Genotypic and phenotypic heterogeneity of Streptococcus thermophilus strains isolated from dairy products

AIMS: Forty strains of Streptococcus thermophilus isolated from dairy products were identified and typed by a polyphasic approach which included phenotypic and genotypic criteria. METHODS AND RESULTS: Strains were identified by sugar fermentation pattern and species-specific PCR. Phenotypic diversity was evaluated by a chemometric model taking into account some biochemical characteristics (e.g. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by PCR fingerprinting. The overall phenotypic and genotypic information was elaborated on a multivariate statistical basis by principal components analysis and cluster analysis, respectively. When acidifying and peptidase activities were considered, PCA indicated that most of the strains isolated from Pecorino Toscano cheese were separable from the others. Similarly, most of the starter culture strains tended to separate from the cheese isolates. CONCLUSIONS: A wide strain heterogeneity among Strep. thermophilus strains isolated from dairy products was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: A computerized analysis of genotypic and phenotypic information could be applied successfully to differentiate and characterize reliably and rapidly isolates occurring in different dairy products and to comprehend the technological role of specific Strep. thermophilus strains in dairy technology.     

Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products

AIMS: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods METHODS AND RESULTS: Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. CONCLUSIONS: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.     

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption

Aims: To determine whether the presence and type of exopolysaccharides (EPS), slime‐EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. Methods and Results: Phage–host interactions between eight EPS‐producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (φYsca, φ3, φ5, φ6, φ8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular‐producing strains were more sensitive to phage attacks than the noncapsular‐producing strains. Streptococcus thermophilus CRL1190 (capsular‐producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular‐negative, EPS low‐producing mutant of strain CRL1190 was reduced, especially for φYcsa and φ8. Conclusions: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. Significance and Impact of the Study: Capsular‐producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.     

Comparison of Lactococcus garvieae strains isolated in northern Italy from dairy products and fishes through molecular typing

Aims:  To investigate the genetic relatedness between Lactococcus garvieae strains isolated from fish and dairy samples collected in northern Italy, using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR), Sau -PCR and amplified fragment length polymorphism (AFLP).
Methods and Results:  Eighty-one isolates from bovine and caprine dairy products ( n  = 53) and from diseased rainbow trouts and other fishes ( n  = 28) were examined. All methods showed a typeability of 100%, repeatability ranging from 84·4% to 97·5% and discriminatory powers from 0·798 to 0·986. Dairy and fish strains revealed a low genetic relatedness as they are often grouped into distinct clusters. RAPD analysis discriminated 52 genotypes when primer M13 was used, whereas with primer P5 only 27 genotypes were identified. When Sau -PCR was performed, 13 genotypes were detected while AFLP analysis allowed the differentiation of 32 genotypes.
Conclusion:  L. garvieae strains isolated from dairy samples are generally not related to those collected from fish lactococcosis outbreaks.
Significance and Impact of the Study:  L. garvieae strains exhibit a genetic diversity related to the specific animal host they colonize. RAPD M13 fingerprinting proved to be a molecular tool for comparing isolates, whereas Sau -PCR and AFLP analyses were useful techniques to investigate the distribution of L. garvieae populations in the environment.     

Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches

Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro‐intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low‐resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum‐marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism.     

Diversity of streptomycetes among specific Greek terrestrial ecosystems

The diversity of streptomycetes isolated from different Greek terrestrial ecosystems using phenotypic identification, and the relationship between the number of species and the number of isolates as a diversity index, was studied. A total of 344 Streptomyces strains have been isolated and identified from diverse sites in the Greek territory, such as heavily disturbed agricultural areas and preserved forest areas, and from specific rhizosphere ecosystems. According to phenotypic identification, these strains belonged to 19 different cluster groups with a Willcox probability > 0·8. Streptomyces cyaneus , Strep. albidoflavus , Strep. diastaticus and Strep. exfoliatus were the most common cluster groups isolated from at least six different habitats. On the other hand, there were cluster groups that appeared in only one or two habitats, such as Strep. griseoflavus , Strep. rimosus , Streptoverticillium blastmyceticum , Nocardia mediterranea and Strep. fulvissimus . The diversity indices among the different cluster groups of each sampling area indicated that the different habitats can be sub-divided into two main groups: rhizosphere habitats and non-rhizosphere habitats, showing that the rhizosphere is one of the most important factors which determines the population structure of a specific soil area.     

