Gisèle LaPointe

MICs of Mutacin B-Ny266, Nisin A, Vancomycin, and Oxacillin against Bacterial Pathogens

ABSTRACT Peptide antibiotics, particularly lantibiotics, are good candidates for replacing antibiotics to which bacteria have become resistant. In order to compare two such lantibiotics with two antibiotics, the MICs of nisin A, mutacin B-Ny266, vancomycin, and oxacillin against various bacterial pathogens were determined. The results indicate that nisin A and mutacin B-Ny266 are as active as vancomycin and oxacillin against most of the strains tested. Furthermore, mutacin B-Ny266 remains active against strains that are resistant to nisin A, oxacillin, or vancomycin. The wide spectrum of activity of mutacin B-Ny266, its low MICs against bacterial pathogens, and its activity against bacteria resistant to other inhibitors support the development of this substance for therapeutic use.

Molecular analysis of bacterial population structure and dynamics during cold storage of untreated and treated milk

Spoilage bacteria in milk are controlled by treatments such as thermization, microfiltration and addition of carbon dioxide. However, little information is known about the changes in microbial communities during subsequent cold storage of treated milk. Culture-dependent methods and a direct molecular approach combining 16S rRNA gene clone libraries and quantitative PCR (Q-PCR) were applied to obtain a better overview of the structure and the dynamics of milk microbiota. Raw milk samples were treated by the addition of carbon dioxide (CO(2)), thermization (TH) or microfiltration (MF) and stored at 4 degrees C or 8 degrees C up to 7d. Untreated milk (UT) was used as a control. Psychrotrophic and staphylococci bacteria were enumerated in the milk samples by culture methods. For the molecular approach, DNA was extracted from milk samples and 16S rRNA gene was amplified by PCR with universal primers prior to cloning. The Q-PCR method was used to evaluate the dynamics of dominant bacterial species revealed by clone library analysis of 16S rRNA gene. Comparison of the 16S rRNA gene sequence indicated that the two most abundant operational taxonomic units (OTU), determined at 97% identity, belonged to the class Gammaproteobacteria (40.3% of the 1415 sequences) and Bacilli (40%). Dominant bacterial species in UT, CO(2) and TH milk samples at day 3 were affiliated with Staphylococcus, Streptococcus, Clostridia, Aerococcus, Facklamia, Corynebacterium, Acinetobacter and Trichococcus. Dominant bacterial species detected in MF milk were Stenotrophomonas, Pseudomonas and Delftia, while Pseudomonas species dominated the bacterial population of UT, CO(2) and MF milk samples at day 7. Staphylococcus and Delftia were the dominant bacterial species in thermized milk. Q-PCR results showed that populations of S. aureus, A. viridans, A. calcoaceticus, C. variabile and S. uberis were stable during 7d of storage at 4 degrees C. Populations of P. fluorescens, S. uberis and total bacteria increased in UT and CO(2) milk samples during 7d of storage at 8 degrees C and were noticeable from day 3. This study shows new microbial species which can develop during cold storage after milk treatment and contributes to identifying causes of reduced shelf life and deterioration of technological properties of milk during storage.

Purification and structure of mutacin B-Ny266: a new lantibiotic produced by Streptococcus mutans

Mutacins are bactericidal substances of proteinaceous nature produced by Streptococcus mutans. Lantibiotics are antibacterial substances containing post‐translationally modified amino acids such as lanthionine. Mutacin B‐Ny266 was purified from the cell pellet of S. mutans strain Ny266 by ethanol extraction at pH 2.0 followed by reversed‐phase chromatography (Sep‐Pak® cartridge) and by HPLC on a C18 column. The mean purification factor was 3240±81 and the mean yield was 1.0±0.1%. Molecular mass of mutacin B‐Ny266 as determined by mass spectroscopy is 2270.29±0.21 Da. The amino acid sequence of the purified active fraction was obtained by Edman degradation after treatment with alkaline ethanethiol. Twenty‐one amino acids were detected in this analysis. Mutacin B‐Ny266 belongs to the type A lantibiotics. The proposed sequence is: F–K– –W–U–F– – –P–G– –A–K–O–G– –F–N– –Y– . The molecule differs from that of epidermin/staphylococcin 1580 and gallidermin at positions 1, 2, 4, 5 and 6.

