Effects of yoghurt intake on plasmid transfer and colonisation with transconjugants in the digestive tract of mice associated with human faecal flora

This study deals with the effects of yoghurt intake on wild-type and recombinant plasmid transfer from an exogenous Escherichia coli K12-derivative donor strain to an endogenous recipient strain in the digestive tract of mice associated with human faecal flora. We showed that the self-transmissible plasmid R388 was efficiently transferred to recipient strain PG1 in mice associated with human faecal flora (HFF-PG1) and that the resulting transconjugants (PG1-R388) became established at a high and maximal population level without any selective pressure. Using HFF-PG1 mice made it possible to determine whether yoghurt consumption decreases R388 transfer efficiency and the ability of transconjugant PG1-R388 to successfully colonise the digestive tract. Results indicated that yoghurt consumption had two effects: it reduced the efficacy of plasmid transfer 10-fold and decreased the transconjugant PG1-R388 population density 100-fold, compared to the control group. We also describe another HFF mouse model in which recipient PG1 was replaced by EM0 with which no plasmid transfer was observed. This model made it possible to demonstrate the potential promoting effect of yoghurt intake on transconjugant formation and establishment. Our results indicated no yoghurt effect; no transconjugants appeared in the digestive tract of HFF-EM0 mice fed on yoghurt or on standard food. In both mouse models, HFF-PG1 and HFF-EM0, yoghurt intake did not promote the mobilisation of either the non-self-transmissible plasmid pUB2380 or the recombinant plasmid pCE325.     

Survival of Mycobacterium avium ssp. paratuberculosis in yoghurt and in commercial fermented milk products containing probiotic cultures

Aims: To assess the survival of Mycobacterium avium ssp. paratuberculosis (MAP) in yoghurt and commercial fermented milk products containing probiotic strains. Methods and Results: Whole and skimmed UHT milk artificially inoculated with MAP were used to manufacture yoghurt, using two different yoghurt starter cultures. Five commercial fermented milk products were inoculated with MAP. Two different MAP strains were studied. The survival of MAP in all products was monitored by culture over a 6‐week storage period at 6°C. In yoghurt, MAP counts did not change appreciably during the storage period. Fat content and type of yoghurt starter culture had no consistent effect on the survival of MAP. In the fermented milk products, survival patterns varied but resulted in a 1·5 to ≥3·8 log reduction for the Niebüll strain and a 1·2–2·2 log reduction for the NIZO strain after 6 weeks, depending on the probiotic starters present in the product. Conclusions: MAP easily survived in yoghurt but MAP numbers decreased in fermented milk products containing probiotic cultures. Significance and Impact of the Study: The results contribute to the lack of knowledge on the behaviour of MAP in yoghurt and fermented milk products containing probiotic cultures. This knowledge is valuable in the context of the risk of MAP transmission to humans via yoghurt and the possible contribution of probiotic fermented milk products to the elimination of MAP.     

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

G arvie , E.I. C ole , C.B., F uller , R. & H ewitt , D. 1984. The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat. Journal of Applied Bacteriology 56 , 237–245
Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro . Two enzyme systems (β-galactosidase and lactase) were studied using, respectively, o -nitrophenyl-β-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates. increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly hetero-fermentative ( Lact. reuteri ) to one which was predominantly homofermentative ( Lact. salivarius ).     

Effect of fermented milk intake on plasmid transfer and on the persistence of transconjugants in the digestive tract of gnotobiotic mice

Plasmid transfer occurs in the digestive tract and the transconjugants may become durably established. The aim of the present work is to investigate the effect of probiotics on plasmid transfer and on establishment of transconjugants in the gut. Plasmid transfers were carried out in the digestive tract of germ free mice associated with an E. coli K12 donor strain harboring three plasmids (R388, self-transmissible, pCE325 and pUB2380, mobilisable,) and an E. coli recipient strain, PG1, of human origin (Duval-Iflah et al., 1994). Milks fermented with either Lactobacillus bulgaricus or Streptococcus thermophilus or symbiosis, S85, of both strains were given daily as 1/3 of food diet. Fermented milks have no effect on the transfer of R388 and pUB2380 except a slight increase of TC(R388) with milk fermented with S85. Long term ingestion of milk fermented with S85 inhibited the formation and the establishment of transconjugants TC(pCE325). Milk fermented with L. bulgaricus lowered the population density of TC(pCE325) in animals where they were already established. This phenomenon was reversible, since the density of TC(pCE325) increased in the same animals after cessation of supplementation. Bacterial cultures obtained in MRS broth and given in state of drinking water were compared with fermented milks. Bacterial cultures with L. bulgaricus and with S85 favoured the establishment of TC(pCE325). These results indicate for the first time that probiotics have various effects on the formation and/or establishment of transconjugants in the gut of axenic mice. The effects depend on whether the probiotics were cultivated in milk or in MRS, indicating that bacterial metabolites and viable bacteria can be involved.     