High resolution genotyping of Bacillus anthracis outbreak strains using four highly mutable single nucleotide repeat markers

Aims: Bacillus anthracis is a genetically monomorphic bacterium with little diversity to be expected during an outbreak. This study used more rapidly evolving genetic markers on outbreak samples to ascertain genetic diversity. Methods and Results: Forty‐seven isolates from a B. anthracis outbreak during the summer of 2005 in South Dakota were analysed using single nucleotide polymorphisms (SNP) and multi‐locus VNTR analysis (MLVA). Results indicated that all of the outbreak strains belonged to a single clonal lineage. However, analysis of four single nucleotide repeat (SNR) markers resolved these isolates into six distinct genotypes providing insights into disease transmission. Conclusions: Strain determination of unknown B. anthracis samples can be ascertained by SNP and MLVA markers. However, comparison of many samples obtained during an outbreak will require markers with higher rates of mutation to ascertain genetic diversity. Significance and Impact of the Study: SNR4 analysis allowed discrimination of closely related B. anthracis isolates and epidemiological tracking of the outbreak. When used in conjunction with other genotyping schemes that allow broad genetic relationships to be determined, SNR markers are powerful tools for detailed tracking of natural B. anthracis outbreaks and could also prove useful in forensic investigations.     

A food‐grade site‐directed mutagenesis system for Streptococcus thermophilus LMG 18311

Aims: To develop a general method for site‐directed mutagenesis in the dairy starter strain Streptococcus thermophilus LMG 18311 which does not depend on antibiotic‐resistance genes or other selection markers for the identification of transformants. Methods and Results: In a previous study, we demonstrated that Strep. thermophilus LMG 18311 can be made competent for natural genetic transformation by overexpression of the alternative sigma factor ComX. In the present study, we wanted to investigate whether the natural transformation mechanism of Strep. thermophilus LMG 18311 is efficient enough to make it feasible to perform site‐directed mutagenesis in this strain without the use of a selection marker. Competent bacteria were mixed with a DNA fragment engineered to contain a nonsense and a frameshift mutation in the middle of the target gene (lacZ) and subsequently seeded on agar plates. By performing colony‐lift hybridization using a digoxigenin‐labelled oligonucleotide probe, we succeeded in identifying transformants containing the sought after mutation. Conclusions: By exploiting the natural transformability of Strep. thermophilus LMG 18311 and standard molecular methods, we have demonstrated that the genome of this bacterium can be altered at preselected sites without introduction of any foreign DNA. Significance and Impact of the Study: A food‐grade site‐directed mutagenesis system has been developed for Strep. thermophilus LMG 18311 that can be used by the dairy industry to construct starter strains with novel and/or improved properties.     

Carbamoylphosphate synthetase activity is essential for the optimal growth of Streptococcus thermophilus in milk

Aim:  The aim of the study was to study the role of carbon dioxide metabolism in Streptococcus thermophilus through investigation of the phenotype of a carbamoylphosphate synthetase-negative mutant.
Methods and results:  The effect of carbon dioxide on the nutritional requirements of Strep. thermophilus DSM20617^T and its derivative, carbamoylphosphate synthetase-negative mutant A17( ΔcarB ), was investigated by cultivating the strain in a chemically defined medium under diverse gas compositions and in milk. The results obtained revealed that CO₂ depletion or carB gene inactivation determined the auxotrophy of Strep. thermophilus for l -arginine and uracil. In addition, the parent strain grew faster than the mutant, even when milk was supplemented with uracil or arginine.
Conclusions:  Milk growth experiments underlined that carbamoylphosphate synthetase activity was essential for the optimal growth of Strep. thermophilus in milk.
Significance and impact of the study:  The study of the carbon dioxide metabolism in Strep. thermophilus revealed new insights with regard to the metabolism of this species, which could be useful for the optimization of dairy fermentation processes.     

