Distinct differences were found between Asian and South American strains of biovar N2, and between Asian biovar N2 and biovar 2 strains with respect to their ability to utilize several carbon sources. Using cluster analysis based on repetitive sequence‐based polymerase chain reaction (rep‐PCR) genomic fingerprints, the Asian biovar N2 strains were divided into two groups, group 1 containing Japanese strains and group 2 containing Indonesian and Philippine strains. The fingerprints showed the genetic diversity of biovar N2 strains in Asia.     

Cluster analysis of AP-PCR generated DNA fingerprints of Vibrio vulnificus isolates from patients fatally infected after consumption of raw oysters

Arbitrarily-primed-polymerase chain reaction (AP-PCR) DNA fingerprints were generated for 10 Vibrio vulnificus strains isolated from patients who became infected and died between 1993 and 1996 as a result of consuming raw oysters. Analysis of the DNA fingerprints with gel imaging and cluster analysis software revealed significant genetic heterogeneity among these strains, suggesting that V. vulnificus has a high degree of variation in its genomic organization, and that multiple pathogenic strains with greatly diverse genomic arrangements, rather than a single type of infective strain or serogroup, caused these infections.     

Coaggregation amongst aquatic biofilm bacteria

In a comparative study, the PCR-based RAPD and ERIC fingerprint methods were evaluated for their resolving power to discriminate among 21 isolates of a natural Rhizobium meliloti population. PCR fingerprint patterns were analysed by using an automated laser fluorescent (ALF) DNA sequencer, thus allowing the automated on-line storage of data. Results obtained were compared to a classification system using insertion sequence (IS) fingerprinting. Both PCR fingerprint methods were comparable in their ability to resolve differences amongst Rh. meliloti isolates. Grouping of strains on the basis of their RAPD as well as their ERIC fingerprints correlated with grouping of strains according to their IS fingerprints. Moreover, strains displaying identical PCR patterns could be further differentiated according to their IS fingerprints, thus allowing a detailed insight into phylogenetic relationship among strains. The automated evaluation of strain-specific fingerprint patterns has the potential to become a valuable tool for studies of bacterial population genetics. Moreover, the rapid identification of single strains, e.g. pathogens in epidemiological studies seems feasible.     

High density whole genome fingerprinting of methicillin-resistant and -susceptible strains of Staphylococcus aureus in search of phenotype-specific molecular determinants

AFLP is a selective restriction fragment amplification method generating DNA fingerprints for microbial isolates. We present high-throughput AFLP (htAFLP) to characterize molecular markers associated with bacterial phenotypes. Methicillin-resistant and -susceptible isolates of Staphylococcus aureus have been used for this model study in conjunction with the available S. aureus genome sequences. This facilitates the calculation of theoretical AFLP fingerprints, comparison of these fingerprints with genuine experimental fingerprints, and the subsequent identification of polymorphic AFLP markers without sequence analysis. Analysis of 46 MRSA and 46 MSSA strains by 39 different AFLP reactions generated more than 2500 fragments per strain and an overall number of 6180 scorable markers within all strains. We successfully identify MRSA specific markers and elaborate on the general applicability of the htAFLP approach. This method can be applied to any microbial species for which at least one full-genome sequence is available.     

Fingerprinting genomes using PCR with arbitrary primers.

Simple and reproducible fingerprints of complex genomes can be generated using single arbitrarily chosen primers and the polymerase chain reaction (PCR). No prior sequence information is required. The method, arbitrarily primed PCR (AP-PCR), involves two cycles of low stringency amplification followed by PCR at higher stringency. We show that strains can be distinguished by comparing polymorphisms in genomic fingerprints. The generality of the method is demonstrated by application to twenty four strains from five species of Staphylococcus, eleven strains of Streptococcus pyogenes and three varieties of Oryza sativa (rice).     