Conjugal transfer of lactose-fermenting ability fromStreptococcus lactis C2 toLeuconostoc cremoris CAF7 yieldsLeuconostoc that ferment lactose and produce diacetyl

Summary Conjugation between lactose-fermenting (Lac⁺)Streptococcus lactis C2 and Lac^– Leuconostoc cremoris CAF7 was performed. The frequency of Lac⁺ transfer was 1.5 · 10^–2 per donor cell. Lac⁺ Leuconostoc transconjugants could ferment lactose significantly faster than wild-type cells. When grown in litmus milk fortified with 0.2% yeast extract, Lac⁺ transconjugants reached pH 4.68 within 24 h at 30°C and produced diacetyl. The identity of the transconjugants asLeuconostoc derivatives was confirmed by their resistance to phage c2 and to vancomycin (>500 g/ml), and by growth on selective medium containing azide. Plasmid profiles of 10 transconjugants showed two unique patterns. A novel enlarged plasmid was found. Southern blot hybridization revealed some homology with the 30 Md Lac⁺ plasmid of donor, recipient and the transconjugants, as well as with some of the remaining plasmids of the donor.Technical Paper No. 7953, Oregon Agricultural Experiment Station.     

Recombinant plasmid associated cell aggregation and high-frequency conjugation of Streptococcus lactis ML3

Lactose-positive (Lac+) transconjugants resulting from matings between Streptococcus lactic ML3 and S. lactis LM2301 possess a single plasmid of approximately 60 megadaltons (Mdal) which is nearly twice the size of the lactose plasmid of the donor. The majority of these Lac+ transconjugants aggregated in broth and were able to transfer lactose-fermenting ability at a frequency higher than 10(-1) per donor on milk agar plates or in broth. Lac+ transconjugants which did not clump conjugated at a much lower frequency. Lactose-negative derivatives of Lac+ clumping transconjugants did not aggregate in broth and were missing the 60-Mdal plasmid. The ability to aggregates in broth was very unstable. Strains could lose the ability to clump but retain lactose-fermenting ability. The majority of these Lac+ nonclumping derivatives of clumping transconjugants contained a plasmid of approximately 33 Mdal, the size of the lactose plasmid of the original donor ML3. These strains transferred lactose-fermenting ability at a frequency of approximately 10(-6) per donor, resulting in both Lac+ clumping transconjugants which contained a 60-Mdal plasmid and Lac+ nonclumping transconjugants which possessed a 33-Mdal plasmid. Our results suggest that the genes responsible for cell aggregation and high-frequency conjugation are on the segment of deoxyribonucleic acid which recombined with the 33-Mdal lactose plasmid in S. lactis ML3.     

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat

Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Continuous yoghurt production withLactobacillus bulgaricus andStreptococcus thermophilus entrapped in Ca-alginate

Summary The principles of a new process for the continuous manufacture of yoghurt withLactobacillus bulgaricus andStreptococcus thermophilus entrapped in Ca-alginate beads were given. Characteristics of non-optimized pH-stat continuous stirred tank reactors withL. bulgaricus andS. thermophilus entrapped in the same or separate particles were examined. In these reactors the highest production rate were 9.4 g.l^–1.h^–1 for lactic acid and 3.4×10¹¹ C.F.U. 1^–1.h^–1 for inoculation when microorganisms were entrapped separately. A stable balance of the yoghurt bacteria liberated in continuously prefermented milk was observed.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Selection and Study of Potent Lactose-Fermenting Yeasts

Whey-fermenting Kluyveromyces cultures were revealed among 105 yeast strains assimilating lactose. Eighteen strains from milk products, showing maximum potency, fermented galactose, sucrose, and raffinose, in addition to lactose. Many yeast strains fermented inulin. Most strains were resistant to cycloheximide and grew in medium containing glucose, NaCl, and ethanol at concentrations of up to 50, 11–12, and 10–12%, respectively (4°C). Three strains had mycocinogenic activity. After fermentation of whey with selected yeast strains at 30°C for 2–3 days, the ethanol concentration was 4–5%.     

