rep-PCR Genomic Fingerprinting Research Articles

Adjunct Culture of Non-Starter Lactic Acid Bacteria for the Production of Provola Dei Nebrodi PDO Cheese: In Vitro Screening and Pilot-Scale Cheese-Making.

The present study aimed at selecting non-starter lactic acid bacteria strains, with desirable technological and enzymatic activities, suitable as adjunct culture for the Provola dei Nebrodi cheese production. One hundred and twenty-one lactic acid bacteria, isolated from traditional Provola dei Nebrodi cheese samples, were genetically identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-seven strains, included in the qualified presumption of safety (QPS) list, were tested for technological and proteinase/peptidase activities. Results showed that technological features and flavour formation abilities were strain-dependent. Among the selected strains, Lacticaseibacillus paracasei PN 76 and Limosilactobacillus fermentum PN 101 were used as adjunct culture in pilot-scale cheese-making trials. Data revealed that adjunct cultures positively affected the flavour development of cheese, starting from 30 days of ripening, contributing to the formation of key flavour compounds. The volatile organic compound profiles of experimental cheeses was significantly different from those generated in the controls, suggesting that the selected adjunct strains were able to accelerate the flavour development, contributing to a unique profile of Provola dei Nebrodi cheese.

Piacentinu Ennese PDO Cheese as Reservoir of Promising Probiotic Bacteria.

Piacentinu Ennese is a protected designation of origin (PDO) cheese produced in the surrounding area of Enna (Sicily, Italy), using raw ewe’s milk without the addition of any starter cultures. In the present study, the Lactobacillus population of Piacentinu Ennese PDO cheese was in vitro screened in order to select promising probiotic strains to be further used in humans. One hundred and sixty-nine lactic acid bacteria (LAB) were isolated from 90 days ripened cheeses and identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by MALDI-TOF MS. One hundred and thirteen (113) isolates belonging to QPS-list species were characterized for both safety and functional properties. All tested isolates were considered safe because none showed either gelatinase, DNase, mucinase, or hemolytic activity. Tolerance to lysozyme, bile salts, and acidic conditions, along with ability to survive under simulated gastrointestinal digestion, were observed. In addition, based on antimicrobial activity against pathogens, cell surface characteristics, Caco-2 adhesion abilities, and anti-inflammatory potential, it was possible to confirm the strain-dependent functional aptitude, suggesting that Piacentinu Ennese PDO cheese may be considered a precious source of probiotic candidates.

Evolution of Yeast Consortia during the Fermentation of Kalamata Natural Black Olives upon Two Initial Acidification Treatments.

The objective of this study was to elucidate the yeast consortia structure and dynamics during Greek-style processing of Kalamata natural black olives in different brine solutions. Olives were subjected to spontaneous fermentation in 7% (w/v) NaCl brine solution (control treatment) or brine acidified with (a) 0.5% (v/v) vinegar, and (b) 0.1% (v/v) lactic acid at the onset of fermentation. Changes in microbial counts, pH, acidity, organic acids, sugars, and alcohols were analyzed for a period of 187 days. Yeast consortia diversity was evaluated at days 4, 34, 90, 140, and 187 of fermentation. A total of 260 isolates were characterized at sub-species level by rep-PCR genomic fingerprinting with the oligo-nucleotide primer (GTG)5. The characterization of yeast isolates at species level was performed by sequencing of the D1/D2 domain of 26S rRNA gene. Results showed that yeasts dominated the process presenting a relatively broad range of biodiversity composed of 11 genera and 21 species. No lactic acid bacteria (LAB) or Enterobacteriaceae could be enumerated after 20 and 10 days of fermentation, respectively. The dominant yeast species at the beginning were Aureobasidium pullulans for control and vinegar acidification treatments, and Candida naeodendra for lactic acid treatment. Between 34 and 140 days the dominant species were Candida boidinii, Candida molendinolei and Saccharomyces cerevisiae. In the end of fermentation the dominant species in all processes were C. boidinii and C. molendinolei, followed by Pichia manshurica and S. cerevisiae in lactic acid acidification treatment, P. manshurica in vinegar acidification treatment, and Pichia membranifaciens in control fermentation.