Molecular Characterization of a Theta Replication Plasmid and Its Use for Development of a Two-Component Food-Grade Cloning System for Lactococcus lactis

ABSTRACT pCD4, a small, highly stable theta-replicating lactococcal plasmid, was used to develop a food-grade cloning system. Sequence analysis revealed five open reading frames and two putativecis-acting regions. None appears to code for undesirable phenotypes with regard to food applications. Functional analysis of the replication module showed that only the cis-actingori region and the repB gene coding for the replication initiator protein were needed for the stable replication and maintenance of pCD4 derivatives in Lactococcus lactis. A two-component food-grade cloning system was derived from the pCD4 replicon. The vector pVEC1, which carries the functional pCD4 replicon, is entirely made up of L. lactis DNA and has no selection marker. The companion pCOM1 is arepB-deficient pCD4 derivative that carries an erythromycin resistance gene as a dominant selection marker. The pCOM1 construct can only replicate in L. lactis iftrans complemented by the RepB initiator provided by pVEC1. Since only the cotransformants that carry both pVEC1 and pCOM1 can survive on plates containing erythromycin, pCOM1 can be used transiently to select cells that have acquired pVEC1. Due to the intrinsic incompatibility between these plasmids, pCOM1 can be readily cured from the cells grown on an antibiotic-free medium after the selection step. The system was used to introduce a phage resistance mechanism into the laboratory strain MG1363 of L. lactisand two industrial strains. The introduction of the antiphage barrier did not alter the wild-type plasmid profile of the industrial strains. The phenotype was stable after 100 generations and conferred an effective resistance phenotype against phages of the 936 and c2 species.

Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

ABSTRACT A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3′ degenerate core based on four highly conserved amino acids and a longer 5′ consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS−) and EPS-producing (EPS+) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS+ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS+ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.

Assessment of Probiotic Viability during Cheddar Cheese Manufacture and Ripening Using Propidium Monoazide-PCR Quantification

The use of a suitable food carrier such as cheese could significantly enhance probiotic viability during storage. The main goal of this study was to assess viability of commercial probiotic strains during Cheddar cheesemaking and ripening (4–6 months) by comparing the efficiency of microbiological and molecular approaches. Molecular methods such as quantitative PCR (qPCR) allow bacterial quantification, and DNA-blocking molecules such as propidium monoazide (PMA) select only the living cells’ DNA. Cheese samples were manufactured with a lactococci starter and with one of three probiotic strains (Bifidobacterium animalis subsp. lactis BB-12, Lactobacillus rhamnosus RO011, or Lactobacillus helveticus RO052) or a mixed culture containing B. animalis subsp. lactis BB-12 and L. helveticus RO052 (MC1), both lactobacilli strains (MC2), or all three strains (MC3). DNA extractions were then carried out on PMA-treated and non-treated cell pellets in order to assess PMA treatment efficiency, followed by quantification using the 16S rRNA gene, the elongation factor Tu gene (tuf) or the transaldolase gene (tal). Results with intact/dead ratios of bacteria showed that PMA-treated cheese samples had a significantly lower bacterial count than non-treated DNA samples (P < 0.005), confirming that PMA did eliminate dead bacteria from PCR quantification. For both quantification methods, the addition of probiotic strains seemed to accelerate the loss of lactococci viability in comparison to control cheese samples, especially when L. helveticus RO052 was added. Viability of all three probiotic strains was also significantly reduced in mixed culture cheese samples (P < 0.0001), B. animalis subsp. lactis BB-12 being the most sensitive to the presence of other strains. However, all probiotic strains did retain their viability (log 9 cfu/g of cheese) throughout ripening. This study was successful in monitoring living probiotic species in Cheddar cheese samples through PMA-qPCR.

Comparative transcriptome analysis of Lactococcus lactis subsp. cremoris strains under conditions simulating Cheddar cheese manufacture.

Gene expression in response to technological variations can influence fermentation and flavor generation in Cheddar cheese, and can vary from one lactococcal strain to another, perceived as differences in starter performance. The aim of this study was to determine the influence of cheese cooking temperature at 38 °C and salting on the transcriptional profiles of four closely related strains of L. lactis subsp. cremoris under simulated conditions of Cheddar cheese manufacture. Two responses could be distinguished, a core gene expression, corresponding to the common response of all strains and strain-specific response during the Cheddar simulating process. For the core gene expression after heating of inoculated milk at 38 °C, two groups of differentially expressed genes were identified: i) stress response and ii) carbohydrate and amino acid metabolism. The response to combined stresses of heat, acid and salt resulted in: i) general decrease of functions linked to cell division and metabolism, ii) specific responses related to stress such as the induction of genes coding for chaperones and proteases and iii) expression of prophage lytic systems for certain strains. Strain-specific responses were mainly observed in three of the four tested strains. These responses were the induction of genes related to osmotic stress or the release of CodY repression leading to the activation of oligopeptide transporters as well as the bcaT gene, related to amino acid degradation for the production of flavor. Comparing transcriptomes provides a core expression profile that contributes to understanding gene expression responses to environmental variations. The strain-specific responses identify predictive markers for the transcriptional state of starter strains before they enter the cheese ripening phase.

