Characterization of the Bacterial Community Naturally Present on Commercially Grown Basil Leaves: Evaluation of Sample Preparation Prior to Culture-Independent Techniques
<p>Overview of the five basil batches used and the experimental set-up of this study to characterization of the epiphytic bacterial community of basil by molecular techniques denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS), both targeting the 16S rRNA gene.</p> "> Figure 2
<p>Denaturing gradient gel electrophoresis (DGGE) patterns of bacterial communities of basil batches I, II and III with different sample preparation methods and with and without cultivation steps.</p> "> Figure 3
<p>Principal component analysis of the NGS data of (<b>A</b>) basil batches I, II and III with different sample preparation methods and (<b>B</b>) storage of basil batches IV and V at different temperatures.</p> "> Figure 4
<p>Changes in the total bacterial density on basil leaves from batch IV (<b>A</b>) and batch V (<b>B</b>) stored in bags at 7 °C, 15 °C and 22 °C for 14 days, determined by plating on TSA and assessment of the overall visual quality of basil leaves from batch IV (<b>C</b>) and batch V (<b>D</b>). Perfect quality corresponds with a score of 9, while the limit of acceptability for consumption lies at score 5 (indicated by the horizontal line), so all scores equal to or below 5 correspond with spoiled basil samples.</p> "> Figure 5
<p>Denaturing gradient gel electrophoresis (DGGE) patterns of bacterial communities on basil leaves from batch IV and batch V stored at 7 °C, 15 °C and 22 °C for 14 days.</p> ">
Abstract
:1. Introduction
2. Experimental Section
2.1. Basil
2.2. Molecular Microbiological Analyses
2.2.1. Sample Preparation
2.2.2. DNA Extraction
2.2.3. PCR Amplification of 16S rRNA Gene
2.2.4. Denaturing Gradient Gel Electrophoresis (DGGE)
2.2.5. Next-Generation Sequencing (NGS)
2.3. Culture-Based Microbiological Analyses
2.4. Storage Experiments
3. Results
3.1. Culture-Independent Characterisation of the Bacterial Community
Sample | Basil I, Stomacher | Basil I, Wash | Basil II, Stomacher | Basil II, Wash | Basil III, Stomacher | Basil III, Wash |
---|---|---|---|---|---|---|
Total number of reads | 36,887 | 40,630 | 33,794 | Failed | 64,772 | 10,772 |
Median length of reads (bp) | 473 | 474 | 473 | 473 | 473 | |
Not assigned | 29 | 694 | 51 | 20 | 6 | |
Eukaryota | 33,809 | 22,490 | 28,242 | 63,837 | 10,202 | |
Bacteria (rescaled to 100% below) | 3,050 | 17,446 | 5,501 | 915 | 564 | |
Bacteroidetes | 1% | 1% | 74% | |||
Arcicella | 1% | |||||
Chryseobacterium | 1% | 5% | ||||
Flavobacterium | 11% | |||||
Sphingobacterium | 56% | |||||
Alphaproteobacteria | 87% | 5% | 83% | 71% | 17% | |
Altererythrobacter | 2% | |||||
Novosphingobium | 86% | 4% | 81% | 71% | 9% | |
Sphingobium | 1% | |||||
Sphingomonas | 1% | 2% | ||||
Betaproteobacteria | 1% | 6% | 1% | |||
Herbaspirillum | 4% | |||||
Gammaproteobacteria | 10% | 88% | 16% | 26% | 5% | |
Acinetobacter | 4% | 18% | 5% | |||
Pseudomonas | 1% | 40% | 4% | 5% | ||
Rheinheimera | 1% | |||||
Enterobacteriaceae | 8% | 43% | 11% | 2% | ||
Enterobacter | 15% | 2% | ||||
Erwinia | 1% | 2% | ||||
Klebsiella | 3% | 11% | 1% | |||
Kluyvera | 1% | |||||
Pantoea | 6% | 6% | ||||
Rahnella | 4% | |||||
Raoultella | 2% | 2% | ||||
Unclassified | 1% | 1% | 1% | 2% |
3.2. Culturing of the Bacterial Community