Analysis of the Molecular Diversity of Common Bacterial Blight (Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscansi) Strains from Ethiopia Revealed by Rep-PCR Genomic Fingerprinting

Common bacterial blight (CBB) disease of the common bean (Phaseolus vulgaris) caused by Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, is one of the most damaging foliar diseases of common bean production in Ethiopia. CBB causes economic losses due to reduction in seed quality and yield in common bean producing regions of Ethiopia. Currently, information on the genetic diversity of CBB strains in Ethiopia has been lacking. Here, for this specific study common bean bacterial blight strains were obtained from infected leaves collected from diverse bean growing areas. The collected strains of CBB were characterized to study the genetic diversity and relatedness of the CBB strains using repetitive extragenic elements polymerase chain reaction (rep-PCR) genomic fingerprinting technique. Analysis of Molecular Variance (AMOVA) revealed the existence of genetic diversity among bacterial strains and confirmed the presence of genetically distinct strains in Ethiopia. CBB pathogens are seed-borne so the lack of geographic differentiation among the six-different common bean growing localities could be the result of the distribution of one or some limited bacterial genotypes. Common bean improvement programs that develop CBB-resistant bean varieties for higher production should consider this information to determine the relevance and extent of resistance of improved bean cultivars.

Rep-PCR Genomic Fingerprinting Revealed Genetic Diversity and Population Structure among Ethiopian Isolates of Pseudocercospora griseola Pathogen of the Common Bean (Phaseolus vulgaris L.)

Angular leaf spot (Pseudocercospora griseola) is one of the most devastating diseases affecting common bean production in most parts of Ethiopia. Thus, use of common bean varieties with durable resistance is the most effective and economical control measure. Knowledge about the genetic variability and population structure of the pathogen populations is important for a successful common bean improvement program. The objective of this study was to determine the genomic diversity existing among and between P. griseola isolates which were obtained from the field survey collection of diverse common-bean growing areas of Ethiopia. The study used the repetitive extragenic palindromic elements-polymerase chain reaction protocol to fingerprinting DNA sequence diversity. To study the genetic diversity, we analysed molecular data from 79 single-spore isolates of the P. griseola pathogen. Hence, Molecular Analysis of Variance (AMOVA) and cluster analysis revealed the existence of high genetic diversity within and among P. griseola isolates. ERIC PCR produced 21 different patterns of clusters, whereas, REP-PCR and BOX PCR produced 11 and 5 different patterns of clusters respectively. This is because of some isolates that shared the same BOX patterns could be distinguished by the ERIC and REP finger printing patterns. The ERIC-, BOX- and REP-PCR combined fingerprinting patterns discriminated 25 different patterns among the 79 monosporic P. griseola isolates were produced at cut-off 77% genetic similarity matrix. These discriminated clusters revealed the existence of genetic diversity within and among the isolates of P. griseola collected from the diverse common bean growing regions of Ethiopia.

Clavibacter michiganensis subsp. michiganensis strains virulence and genetic diversity. a first study in Argentina

Tomato bacterial wilt and canker, caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is considered one of the most important bacterial diseases of tomato worldwide. During the last two decades, severe outbreaks have occurred in greenhouses in the horticultural belt of Buenos Aires-La Plata, Argentina. Cmm strains collected in this area over a period of 14 years (2000–2013) were characterized for genetic diversity by rep-PCR genomic fingerprinting and level of virulence in order to have a better understanding of the source of inoculum and virulence variability. Analyses of BOX-, ERIC- and REP-PCR fingerprints revealed that the strains were genetically diverse; the same three fingerprint types were obtained in all three cases. No relationship could be established between rep-PCR clustering and the year, location or greenhouse origin of isolates, which suggests different sources of inoculum. However, in a few cases, bacteria with identical fingerprint types were isolated from the same greenhouse in different years. Despite strains differing in virulence, particularly within BOX-PCR groups, putative virulence genes located in plasmids (celA, pat-1) or in a pathogenicity island in the chromosome (tomA, chpC, chpG and ppaA) were detected in all strains. Our results suggest that new strains introduced every year via seed importation might be coexisting with others persisting locally. This study highlights the importance of preventive measures to manage tomato bacterial wilt and canker.

