Geno- and phenotypic characterization of lactic acid bacteria and Bacillus spp. strains isolated from African indigenous fermented food products and their applications in the food and feed industries

TY - BOOK

T1 - Geno- and phenotypic characterization of lactic acid bacteria and Bacillus spp. strains isolated from African indigenous fermented food products and their applications in the food and feed industries

AU - Adimpong, David Bichala

PY - 2013

Y1 - 2013

N2 - African indigenous fermented food products are characterized by complex and diverse groups of microorganisms and therefore offer a rich source for selection of microbial strains for various applications in the biotechnology and food bio-processing sectors. There is however, a global public health concern regarding the use of live microbial strains for biotechnological and food bioprocessing applications due to the risk of dissemination of multiple antimicrobial resistance genesand virulence factors to pathogenic microorganisms. In view of this, the safety of microbial strains with potential biotechnological and food bio-processing applications especially as live culture is the responsibility of the producer. This requires careful safety evaluations such as sensitivity to antimicrobial agents and production of virulence factors. The aim of this PhD Thesis was to characterize lactic acid bacteria (LAB) and Bacillus spp. strains isolated from selected African indigenous fermented food products in order to gain an in-depth knowledge on their physiology, safety and genomics in consideration for different biotechnological applications. The study was categorised into the 3 major research areas; microbial identification, antimicrobial susceptibility study and genome sequencing. Subsequently, 33 LAB strains were identified by molecular techniques including; sequencing of the 16S rRNA gene, rep-PCR fingerprinting analyses and species-specific PCR assays (Appendix I). Strain ZN7a-9 was identified as a L. delbrueckii species based on its 16S rRNA gene sequence, DNA-DNA hybridization (DDH) and peptidoglycan cell-wall structure. But it could not be assigned to any of the recognised members of the L. delbrueckii subspecies taxon based on its phenotypic features, Multilocus Sequence Typing and splits-decomposition analyses. In order to be able to accommodate strain ZN7a-9 within the L. delbrueckii subsp. taxon and to additionally distinguish it from the recognised members of this taxon, it was proposed as a new L. delbrueckii subspecies; Lactobacillus delbrueckii subsp. jakobsenii subsp. nov. (Appendix II).By the broth microdilution technique, the LAB strains and 85 Bacillus spp. strains representing 38 B. licheniformis, 29 B. subtilis subsp. subtilis and 18 B. sonorensis strains were characterised for susceptibility to antimicrobial compounds of clinical and veterinary importance. The LAB strains were susceptible to; ampicillin, chloramphenicol, clindamycin and erythromycin but intrinsically resistant to kanamycin, streptomycin and vancomycin. Variable and multiple resistance to tetracycline and gentamicin was observed (Appendix I). All the Bacillus spp. strains were susceptible to tetracycline, vancomycin and gentamicin but resistant to streptomycin. Also, speciesspecific variation in sensitivity of the 3 Bacillus spp. to chloramphenicol, clindamycin, erythromycin and kanamycin was observed. The erythromycin resistance was only present in the B. licheniformis strains (50.0 %) and strongly correlated with the presence of the rRNA methylase gene; erm(D) or erm(K) located on an 11.4-kbp plasmid. Since rRNA methylase genes are known to confer erythromycin resistance phenotype to microbial strains, the observed erythromycin resistance phenotype was attributed to the erm(D) or erm(K) genes detected by PCR (Appendix III). Analyses of the whole genome draft-sequence of Bacillus sonorensis strain L12 which was generated using the Illumina Hiseq platform revealed it encodes gene clusters for de novo biosyntheses of the non-ribosomal lipopeptides metabolites; bacitracin, iturin, plipastatin and fengycin which have potential biotechnological applications (Appendix IV).In conclusion, accurate identification of the LAB strains were established and results of their antimicrobial susceptibility profile and haemolytic activities revealed no safety issues of concern (Appendix I) which was the basis for selecting some of these strains to assess their potential industrial applications. This Thesis provided strong evidence on a high level of genomic heterogeneity among members of the Lb. delbrueckii spp. for which a new subspecies was proposed (Appendix II). The data on antimicrobial susceptibility profiles of the 3 Bacillus species strains (Appendix III) will enable regulatory and public health authorities to accurately proposevii antimicrobial breakpoint values for these species as this Thesis has provided evidence on the inadequacy of the antimicrobial breakpoint values recommended by EFSA for the Bacillus genus. Clindamycin has also been indicated as a potential selective agent, which could be used in a base medium for isolating B. licheniformis spp. strains from B. sonorensis and B. subtilis. Finally, this Thesis has provided a deeper insight into the biosyntheses capabilities of the B. sonorensis spp. which until now was not known and the draft genome sequence will further contribute to future scientific research work on genetic engineering of this species. It would however be worthy to investigate the stress resistance mechanisms of the novel strain ZN7a-9 as such data could be used to develop screening assays. Furthermore, analyses of the full genome draft-sequence of strain L12 highlighted the need for metabolomic and functional analyses of the strain to be performed as it represent a potential candidate which could be used as plants bio-control agent against fungal pathogens causing devastating post-harvest crop losses.