Intra-specific phenotypic and genotypic variation in toxic cyanobacterial Microcystis strains

Aims:  We determined if the intra-specific genetic diversity of Microcystis aeruginosa correlates with phenotypic characteristics.
Methods and Results:  Microcystis aeruginosa isolates from various Japanese waters were characterized using genetic analyses based on the 16S–23S rDNA internal transcribed spacer (ITS) region and DNA-independent RNA polymerase ( rpoC1 ) gene sequences. In addition, morphological and biochemical properties, and the toxicity of M. aeruginosa strains were determined. We found a correlation in phylogenetic clusters of the ITS region and rpoC1 gene sequences. Using a polyphasic approach, genotypic and phenotypic variations in M. aeruginosa showed that the three genetic lineage groups are comprised of a particular phenotype or subgroup of closely related phenotypes. However, some strains had high phenotypic and genotypic diversity compared to the three lineage groups and did not show distinct lineages; therefore, these strains were designated as the 'complex group'.
Conclusions:  The 'complex group' consisted of genetically and phenotypically incoherent and high diverse populations in M. aeruginosa , although some genotypes or lineages displayed consistent phenotypes.
Significance and Impact of the Study:  The polyphasic approach combining phenotypic and genetic characterization was effective for comprehending distinct lineages and discriminating the potential complexity of M. aeruginosa populations at the intra-species level.     

Identification and characterization of Streptococcus thermophilus strains by pulsed-field gel electrophoresis

The chromosomal DNA of a number of strains of the lactic acid bacterium Streptococcus thermophilus was analysed with the aim of rapidly differentiating and assessing the characteristics of each strain. Pulsed-field gel electrophoresis was used to separate large DNA fragments formed by the restriction enzymes Sma I, Sfi I or Apa I. Hybridization with a non-radioactive DNA probe confirmed the identification of strains as Strep. thermophilus and analysis of the electrophoretic patterns differentiated some strains from others. A more extensive study of the pulsed-field electrophoresis restriction patterns of new isolates of Strep. thermophilus may facilitate assessment of their technological properties by comparison of their restriction patterns with those of reference strains.     

Genetic diversity in Swiss cheese starter cultures assessed by pulsed field gel electrophoresis and arbitrarily primed PCR

AIMS: To assess intraspecific genetic heterogeneity among commercial Swiss cheese starter culture strains of Lactobacillus helveticus, Streptococcus thermophilus and Propionibacterium freudenreichii and to compare the efficacy of two genetic typing methods. METHODS AND RESULTS: Two genetic typing methods, pulsed field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR), were used. Nine Strep. thermophilus strains revealed eight PFGE and five AP-PCR genotypes. Seventeen Lactobacillus strains yielded 16 and five genotypes by PFGE and AP-PCR, respectively. Eleven Propionibacterium strains yielded 10 PFGE genotypes. Cluster analysis of PFGE profiles generated similarity coefficients for Strep. thermophilus, Lact. helveticus and Prop. freudenreichii strains of 29.5%, 60.3%, and 30.5%, respectively. Milk acidification rates for Strep. thermophilus and Lact. helveticus were determined. CONCLUSIONS: Pulsed field gel electrophoresis is more discriminatory than AP-PCR. The Lact. helveticus group is more homogeneous than the other species examined. Strains with > 87% similarity by PFGE consistently had the same acidification rate and AP-PCR profile. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial strains sold for Swiss cheese manufacture in the United States are genetically diverse. Clustering of genetically related bacteria may be useful in identifying new strains with industrially relevant traits.     