Genetic diversity of Ralstonia solanacearum strains from China assessed by PCR-based fingerprints to unravel host plant- and site-dependent distribution patterns

Bacterial wilt caused by Ralstonia solanacearum is a serious threat to crop production in China. A collection of 319 R. solanacearum strains isolated from 14 different diseased host plants collected in 15 Chinese provinces was investigated by BOX fingerprints in order to test the influence of the site and the host plant on their genetic diversity. Phylotype, fliC-RFLP patterns and biovar were determined for all strains and the sequevar for 39 representative strains. The majority of strains belonged to the Asian phylotype I, shared identical fliC-RFLP patterns and were assigned to four biovars (bv3:123; bv4:162; bv5:3; and bv6:11). Twenty strains were phylotype II, assigned to biovar 2, and had distinct fliC-RFLP patterns. BOX-PCR fingerprints generated from the genomic DNA of each strain revealed a high diversity of the phylotype I strains, where 28 types of BOX fingerprints could be distinguished. While many BOX clusters comprised isolates from different provinces and several host plants, some groups contained isolates that were plant or site specific. All phylotype II isolates originating from 10 provinces belonged to sequevar 1 and displayed identical BOX patterns as the potato brown rot strains from various regions of the world.     

副溶血性弧菌重复序列-PCR分型研究

目的利用REP—PCR技术对副溶血性弧菌进行分子分型研究和亲缘关系的探讨。方法以基因外重复回文序列（REP）为引物,对20株副溶血性弧菌基因组DNA进行扩增,得到DNA指纹图谱,并利用SPSS11．5统计软件对DNA扩增图谱进行分析,做出聚类图,并与血清型进行比较分析。结果REP-PCR可以把20株菌分为7个型,优势菌型为A型和B型,分辨力指数为0．932。结论REP．PCR方法可以用于副溶血性弧菌分型分析,具有较好的分型能力,如果能将分子分型和血清分型两种方法联用,分辨率会更高。研究推断血清型01群与03群菌株之间亲缘关系密切。     

Characterization of beta-lactam-resistant Bacteroides fragilis isolates by use of PCR fingerprinting

PCR fingerprinting was used for characterization of 35 beta-lactam-resistant Bacteroides fragilis strains isolated in Sweden and Hungary. Ten B. fragilis strains showed unique PCR fingerprints by use of the M13 core primer. Their main product was a DNA fragment with a length of 2000-bp which was absent in the other 25 strains and the reference strain B. fragilis ATCC 25285. The 2000-bp fragment from four imipenem-resistant strains gave rise to positive reactions in a specific PCR for detection of ccrA. Printed by the T3B primer, five B. fragilis strains, including the imipenem-resistant strains showed unique PCR fingerprints. The investigated imipenem-resistant strains produced carbapenem-hydrolysing metallo-beta-lactamases. The study indicates that the unique PCR fingerprinting profiles shown in highly beta-lactam resistant B. fragilis strains are correlated to antimicrobial resistance. The PCR fingerprinting technique is a useful tool for differentiation of Bacteroides fragilis strains with high-level beta-lactam resistance.     

副溶血性弧菌肠细菌基因间共有重复序列-PCR分子分型和耐药性、血清型研究

目的对副溶血性弧菌进行ERIC-PCR分子分型、耐药性和血清型相关性研究。方法肠细菌基因间共有重复序列（ERIC）为引物,对40株菌株基因组DNA进行扩增,得到DNA指纹图谱,并利用SPSS13.0统计软件对DNA扩增图谱进行分析,做出聚类图从而分型,并与菌株血清型、耐药性比较分析。结果40株菌用ERIC-PCR分为5个型,分辨力指数为（DI）为0.5;血清分型分为4个型;对8种抗生素中的萘啶酸、头孢噻亏、头孢西丁出现了不同程度的耐药。耐药菌株均出现在ERIC-PCR方法分型A型和血清分型O3型中。结论研究显示ERIC-PCR方法可以用于该菌分型分析,具有较好的分型能力。血清分型与ERIC-PCR方法分型一致。通过ERIC-PCR分型的树状图和血清分型结果推断,血清型O3群菌株很可能起源于血清型O1群菌株,血清型O3群和O1群密切相关。     

Evaluation of arbitrarily primed PCR for typing of Desulfovibrio desulfuricans strains

Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard's coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.     

应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析

采用RAPD技术对采集不同地区的8个斜生褐孔菌野生菌核分离得到的菌株亲缘关系进行研究,获得了斜生褐孔菌不同菌株的DNA指纹图谱。结果显示:12个引物共扩增出167条带,其中101条为多态性带,多态性比率为60.5%,同一培养时期的各菌株间RAPD图谱表明菌株间存在一定的种内及地理来源差异。若以遗传距离0.508为结合线,可将供试菌株划分为三大类,BCX01、BCX02归为一类,JL01、JL02、JL03、JL04、JL05归为一类,HLJ01单独聚为一类。