Showing papers by "Gerald F. Fitzgerald published in 2001"

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy.

Abstract: The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.

Improvement and Optimization of Two Engineered Phage Resistance Mechanisms in Lactococcus lactis

Abstract: Homologous replication module genes were identified for four P335 type phages DNA sequence analysis revealed that all four phages exhibited more than 90% DNA homology for at least two genes, designated rep2009 and orf17 One of these genes, rep2009, codes for a putative replisome organizer protein and contains an assumed origin of phage DNA replication (ori2009), which was identical for all four phages DNA fragments representing the ori2009 sequence confer a phage-encoded resistance (Per) phenotype on lactococcal hosts when they are supplied on a high-copy-number vector Furthermore, cloning multiple copies of the ori2009 sequence was found to increase the effectiveness of the Per phenotype conferred A number of antisense plasmids targeting specific genes of the replication module were constructed Two separate plasmids targeting rep2009 and orf17 were found to efficiently inhibit proliferation of all four phages by interfering with intracellular phage DNA replication These results represent two highly effective strategies for inhibiting bacteriophage proliferation, and they also identify a novel gene, orf17, which appears to be important for phage DNA replication Furthermore, these results indicate that although the actual mechanisms of DNA replication are very similar, if not identical, for all four phages, expression of the replication genes is significantly different in each case

Naturally occurring lactococcal plasmid pAH90 links bacteriophage resistance and mobility functions to a food-grade selectable marker.

Abstract: The bacteriophage resistance plasmid pAH90 (26,490 bp) is a natural cointegrate plasmid formed via homologous recombination between the type I restriction-modification specificity determinants (hsdS) of two smaller lactococcal plasmids, pAH33 (6,159 bp) and pAH82 (20,331 bp), giving rise to a bacteriophage-insensitive mutant following phage challenge (D. O9Sullivan, D. P. Twomey, A. Coffey, C. Hill, G. F. Fitzgerald, and R. P. Ross, Mol. Microbiol. 36:866–876; 2000). In this communication we provide evidence that the recombination event is favored by phage infection. The entire nucleotide sequence of plasmid pAH90 was determined and found to contain 24 open reading frames (ORFs) responsible for phenotypes which include restriction-modification, phage adsorption inhibition, plasmid replication, cadmium resistance, cobalt transport, and conjugative mobilization. The cadmium resistance property, encoded by the cadA gene, which has an associated regulatory gene (cadC), is of particular interest, as it facilitated the selection of pAH90 in other phage-sensitive lactococci after electroporation. In addition, we report the identification of a group II self-splicing intron bounded by two exons which have the capacity to encode a relaxase implicated in conjugation in gram-positive bacteria. The functionality of this intron was evident by demonstrating splicing in vivo. Given that pAH90 encodes potent phage defense systems which act at different stages in the phage lytic cycle, the linkage of these with a food-grade selectable marker on a replicon that can be mobilized among lactococci has significant potential for natural strain improvement for industrial dairy fermentations which are susceptible to phage inhibition.

The application of a fermented food ingredient containing 'variacin', a novel antimicrobial produced by Kocuria varians, to control the growth of Bacillus cereus in chilled dairy products.

Abstract: T. O’MAHONY, N. REKHIF, C. CAVADINI AND G.F. FITZGERALD. 2001. Aims: The feasibility of applying variacin, a lantibiotic produced by Kocuria varians in the form of a spray-dried fermented ingredient to control the growth of psychrotrophic Bacillus cereus strains in chilled dairy foods, was evaluated. Methods and Results: A range of chilled dairy food formulations modelling commercially-available products were fabricated, to which were added varying amounts of active ingredient. These were subsequently challenged with a B. cereus spore cocktail over a range of abuse temperatures. This work was validated by the inclusion of the fermented ingredient to commercial products. Conclusions: Results demonstrate the functionality of the bacteriocin at refrigeration abuse temperatures, and indicate the robust nature of the proteinaceous antimicrobial agent with regard to processing. Significance and Impact of the Study: This study indicates the applicability of fermented food ingredients containing naturally-occurring antimicrobials as additional hurdles in food preservation.

Transcriptional Analysis and Regulation of Expression of the ScrFI Restriction-Modification System of Lactococcus lactis subsp.cremoris UC503