Chelatococcus thermostellatus sp. nov., a new thermophile for bioplastic synthesis: comparative phylogenetic and physiological study

The poly(3-hydroxybutyrate), PHB, accumulating thermophilic strain MW9T, isolated from an aerobic organic waste treatment plant, was characterized by detailed physiological and phylogenetic studies. The strain is a Gram-stain-negative, rod shaped, non-spore forming member of Alphaproteobacteria. It shows optimum growth at 50 °C. Based on 16S rRNA gene sequence similarity, the strain together with five very similar isolates, was affiliated to the genus Chelatococcus (Ibrahim et al. in J Appl Microbiol 109:1579–1590, 2010). Rep-PCR genomic fingerprints and partial dnaK gene sequence also revealed that these isolates are very similar, but differ from other Chelatococcus type strains. The major fatty acids were similar to those of other strains of the genus Chelatococcus. DNA–DNA hybridization of strain MW9T with Chelatococcus species type strains revealed 11.0–47.7 % relatedness. G+C content of DNA was 67.1 mol%, which is comparable with the other strains of Chelatococcus species. The physiological and phenotypic characteristics of the new strain MW9T are sufficient to differentiate it from previously described species in the genus Chelatococcus. Strain MW9T is considered to represent a novel species of the genus Chelatococcus, for which the name Chelatococcus thermostellatus is proposed. The type strain is MW9T (=LMG 27009T = DSM 28244T). Compared to known Chelatococcus strains, strain MW9T could be a potent candidate for bioplastic production at elevated temperature.

Evaluation of native bacteria and manganese phosphite for alternative control of charcoal root rot of soybean

Plant growth promoting rhizobacteria (PGPR) are potential agents to control plant pathogens and their combined use with biopesticides such as phosphites may constitute a novel strategy to incorporate in disease management programs. In the present study, 11 bacterial isolates were selected on the basis of their antagonistic activity against Macrophomina phaseolina in dual-culture tests, and their plant growth promoting traits. Selected isolates were characterised on the basis of auxin and siderophore production, phosphate solubilisation and rep-PCR genomic fingerprinting. Two of these isolates, identified as Pseudomonas fluorescens 9 and Bacillus subtilis 54, were further evaluated for their inhibitory capacity against M. phaseolina using in vitro (on soybean seeds) and in vivo (greenhouse assay) tests. Both bacteria were applied individually as well as in combined treatment with manganese phosphite as seed treatments. Damage severity on soybean seeds was significantly reduced, compared with the untreated control, by both bacterial strains; however, the individual application of phosphite showed to be least effective in controlling M. phaseolina. Interestingly, the phosphite treatment improved its performance under greenhouse conditions compared to the results from the in vitro assays. In the greenhouse trials, the greatest reductions in disease severity were achieved when strain P. fluorescens 9 was applied singly or when strain B. subtilis 54 was combined with manganese phosphite, achieving 82% of control in both cases. This work is the first to report the control of M. phaseolina using combined treatment with PGPR and phosphite under greenhouse conditions.

Genotypic Characterization of Azotobacteria Isolated from Argentinean Soils and Plant‐Growth‐Promoting Traits of Selected Strains with Prospects for Biofertilizer Production

The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA), gibberellin (GA3) and zeatin (Z) biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2–18.2 μg IAA mL−1, 0.3–0.7 μg GA3 mL−1, and 0.5–1.2 μg Z mL−1. Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

Characterization of phenotypic variants of Clavibacter michiganensis subsp. michiganensis isolated from Capsicum annuum