3.3. The Bacterial Community on Basil Leaves throughout Storage and Spoilage
Sample | Basil I, BPW 3d | Basil II, BPW 5h | Basil II, BPW 28h | Basil I, TSA | Basil II, TSA | Basil III, TSA | Basil II, VRBL | Basil III, VRBL | Basil II, XLD | Basil III, XLD |
---|---|---|---|---|---|---|---|---|---|---|
Total number of reads | 42,703 | 29,757 | 36,870 | 13,533 | 47,182 | 39,389 | 23,565 | 38,841 | 40,201 | 31,964 |
Median length of reads (bp) | 509 | 494 | 500 | 503 | 502 | 496 | 500 | 496 | 499 | 503 |
Not assigned | 1,829 | 1,985 | 2,294 | 299 | 3,839 | 378 | 114 | 712 | 226 | 198 |
Eukaryota | 0 | 14,201 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
Bacteria (rescaled to 100% below) | 40,874 | 13,572 | 34,576 | 13,234 | 43,343 | 39,011 | 23,451 | 38,129 | 39,975 | 31,766 |
Actinobacteria | 12% | |||||||||
Arthrobacter | 8% | |||||||||
Kocuria | 2% | |||||||||
Bacteroidetes | 55% | 4% | ||||||||
Bacteroides | 42% | |||||||||
Macellibacteroides | 3% | |||||||||
Parabacteroides | 11% | |||||||||
Chryseobacterium | 3% | |||||||||
Clostridia | 6% | 1% | ||||||||
Clostridium | 5% | |||||||||
Alphaproteobacteria | 5% | |||||||||
Novosphingobium | 5% | |||||||||
Betaproteobacteria | 6% | 3% | 2% | |||||||
Comamonas | 5% | 2% | 2% | |||||||
Gammaproteobacteria | 38% | 95% | 93% | 83% | 100% | 97% | 100% | 98% | 100% | 100% |
Aeromonas | 38% | 31% | 36% | |||||||
Alishewanella | 6% | |||||||||
Shewanella | 1% | 1% | ||||||||
Rheinheimera | 13% | |||||||||
Acinetobacter | 4% | 1% | 45% | 17% | 6% | |||||
Pseudomonas | 1% | 37% | 14% | 16% | 13% | 6% | 23% | 1% | 55% | |
Stenotrophomonas | 1% | 1% | ||||||||
Enterobacteriaceae | 34% | 94% | 55% | 23% | 84% | 10% | 92% | 36% | 99% | 7% |
Aranicola | 2% | |||||||||
Cedecea | 2% | |||||||||
Citrobacter | 17% | 1% | 1% | 2% | ||||||
Enterobacter | 8% | 10% | 15% | 5% | 5% | 1% | 11% | 16% | 11% | 3% |
Erwinia | 10% | 5% | 6% | 13% | 28% | 28% | ||||
Klebsiella | 3% | 1% | 7% | 4% | 5% | 7% | 6% | |||
Kluyvera | 1% | 1% | 1% | 1% | 4% | 6% | ||||
Pantoea | 2% | 70% | 21% | 3% | 61% | 1% | 41% | 7% | 45% | 1% |
Pectobacterium | 3% | 7% | ||||||||
Raoultella | 1% | 2% |
Sample | Basil IV Day0 | Basil IV 7d 4°C | Basil IV 7d 14°C | Basil IV 15d 4°C | Basil IV 15d 14°C | Basil IV 22d 4°C | Basil IV 22d 14°C | Basil V Day0 | Basil V 7d 4°C | Basil V 7d 14°C | Basil V 15d 4°C | Basil V 15d 14°C | Basil V 22d 4°C | Basil V 22d 14°C |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Total number of reads | 36.746 | 35.279 | 63.993 | 11.892 | 24.615 | 49.852 | 49.347 | 60.524 | 57.824 | 83.574 | 37.559 | 51.080 | 70.829 | 64.761 |
Median length of reads (bp) | 438 | 456 | 404 | 437 | 405 | 464 | 393 | 417 | 459 | 402 | 437 | 457 | 453 | 393 |
Not assigned | 56 | 40 | 354 | 109 | 788 | 32 | 700 | 94 | 90 | 928 | 181 | 151 | 208 | 3.815 |
Eukaryota | 35.626 | 34.556 | 26.747 | 9.870 | 51 | 49.363 | 222 | 46.475 | 53.834 | 1.735 | 27.767 | 48.859 | 56.755 | 9.132 |
Bacteria (rescaled to 100 % below) | 1.065 | 682 | 36.892 | 1.913 | 23.776 | 457 | 48.425 | 13.954 | 3.900 | 80.911 | 9.611 | 2.070 | 13.867 | 51.815 |
Actinobacteria | 14% | 1% | 1% | 14% | 1% | |||||||||
Microbacterium | 2% | 1% | ||||||||||||
Arthrobacter | 10% | 1% | 8% | |||||||||||
Bacteroidetes | 4% | 2% | 17% | 29% | 18% | 14% | 16% | 19% | ||||||
Chryseobacterium | 2% | 1% | 11% | 3% | 1% | |||||||||