Abstract: ScrFI is a type II restriction-modification system from Lactococcus lactis which recognizes the nucleotide sequence 5′-CC↓ NGG-3′, cleaving at the point indicated by the arrow, and it comprises an endonuclease gene that is flanked on either side by genes encoding two 5-methylcytosine methylases. An open reading frame (orfX) of unknown function is located immediately upstream of these genes. In this study Northern analysis was performed, and it revealed that orfX, scrFIBM, and scrFIR are cotranscribed as a single polygenic mRNA molecule, while scrFIAM is transcribed independently. 5′ extension analysis indicated that the start site for the scrFIAM promoter was a thymine located 4 bp downstream of the −10 motif. The transcriptional start site for the orfX promoter was also found to be a thymine which is more atypically located 24 bp downstream of the −10 motif proximal to the start codon. A helix-turn-helix motif was identified at the N-terminal end of one of the methylases (M.ScrFIA). In order to determine if this motif played a role in regulation of the ScrFI locus, M.ScrFIA was purified. It was then employed in gel retardation assays using fragments containing the two promoters found on the ScrFI operon, one located upstream of orfX and the other located just upstream of scrFIAM. M.ScrFIA was found to bind to the promoter region upstream of the gene encoding it, indicating that it may have a regulatory role. In further studies the two putative promoters were introduced into a vector (pAK80) upstream of a promoterless lacZ gene, and cloned fragments of the ScrFI locus were introduced in trans with each of these promoter constructs to investigate the effect on promoter activity. These results implicated M.ScrFIA in regulation of both promoters on the ScrFI locus.

The use of cadmium resistance on the phage-resistance plasmid pNP40 facilitates selection for its horizontal transfer to industrial dairy starter lactococci.

Abstract: Aims: To facilitate the horizontal transfer and selection of phage-resistance plasmids in industrial lactococci. Methods and Results: Cadmium-resistance properties similar to those previously identified in Lactococcus were linked to the well-known phage-resistance plasmid pNP40. This finding was exploited to facilitate delivery of the plasmid to an industrial cheese starter Lactococcus lactis DPC4268. Additionally, 25 different cadmium-sensitive cheese starter lactococci were also identified as potential recipients for the phage-resistance plasmid pNP40, and also the plasmids pAH90/pAH82 which also encode cadmium resistance. All three plasmids were successfully conjugated to strain DPC4268. Cheddar cheese was manufactured in industry with the pNP40 phage-resistant transconjugant. Significance and Impact of the Study: Food-grade enhancement of phage resistance in industrial starter strains has been made simpler by the use of this selection, especially since the majority of potential recipient starter strains analysed were cadmium sensitive.

Application of Streptococcus thermophilus DPC1842 as an adjunct to counteract bacteriophage disruption in a predominantly lactococcal Cheddar cheese starter: use in bulk starter culture systems

Abstract: A significant amount of Cheddar cheese manufactured world-wide relies on bulk starter cultures instead of direct vat set (DVS) cultures While the inclusion of S thermophilus is some- times used to counteract failure due to lactococcal phage in the latter system, it is considered difficult to implement in bulk starter systems and is normally avoided This stems from the problem in con- trolling the ratio of S thermophilusto lactococci during the bulk starter preparation such that suitable acidification rates can be achieved The current study demonstrates how S thermophilusnumbers can be controlled during growth in the bulk starter medium prior to inoculation of a culture, based on three lactococcal strains and S thermophilusDPC1842, into the cheese vat The concentration of inor- ganic phosphate necessary to inhibit the growth of strain DPC1842 in a whey-based bulk starter medium was found to be 018 molL -1 Since higher levels of phosphate exist in different commer- cial bulk starter media used for mesophilic cultures, a number of these media can be used for prop- agation of this starter blend without domination of strain DPC1842 over its lactococcal counter- parts Strain DPC1842 is highly phage resistant and is particularly acid-fast in the cheese milk and reduces the pH efficiently at very low inocula Cheddar cheese was manufactured in a commercial plant with this system and the resulting cheese had good flavour characteristics This study demonstrates the effectiveness of the S thermophilus DPC1842 component in rescuing the fermentation in the event of severe lactococcal phage attack

Insertional mutagenesis of an industrial strain of Streptococcus thermophilus.

Abstract: Random mutagenesis of an industrial strain of Streptococcus thermophilus was achieved through an adapted version of a two-plasmid system. The mutagenesis strategy is based on random integration of derivatives of the non-replicative (Rep−) plasmid pORI19 by means of homologous recombination following a temperature shift that eliminates replication of the temperature-sensitive (Repts) helper plasmid pVE6007. In this way mutants were generated which were affected in bacteriophage sensitivity or sucrose metabolism. Homologues were identified of a protein related to folate metabolism from a bacteriophage-resistant mutant and of two subunits of an oligopeptide transport system from a mutant deficient in sucrose utilisation.

Traditional and molecular approaches to improving bacteriophage resistance of Cheddar and Mozzarella cheese starters.

Abstract: Infection by bacteriophage (bacterial viruses) during dairy fermentations remains a major cause of starter culture failure in Cheddar and Mozzarella manufacture, often resulting in substantial economic losses. A variety of practical measures to alleviate the problem of phage infection have been adopted over the years. The application of genetic techniques to improve the phage resistance of starter cultures for dairy fermentations is currently being explored and this approach has significant potential to alleviate the problem. This review highlights the significant developments that have been made in understanding the interaction between dairy starter cultures and bacteriophage in industry. It also describes the exploitation of molecular methodology to genetically defend these bacteria from the ever-present threat of bacteriophage and the scientific advances, which formed the basis for these achievements. Attention is also given to the development of food-grade approaches to improve genetic traits of industrial starter strains in the context of phage resistance.