AB - African indigenous fermented food products are characterized by complex and diverse groups of microorganisms and therefore offer a rich source for selection of microbial strains for various applications in the biotechnology and food bio-processing sectors. There is however, a global public health concern regarding the use of live microbial strains for biotechnological and food bioprocessing applications due to the risk of dissemination of multiple antimicrobial resistance genesand virulence factors to pathogenic microorganisms. In view of this, the safety of microbial strains with potential biotechnological and food bio-processing applications especially as live culture is the responsibility of the producer. This requires careful safety evaluations such as sensitivity to antimicrobial agents and production of virulence factors. The aim of this PhD Thesis was to characterize lactic acid bacteria (LAB) and Bacillus spp. strains isolated from selected African indigenous fermented food products in order to gain an in-depth knowledge on their physiology, safety and genomics in consideration for different biotechnological applications. The study was categorised into the 3 major research areas; microbial identification, antimicrobial susceptibility study and genome sequencing. Subsequently, 33 LAB strains were identified by molecular techniques including; sequencing of the 16S rRNA gene, rep-PCR fingerprinting analyses and species-specific PCR assays (Appendix I). Strain ZN7a-9 was identified as a L. delbrueckii species based on its 16S rRNA gene sequence, DNA-DNA hybridization (DDH) and peptidoglycan cell-wall structure. But it could not be assigned to any of the recognised members of the L. delbrueckii subspecies taxon based on its phenotypic features, Multilocus Sequence Typing and splits-decomposition analyses. In order to be able to accommodate strain ZN7a-9 within the L. delbrueckii subsp. taxon and to additionally distinguish it from the recognised members of this taxon, it was proposed as a new L. delbrueckii subspecies; Lactobacillus delbrueckii subsp. jakobsenii subsp. nov. (Appendix II).By the broth microdilution technique, the LAB strains and 85 Bacillus spp. strains representing 38 B. licheniformis, 29 B. subtilis subsp. subtilis and 18 B. sonorensis strains were characterised for susceptibility to antimicrobial compounds of clinical and veterinary importance. The LAB strains were susceptible to; ampicillin, chloramphenicol, clindamycin and erythromycin but intrinsically resistant to kanamycin, streptomycin and vancomycin. Variable and multiple resistance to tetracycline and gentamicin was observed (Appendix I). All the Bacillus spp. strains were susceptible to tetracycline, vancomycin and gentamicin but resistant to streptomycin. Also, speciesspecific variation in sensitivity of the 3 Bacillus spp. to chloramphenicol, clindamycin, erythromycin and kanamycin was observed. The erythromycin resistance was only present in the B. licheniformis strains (50.0 %) and strongly correlated with the presence of the rRNA methylase gene; erm(D) or erm(K) located on an 11.4-kbp plasmid. Since rRNA methylase genes are known to confer erythromycin resistance phenotype to microbial strains, the observed erythromycin resistance phenotype was attributed to the erm(D) or erm(K) genes detected by PCR (Appendix III). Analyses of the whole genome draft-sequence of Bacillus sonorensis strain L12 which was generated using the Illumina Hiseq platform revealed it encodes gene clusters for de novo biosyntheses of the non-ribosomal lipopeptides metabolites; bacitracin, iturin, plipastatin and fengycin which have potential biotechnological applications (Appendix IV).In conclusion, accurate identification of the LAB strains were established and results of their antimicrobial susceptibility profile and haemolytic activities revealed no safety issues of concern (Appendix I) which was the basis for selecting some of these strains to assess their potential industrial applications. This Thesis provided strong evidence on a high level of genomic heterogeneity among members of the Lb. delbrueckii spp. for which a new subspecies was proposed (Appendix II). The data on antimicrobial susceptibility profiles of the 3 Bacillus species strains (Appendix III) will enable regulatory and public health authorities to accurately proposevii antimicrobial breakpoint values for these species as this Thesis has provided evidence on the inadequacy of the antimicrobial breakpoint values recommended by EFSA for the Bacillus genus. Clindamycin has also been indicated as a potential selective agent, which could be used in a base medium for isolating B. licheniformis spp. strains from B. sonorensis and B. subtilis. Finally, this Thesis has provided a deeper insight into the biosyntheses capabilities of the B. sonorensis spp. which until now was not known and the draft genome sequence will further contribute to future scientific research work on genetic engineering of this species. It would however be worthy to investigate the stress resistance mechanisms of the novel strain ZN7a-9 as such data could be used to develop screening assays. Furthermore, analyses of the full genome draft-sequence of strain L12 highlighted the need for metabolomic and functional analyses of the strain to be performed as it represent a potential candidate which could be used as plants bio-control agent against fungal pathogens causing devastating post-harvest crop losses.

UR - https://rex.kb.dk/primo-explore/fulldisplay?docid=KGL01009115599&context=L&vid=NUI&search_scope=KGL&tab=default_tab&lang=da_DK

M3 - Ph.D. thesis

BT - Geno- and phenotypic characterization of lactic acid bacteria and Bacillus spp. strains isolated from African indigenous fermented food products and their applications in the food and feed industries

PB - Department of Food Science, University of Copenhagen

ER -