Flavobacterium | 2% | 17% | 25% | 1% | 3% | |||||||||
Pedobacter | 2% | 18% | 2% | |||||||||||
Sphingobacterium | 9% | 18% | ||||||||||||
Bacilli | 2% | 1% | ||||||||||||
Bacillus | 2% | |||||||||||||
Clostridia | 3% | |||||||||||||
Clostridium | 2% | |||||||||||||
Alphaproteobacteria | 5% | 20% | 8% | 11% | 21% | 8% | 2% | 8% | 2% | 23% | 4% | 8% | ||
Methylobacterium | 9% | 1% | ||||||||||||
Agrobacterium | 1% | 1% | 8% | 1% | ||||||||||
Rhizobium | 2% | 2% | 1% | 2% | 2% | |||||||||
Novosphingobium | 5% | 19% | 4% | 6% | 8% | 3% | 2% | 7% | 1% | 11% | 3% | 3% | ||
Sphingomonas | 1% | 1% | 1% | 1% | 1% | |||||||||
Betaproteobacteria | 28% | 15% | 2% | 9% | 15% | 36% | 20% | 6% | 28% | |||||
Achromobacter | 3% | 1% | 1% | |||||||||||
Burkholderia | 2% | 1% | 1% | |||||||||||
Acidovorax | 5% | |||||||||||||
Comamonas | 1% | 1% | ||||||||||||
Delftia | 1% | 8% | ||||||||||||
Variovorax | 2% | |||||||||||||
Duganella | 1% | 3% | ||||||||||||
Herbaspirillum | 5% | 2% | 6% | |||||||||||
Herminiimonas | 1% | |||||||||||||
Janthinobacterium | 17% | 1% | 2% | 2% | ||||||||||
Oxalicibacterium | 1% | 2% | 5% | |||||||||||
Methylobacillus | 12% | |||||||||||||
Methylophilus | 2% | 4% | 32% | 4% | ||||||||||
Zoogloea | 2% | |||||||||||||
Gammaproteobacteria | 66% | 44% | 94% | 64% | 44% | 7% | 54% | 97% | 91% | 100% | 97% | 54% | 95% | 45% |
Pseudoalteromonas | 3% | 1% | ||||||||||||
Acinetobacter | 4% | |||||||||||||
Pseudomonas | 56% | 30% | 71% | 42% | 10% | 3% | 22% | 96% | 85% | 95% | 95% | 44% | 89% | 24% |
Luteibacter | 1% | 6% | 1% | |||||||||||
Stenotrophomonas | 1% | 22% | 2% | 17% | 4% | 12% | ||||||||
Xanthomonas | 1% | 2% | 1% | |||||||||||
Enterobacteriaceae | 8% | 7% | 22% | 21% | 5% | 10% | 1% | 6% | 3% | 2% | 5% | 5% | 8% | |
Buchnera | 2% | |||||||||||||
Enterobacter | 3% | 3% | 2% | 7% | 1% | 6% | ||||||||
Erwinia | 3% | |||||||||||||
Ewingella | 4% | 11% | ||||||||||||
Pantoea | 4% | 1% | 1% | 5% | 2% | 1% | 5% | 3% | 1% | |||||
Rahnella | 4% | 10% | 5% | 1% | 1% | |||||||||
Serratia | 5% | 1% | ||||||||||||
Unclassified | 2% |
4. Discussion
Genus | Gram Staining | Respiratory Metabolism | Motility | Temperature Range Growth | Habitat | Pathogenicity |
---|---|---|---|---|---|---|
Flavobacterium | Gram negative | Aerobic | Nonmotile or motile by gliding | −7 to 45 °C | Soil, freshwater, marine and saline environments | Some species, such as F. columnare, F. psychrophilum and F. branchiophilum, are pathogenic for freshwater fish. Some strains of F. johnsoniae are plant pathogens causing soft rot in various plants. |
Sphingobacterium | Gram negative | Aerobic | Sliding motility | 2 to 45 °C | Soil and composted manure | Some species are opportunistic pathogens for humans. |
Acinetobacter | Gram negative | Aerobic | Twitching motility by fimbriae | 20 to 37 °C | Soil, water, sewage and plants | Although considered normally nonpathogenic, they may cause nosocomial infections such as bacteremia, secondary meningitis, pneumonia, and urinary tract infections in humans. |
Pseudomonas | Gram negative | Aerobic | Motile by one or several polar flagella and fimbriae | 4 to 45 °C | Plants (rhizospheres and leave surfaces) and soil | Some species are pathogenic for humans, animals, or plants. Plant pathogenic species such as P. syringae may cause tumorous outgrowth, rot, blight or chlorosis, and necrosis in plants due to secretion of substances (such as toxins, plant hormones and enzymes) which alter the normal metabolism of plant cells. Others are opportunistic pathogens for animals and humans, such as P. aeruginosa. |