Phenotypic variants of Clavibacter michiganensis subsp. michiganensis (Cmm) were isolated from pepper fields and from pepper seeds during quarantine inspections. All strains isolated from pepper (pepper isolates) produced orange-coloured colonies with lower mucoidy than typical Cmm strains isolated from tomato (tomato isolates). However, the results of ELISA, fatty acid analysis, 16S rDNA sequencing, and PCR analysis showed that all pepper isolates were similar enough to be identified as Cmm. In addition to phenotypic variations, the pepper isolates showed different pathogenic and genetic characteristics from tomato isolates from the USA, Europe, or other countries. They could be clearly distinguished in terms of pathogenicity, as they showed increased pathogenicity to pepper but reduced pathogenicity to tomato. Tomato isolates caused strong wilting and canker in tomato, but caused only canker and no wilting in pepper and bell pepper. However, pepper isolates caused no wilting, even in tomato, and only caused canker in the three host plants. In addition, compared to tomato isolates, pepper isolates showed increased colonization efficiency and caused a greater reduction in shoot dry weight in pepper. Pepper and tomato isolates could be separated into two groups according to host origin on the basis of 16S rDNA and ITS sequence analysis. They also showed different rep-PCR genomic fingerprints. All pepper isolates showed higher cellulase activity than tomato isolates on M9CMC plates. However, two plasmid-borne virulence genes of Cmm, pat-1, and celA, were not detected in any pepper isolates by PCR. Furthermore, PCR for pathogenicity-related genes located on a pathogenicity island (PAI) revealed that all tomato isolates were positive for these genes, whereas the pepper isolates did not show any PCR products for the chpC, chpG, ppaA, or tomA genes. Therefore, we suggest that the pepper isolates may represent a separate Cmm population that has evolved within the limits of this host.

Antimicrobial and Safety Properties of Lactobacilli Isolated from two Cameroonian Traditional Fermented Foods.

Twenty-one Lactobacillus isolates from “Sha’a” (a maize – based fermented beverage) and “Kossam” (traditionally fermented cow milk) were selected in accordance with their antagonistic activities and tested for their bacteriocinogenic potential as well as safety properties. These isolates were preliminarily identified as Lactobacillus plantarum (62%), Lactobacillus rhamnosus (24%), Lactobacillus fermentum (10%) and Lactobacillus coprophilus (4%) based on phenotypic characteristics and rep-PCR genomic fingerprinting. Twelve (57.1%) out of the 21 strains tested were found to be bacteriocin producers, as revealed by the sensitivity of their antimicrobial substances to proteolytic enzymes (Trypsin, Proteinase K) and inhibition of other Lactobacillus spp. These bacteriocinogenic strains showed no positive haemolytic and gelatinase activities and proved to be sensitive to penicillin G, ampicillin, tetracycline, erythromycin, amoxicillin, chloramphenicol, co-trimoxazole and doxycyclin, but resistant to ciprofloxacin and gentamicin. The bacteriocins showed a broad inhibitory activity against Gram-positive and Gram-negative pathogenic bacteria, several of which are classified as especially dangerous by the World Health Organization, as well as Multidrug-resistant strains. These include Staphylococcus aureus, Salmonella enterica subsp. enterica serovare Typhi, Bacillus cereus, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Shigella flexneri. These Lactobacillus strains are promising candidates for use as protective cultures in food fermentation.

Genetic relationships of Edwardsiella strains isolated in China aquaculture revealed by rep-PCR genomic fingerprinting and investigation of Edwardsiella virulence genes

The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity-related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR-based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus-PCR and BOX-PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain-specific or origin-specific clades. Biochemical characterization was sensitive for interspecific typing, while PCR-based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. PCR-based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.

Molecular Characterization of Burkholderia cepacia Complex Isolates Causing Bacterial Fruit Rot of Apricot

The Burkholderia cepacia complex isolates causing bacterial fruit rot of apricot were characterized by speciesspecific PCR tests, recA-HaeIII restriction fragment length polymorphism (RFLP) assays, rep-PCR genomic fingerprinting, recA gene sequencing, and multilocus sequence typing (MLST) analysis. Results indicated that the isolates Bca 0901 and Bca 0902 gave positive amplifications with primers specific for B. vietnamiensis while the two bacterial isolates showed different recA-RFLP and rep-PCR profiles from those of B. vietnamiensis strains. In addition, the two bacterial isolates had a higher proteolytic activity compared with that of the non-pathogenic B. vietnamiensis strains while no cblA and esmR marker genes were detected for the two bacterial isolates and B. vietnamiensis strains. The two bacterial isolates were identified as Burkholderia seminalis based on recA gene sequence analysis and MLST analysis. Overall, this is the first characterization of B. seminalis that cause bacterial fruit rot of apricot.