Comparison of genotypic and biochemical characteristics of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> strains isolated from sour milk products

Overall, 72 strains of lactic acid thermophilic streptococci isolated from sour milk products manufactured in various regions of Russia and European countries were analyzed using classical microbiological and molecular biological methods. Physiological and biochemical properties and genetic diversity of these Streptococcus thermophilus strains were studied, and a comparative analysis of the nucleotide sequences of the 16S rRNA gene was conducted. It has been demonstrated that the homology of proximal parts of the 16S rRNA gene of all the strains studied towards one another and towards the reference strain ATCC19258 amounts to 100%. As for the sugar fermentation, some strains display the characteristics untypical of the S. thermophilus members. The data obtained suggest that it is preferable to use gene 16S rRNA sequencing data for identification of natural isolates of closely related lactic acid bacterial species; moreover, this method is recommended for a precise species identification of industrial bacterial strains used in the food industry.     

A combination of direct viable count and fluorescence in situ hybridization for specific enumeration of viable Lactobacillus delbrueckii subsp.bulgaricus and Streptococcus thermophilus

Aims: We have developed a direct viable count (DVC)‐FISH procedure for quickly and easily discriminating between viable and nonviable cells of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains, the traditional yogurt bacteria. Methods and Results: direct viable count method has been modified and adapted for Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus analysis by testing different times of incubation and concentrations of DNA‐gyrase inhibitors. DVC procedure has been combined with fluorescent in situ hybridization (FISH) for the specific detection of viable cells of both bacteria with specific rRNA oligonucleotide probes (DVC‐FISH). Of the four antibiotics tested (novobiocin, nalidixic acid, pipemidic acid and ciprofloxacin), novobiocin was the most effective for DVC method and the optimum incubation time was 7 h for both bacteria. The number of viable cells was obtained by the enumeration of specific hybridized cells that were elongated at least twice their original length for Lactobacillus and twice their original size for Streptococcus. Conclusions: This technique was successfully applied to detect viable cells in inoculated faeces. Significance and Impact of the Study: Results showed that this DVC‐FISH procedure is a quick and culture‐independent useful method to specifically detect viable Lact. delbrueckii subsp. bulgaricus and Strep. thermophilus in different samples, being applied for the first time to lactic acid bacteria.     

Comparative genotyping of Clostridium thermocellum strains isolated from biogas plants: Genetic markers and characterization of cellulolytic potential

Clostridium thermocellum is among the most prevalent of known anaerobic cellulolytic bacteria. In this study, genetic and phenotypic variations among C. thermocellum strains isolated from different biogas plants were determined and different genotyping methods were evaluated on these isolates. At least two C. thermocellum strains were isolated independently from each of nine different biogas plants via enrichment on cellulose. Various DNA-based genotyping methods such as ribotyping, RAPD (Random Amplified Polymorphic DNA) and VNTR (Variable Number of Tandem Repeats) were applied to these isolates. One novel approach – the amplification of unknown target sequences between copies of a previously discovered Random Inserted Mobile Element (RIME) – was also tested. The genotyping method with the highest discriminatory power was found to be the amplification of the sequences between the insertion elements, where isolates from each biogas plant yielded a different band pattern. Cellulolytic potentials, optimal growth conditions and substrate spectra of all isolates were characterized to help identify phenotypic variations. Irrespective of the genotyping method used, the isolates from each individual biogas plant always exhibited identical patterns. This is suggestive of a single C. thermocellum strain exhibiting dominance in each biogas plant. The genotypic groups reflect the results of the physiological characterization of the isolates like substrate diversity and cellulase activity. Conversely, strains isolated across a range of biogas plants differed in their genotyping results and physiological properties. Both strains isolated from one biogas plant had the best specific cellulose-degrading properties and might therefore achieve superior substrate utilization yields in biogas fermenters.     