Citrobacter | Gram negative | Facultatively anaerobic | Usually motile by peritrichous flagella | 5 to >37 °C | Intestinal tract of humans and some animals, soil, water, sewage, plants and food (vegetables, dairy, fish) | Some species are opportunistic pathogens for humans. |
Enterobacter | Gram negative | Facultatively anaerobic | Motile by peritrichous flagella | 4 to 44 °C | Plants (rhizophere and leaves) and the intestinal tract of humans and animals | Some species are plant pathogens, such as E. nimipressuralis (wetwood in elm trees), E. cancerogenus (canker disease of Populus species) and E. pyrinus (brown leaf spot disease in pears). |
Erwinia | Gram negative | Facultatively anaerobic | Motile by peritrichous flagella | 0 to 40 °C | Plants | Plant pathogens which cause mainly blights and wilts. Infection through natural openings and wounds, followed by spread through the vascular tissue. |
Klebsiella | Gram negative | Facultatively anaerobic | Nonmotile (except K. mobilis) | 5 to 45 °C | Intestinal tract of humans and animals, soil, water, sewage and plants | Opportunistic and nosocomial human pathogens, e.g. K. pneumoniae, causing pneumonia, urinary tract infections, bacteremia and sepsis. |
Kluyvera | Gram negative | Facultatively anaerobic | Motile | 4 to 40 °C | Intestinal tract of humans and animals, soil, sewage and food (milk, dairy and other food products of animal origin) | Opportunistic human pathogen. |
Pantoea | Gram negative | Facultatively anaerobic | Most strains are motile by peritrichous flagella | 4 to 41 °C | Plants, seeds, fruits, soil and water | Some strains are opportunistic pathogens for plants, humans and animals. |
Rahnella | Gram negative | Facultatively anaerobic | Motile by peritrichous flagella | 1 to ≥37 °C | Fresh water, soil, plant rhizosphere, intestinal tract of snails | Opportunistic human pathogens causing wound infections, bacteremias, acute gastroenteritis and septicemia. |
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Ceuppens, S.; Delbeke, S.; De Coninck, D.; Boussemaere, J.; Boon, N.; Uyttendaele, M. Characterization of the Bacterial Community Naturally Present on Commercially Grown Basil Leaves: Evaluation of Sample Preparation Prior to Culture-Independent Techniques. Int. J. Environ. Res. Public Health 2015, 12, 10171-10197. https://doi.org/10.3390/ijerph120810171
Ceuppens S, Delbeke S, De Coninck D, Boussemaere J, Boon N, Uyttendaele M. Characterization of the Bacterial Community Naturally Present on Commercially Grown Basil Leaves: Evaluation of Sample Preparation Prior to Culture-Independent Techniques. International Journal of Environmental Research and Public Health. 2015; 12(8):10171-10197. https://doi.org/10.3390/ijerph120810171
Chicago/Turabian StyleCeuppens, Siele, Stefanie Delbeke, Dieter De Coninck, Jolien Boussemaere, Nico Boon, and Mieke Uyttendaele. 2015. "Characterization of the Bacterial Community Naturally Present on Commercially Grown Basil Leaves: Evaluation of Sample Preparation Prior to Culture-Independent Techniques" International Journal of Environmental Research and Public Health 12, no. 8: 10171-10197. https://doi.org/10.3390/ijerph120810171