Prevalence of the gene trzN and biogeographic patterns among atrazine-degrading bacteria isolated from 13 Colombian agricultural soils

The following study evaluated the diversity and biogeography of 83 new atrazine-degrading bacteria and the composition of their atrazine degradation genes. These strains were isolated from 13 agricultural soils and grouped according to rep-PCR genomic fingerprinting into 11 major clusters, which showed biogeographic patterns. Three clusters (54 strains) belonged to the genus Arthrobacter, seven clusters (28 strains) were similar to the genus Nocardioides and only one strain was a gram-negative from the genus Ancylobacter. PCR assays for the detection of the genes atzA, B, C, D, E, F and trzN conducted with each of the 83 strains revealed that 82 strains (all gram positive) possessed trzN, 74 of them possessed the combination of trzN, atzB and atzC, while only the gram-negative strain had atzA. A similar PCR assay for the two analogous genes, atzA and trzN, responsible for the first step of atrazine degradation, was performed with DNA extracted directly from the enrichment cultures and microcosms spiked with atrazine. In these assays, the gene trzN was detected in each culture, while atzA was detected in only six out of 13 soils. These results raise an interesting hypothesis on the evolutionary ecology of the two atrazine chlorohydrolase genes (i.e. atzA and trzN) and about the biogeography of atrazine-degrading bacteria.

Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe

Sixty-five potato strains of the soft rot-causing plant pathogenic bacterium Dickeya spp., and two strains from hyacinth, were characterised using biochemical assays, REP-PCR genomic finger printing, 16S rDNA and dnaX sequence analysis. These methods were compared with nineteen strains representing six Dickeya species which included the type strains. A group of twenty-two potato strains isolated between 2005-2007 in the Netherlands, Poland, Finland and Israel were characterised as belonging to biovar 3. They were 100% identical in REP-PCR, dnaX and 16S rDNA sequence analysis. In a polyphasic analysis they formed a new clade different from the six Dickeya species previously described, and may therefore constitute a new species. The strains were very similar to a Dutch strain from hyacinth. On the basis of dnaX sequences and biochemical assays, all other potato strains isolated in Europe between 1979 and 1994 were identified as D. dianthicola (biovar 1 and 7), with the exception of two German strains classified as D. dieffenbachia (biovar 2) and D. dadantii (biovar 3), respectively. Potato strains from Peru were classified as D. dadantii, from Australia as D. zeae and from Taiwan as D. chrysanthemi bv. parthenii, indicating that different Dickeya species are found in association with potato.

Identification of a bacterium isolated from galls on carrot and weeds

In 2004, bacterial galls were found on the roots of carrots in Shizuoka Prefecture, Japan. Galls were about 0.1–2 cm in diameter, light brown in color and had rough surfaces. In 2005, similar galls were found on the roots of three weeds: henbit (Lamium amplexicaule L.), Persian speedwell (Veronica persica Poir.) and leaf mustard (Brassica juncea L.). A bacterium that forms white, rough colonies was isolated from the carrot and weeds galls. The bacterial isolates had properties identical with Rhizobacter dauci Goto and Kuwata. Phylogenetic analysis based on 16S rDNA sequences showed that the carrot isolate had the highest homology (similarity of 100%) with that of the type strain of R. dauci. Rep-PCR genomic fingerprinting using BOX A1R primer showed that the carrot and weeds isolates were nearly identical. Pathogenicity of the isolates was confirmed by inoculating the roots of carrots and the weeds. After 2–5 weeks, they formed galls on the roots of the original host species and on other plant species tested. The galls were indistinguishable from those formed naturally, and the inoculated bacterium was reisolated. Thus, the causal bacterium of carrot and weeds gall was identified as R. dauci, and the bacterium was found to have a wider host range than previously known. These weed hosts may serve as inoculum sources for carrot bacterial gall disease.

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.