Sugar source modulates exopolysaccharide biosynthesis in Bifidobacterium longum subsp. longum CRC 002.

The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp. longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080+/-120 mg EPS l(-1) (P<0.01) after 24 h of incubation. For fructose, galactose and glucose, EPS production was similar, at 512+/-63, 564+/-165 and 616+/-93 mg EPS l(-1), respectively. The proposed repeating unit composition of the EPS is 2 galactose to 3 glucose. The effect of sugar and fermentation time on expression of genes involved in sugar nucleotide production ( galK, galE1, galE2, galT1, galT2, galU, rmlA, rmlB1 and rmlCD) and the priming glycosyltransferase ( wblE) was quantified using real-time reverse transcription PCR. A significantly higher transcription level of wblE (9.29-fold) and the genes involved in the Leloir pathway (galK, 4.10-fold; galT1, 2.78-fold; and galE2, 4.95-fold) during exponential growth was associated with enhanced EPS production on lactose compared to glucose. However, galU expression, linking glucose metabolism with the Leloir pathway, was not correlated with EPS production on different sugars. Genes coding for dTDP-rhamnose biosynthesis were also differentially expressed depending on sugar source and growth phase, although rhamnose was not present in the composition of the EPS. This precursor may be used in cell wall polysaccharide biosynthesis. These results contribute to understanding the changes in gene expression when different sugar substrates are catabolized by B. longum subsp. longum CRC 002.

Correlation of the Capsular Phenotype in Propionibacterium freudenreichii with the Level of Expression of gtf, a Unique Polysaccharide Synthase-Encoding Gene

ABSTRACT Many food-grade bacteria produce exopolysaccharides (EPS) that affect the texture of fermented food products and that may be involved in probiotic properties. Propionibacterium freudenreichii is a Gram-positive food-grade bacterium with reported probiotic capabilities that is widely used as starter in Swiss-type cheese. In this study, 68 strains of P. freudenreichii were screened for the β-glucan capsular phenotype by immunoagglutination with a specific antibody and for the presence of the gtf gene coding for polysaccharide synthase. All strains were positive for PCR amplification with gtf gene-specific primers, but the presence of β-glucan capsular EPS was detected for only 35% of the strains studied. Disruption of gtf in P. freudenreichii revealed that gtf is a unique gene involved in β-glucan capsular EPS production in P. freudenreichii. The gtf gene was transferred into and expressed in Lactococcus lactis, in which it conferred an agglutination-positive phenotype. Expression of the gtf gene was measured by performing quantitative reverse transcription-PCR assays with RNA from four capsular and three noncapsular strains. A positive correlation was found between the β-glucan capsular phenotype and gtf gene expression. Sequencing of the region upstream of the gtf open reading frame revealed the presence of an insertion element (IS element) in this upstream region in the four strains with the β-glucan capsular phenotype. The role of the IS element in the expression of neighboring genes and its impact on interstrain variability of the P. freudenreichii capsule phenotype remain to be elucidated.

Seasonal and regional diversity of maple sap microbiota revealed using community PCR fingerprinting and 16S rRNA gene clone libraries.

An arbitrary primed community PCR fingerprinting technique based on capillary electrophoresis was developed to study maple sap microbial community characteristics among 19 production sites in Québec over the tapping season. Presumptive fragment identification was made with corresponding fingerprint profiles of bacterial isolate cultures. Maple sap microbial communities were subsequently compared using a representative subset of 13 16S rRNA gene clone libraries followed by gene sequence analysis. Results from both methods indicated that all maple sap production sites and flow periods shared common microbiota members, but distinctive features also existed. Changes over the season in relative abundance of predominant populations showed evidence of a common pattern. Pseudomonas (64%) and Rahnella (8%) were the most abundantly and frequently represented genera of the 2239 sequences analyzed. Janthinobacterium, Leuconostoc, Lactococcus, Weissella, Epilithonimonas and Sphingomonas were revealed as occasional contaminants in maple sap. Maple sap microbiota showed a low level of deep diversity along with a high variation of similar 16S rRNA gene sequences within the Pseudomonas genus. Predominance of Pseudomonas is suggested as a typical feature of maple sap microbiota across geographical regions, production sites, and sap flow periods.