Phenotypic diversity of Edwardsiella tarda isolated from different origins

Aims: To evaluate the diversity of phenotypic characteristics among isolates of Edwardsiella tarda from various origins. Methods and results: A total of 10 E. tarda strains were investigated on biological characteristics including flagella formation, bacterial motility, biofilm formation, extracellular protein and plasmid profiles. All the E. tarda strains (including two previous recognized as nonflagellation strains) were proven to have an average of 1–7 peritrichous flagella with the precise number positively correlated with motility and biofilm formation. All the E. tarda strains exhibited similar protein profiles except ET2034, LMG2793 and ET080814, which lacked the three major bands of approximately 18, 21 and 55 kDa. E. tarda with the same geographic location shared similar plasmid profiles. Conclusions:  Edwardsiella tarda strains exhibited diversities in phenotypic characteristics that may be linked to differences in geographic location or host origin. In addition, the number of flagella is essential for bacterial motility and biofilm formation. Significance and Impact of the Study: This is the first report demonstrating the difference in flagella formation between E. tarda strains, which may broaden the understanding of flagellation trait at intra‐species level. Furthermore, evaluation of virulence‐associated characteristics can provide useful information for unveiling the diverse pathogenic mechanisms of E. tarda.     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep, thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.     

Rapid genotyping assays for the identification and differentiation of Yersinia ruckeri biotype 2 strains

Aims: To establish PCR‐based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype‐causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States. Methods and Results: Novel assays were developed relying on detection of mutant allele‐specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing. Conclusions: The described methods were specific, rapid and simple to perform and interpret. Significance and Impact of the Study: The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.     

中国猪种布鲁氏菌分子流行病学特征分析

【目的】调查猪种布鲁氏菌的基因多态性和分子流行病学特征。【方法】用经典分型方法对菌株的生物型进行鉴定,分析菌株的地理分布特点;用MLVA-16分型方法对60株猪种布鲁氏菌进行基因分型,采用在线软件评估分型方法的分辨率和位点的多态性,用BioNumerics 5.0软件进行聚类分析。【结果】我国流行的猪种布鲁氏菌主要是猪种生物1型(33株)、2型(3株)和3型菌(24株);分布范围较广,包括广东、广西、内蒙古、北京、吉林、宁夏和西藏等地。MLVA-16分型方法对猪种布鲁氏菌具有极高的分辨力,多态性指数为0.992;Panel1、MLVA-11和Panel 2B均具有较高的分辨率,多态性指数分别为0.884、0.916和0.979。60株猪种布鲁氏菌聚为6大类52个基因型,5个共享基因型(GT24,GT25,GT26,GT28,GT29)包括13株布鲁氏菌,各基因型菌株间有潜在的流行病学关联,可能是分别来自相同传染源的暴发流行;另47株布鲁氏菌呈现独特的基因型,表明菌株来自无流行病学关联的零星散发病例。猪种布鲁氏菌的最小生成树表明我国菌株分别与美国、法国和波兰的菌株有完全相同的MLVA-15基因型。【结论】中国猪种布鲁氏菌有较高的遗传多态性,并与美国、法国和阿根廷的菌株有较高的遗传相似性。我国猪种布病以零星散发为主。     

Evidence for the conjugal transfer of the broad host range plasmid pIP501 into strains of Lactobacillus helveticus

The conjugative broad host range plasmid pIP501 was transferred from Streptococcus faecalis to a series of strains of lactic streptococci used commercially as dairy starter cultures. With these transconjugants as donors the plasmid was exconjugated to two strains of Lactobacillus helveticus and a commercially used strain of Strep. thermophilus. There was evidence that the plasmid could transfer between isogenic derivatives of one of the strains of Lact. helveticus. Transfer from Lact. helveticus to Strep. faecalis was also detected but at a low frequency. There was no evidence for the conjugal transfer of plasmid pIP501 into a strain of Lact. bulgaricus by exconjugation from either lactic streptococci or Lactobacillus sp.