Comparative efficacy of five different rep-PCR methods to discriminate Escherichia coli populations in aquatic environments

Development of efficient techniques to discriminate the sources of E. coli in aquatic environments is essential to improve the surveillance of fecal pollution indicators, to develop strategies to identify the sources of fecal contamination, and to implement appropriate management practices to minimize gastrointestinal disease transmission. In this study the robustness of five different rep-PCR methods, such as REP-PCR, ERIC-PCR, ERIC2-PCR, BOX-PCR and (GTG)(5)-PCR were evaluated to discriminate 271 E. coli strains isolated from two watersheds (Lakelse Lake and Okanagan Lake) located in British Columbia, Canada. Cluster analysis of (GTG)(5)-PCR, BOX-PCR, REP-PCR, ERIC-PCR and ERIC2-PCR profiles of 271 E. coli revealed 43 clusters, 35 clusters, 28 clusters, 23 clusters and 14 clusters, respectively. The discriminant analysis of rep-PCR genomic fingerprints of 271 E. coli isolates yielded an average rate of correct classification (watershed-specific) of 86.8%, 82.3%, 78.4%, 72.6% and 55.8% for (GTG)(5)-PCR, BOX-PCR, REP-PCR, ERIC-PCR and ERIC2-PCR, respectively. Based on the results of cluster analysis and discriminant function analysis, (GTG)(5)-PCR was found to be the most robust molecular tool for differentiation of E. coli populations in aquatic environments.

Internal fruit rot of netted melon caused by Pantoea ananatis (=Erwinia ananas) in Japan

An internal fruit rot with a malodor was found in netted melons (Cucumis melo L.) in commercial greenhouses in Kochi Prefecture, Japan, in 1998, despite their healthy appearance and lack of water-soaking or brown spots on the surface. A yellow bacterium was consistently isolated from the affected fruits. To confirm the pathogenicity of eight representative isolates of the yellow bacterium, we stub-inoculated ovaries (immature-fruits) 5–7 days after artificial pollination, with a pin smeared with bacteria. After the melon fruits had grown for 60 more days, an internal fruit rot resembling the natural infection appeared, and the inoculated bacterium was reisolated. The melon isolates had properties identical with Pantoea ananatis, such as gram-negative staining, facultative anaerobic growth, indole production, phenylalanine deaminase absence, and acid production from melibiose, sorbitol, glycerol, and inositol. Phylogenetic analysis based on 16S rDNA sequences showed that the melon bacterium positioned closely with known P. ananatis strains. The melon bacterium had indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). The bacterium could be distinguished from the other ‘Pantoea’ group strains by rep-PCR genomic fingerprinting. From these results, the causal agent of internal fruit rot was identified as a strain of P.ananatis [Serrano in (Philipp J Sci 36:271–305, 1928); Mergaert et al. in (Int J Syst Bacteriol 43:162–173, 1993)].

Comparison of five rep-PCR genomic fingerprinting methods for differentiation of  fecalEscherichia colifrom humans, poultry and wild birds

The development of a methodology to identify the origin of fecal pollution is important both for assessing the degree of risk posed to public health and for developing strategies to mitigate the environmental loading of pathogens associated with waterborne disease transmission. Five rep-PCR genomic fingerprinting methods, such as rep-PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR, ERIC2-PCR, BOX-PCR and (GTG)(5)-PCR, were assessed for their potential in differentiation of 232 fecal Escherichia coli isolates obtained from humans, poultry (chicken, duck and turkey) and wild birds (Canada goose and gull). Based on the results of cluster analysis and discriminant function analysis, (GTG)(5)-PCR was found to be the most suitable method for molecular typing of fecal E. coli, followed by BOX-PCR, REP-PCR, ERIC-PCR and ERIC2-PCR. A discriminant function analysis of (GTG)(5)-PCR fingerprints showed that 94.1%, 79.8%, 80.5%, 74.4%, 86.7% and 88.6% of turkey, chicken, duck, Canada goose, gull and human E. coli isolates were classified into the correct host group, respectively. Subsequently, (GTG)(5)-PCR was tested for its ability to track the origin of 113 environmental E. coli isolated from natural pond water. In conclusion, the (GTG)(5)-PCR genomic fingerprinting method can be considered as a promising genotypic tool for epidemiological surveillance of fecal pollution in aquatic environments.