The survival of Staphylococcus aureus during the fermentation and storage of yoghurt

The effect of yoghurt culture R _x on the survival of Staphylococcus aureus CCM 5984 added to milk in various concentrations was observed during the fermentation and storage of yoghurt. The end of the fermentation process (3.5 h) was only accompanied by a slight reduction. During the storage of yoghurt at 4°C a 1-2 log reduction was observed. No Staph. aureus was detected in yoghurt produced from milk contaminated by 10³ Staph. aureus cells 1^-1 after 48 h of cold storage. When a concentration of 10² Staph. aureus cells was used for milk contamination, the pathogen was not recovered from yoghurt during the fermentation and storage. The fermentation and storage of yoghurt was accompanied by increases in lactic acid and titrimetric acidity, as well as by a decrease in pH value.     

Leaching of Pseudomonas aeruginosa, and transconjugants containing pR68.45 through unsaturated, intact soil columns

Abstract Leaching of genetically engineered microbes (GEMs) through soil is a significant concern related to groundwater quality. The objective of this study was to examine the leaching, survival and gene transfer of a genetically engineered microbe and indigenous recipients of pR68.45 in nonsterile, undisturbed soil columns. Pseudomonas aeruginosa PAO25, containing the plasmid R68.45, was added to the surface of undisturbed soil columns (10 cm diameter × 80 cm length). Unsaturated flow conditions were maintained by 100 ml daily additions of 2 mM CaCl₂ for a period of 70 days. The population of the GEM exhibited a significant ( P = 0.05) linear decline with time. The GEM leached only to a depth of 30–40 cm in 70 days. Transfer of pR68.45 was shown to occur from P. aeruginosa into the indigenous bacterial population although relatively low numbers of transconjugants were observed (log 2 cfu g⁻¹ dry soil). The number of transconjugants also decreased with depth and time. Leaching of transconjugants, however, occured more readily than that of the GEM, probably as a result of plasmid transfer into smaller, more mobile bacteria. At 70 days incubation, no GEMs were detected in the columns, while transconjugants were observed at several depths. These results demonstrate the importance of examining both the survival and movement of GEMs and transconjugants in soil.     

Lowering of ochratoxin A level in milk by yoghurt bacteria and bifidobacteria

A decrease in the content of ochratoxin A (OA) was observed in milk samples fermented by yoghurt bacteria and bifidobacteria. OA was added to the milk before fermentation at a rate of 0.05–1.5 mg/L. No residues of OA were found in samples containing 0.05 and 0.1 mg/L of OA, fermented byS. salivarius subsp.thermophilus, L. delbrueckii subsp.bulgaricus andB. bifidum. Yoghurt bacteria (S. salivarius subsp.thermophilus andL. delbrueckii subsp.bulgaricus) were the most effective since no residues were detected even in fermented samples containing originally 0.5 mg/L OA.     

Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract,and impact on fish intestinal microbiota

The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid‐mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self‐transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4‐mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%–97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.     

Isolation and properties of transfer regulatory mutants of the nopaline Ti-plasmid pTiC58

Summary Conjugal transfer of the nopaline Ti-plasmid pTiC58 is inducible by the phosphorylated opines, agrocinopines A and B. Although the uninduced level of transfer is negligible (< 10^–7 per donor), some transconjugants have been isolated from crosses performed in the absence of agrocinopine. These transconjugants harbour mutant Ti-plasmids that transfer constitutively (>10^–3 per donor). These mutants have several other correlated phenotypes including constitutive uptake of agrocinopine A, supersensitivity to agrocin 84 and the ability to prevent the excretion of agrocin 84 when they are present in the same cell as the agrocin 84 biosynthetic plasmid pAt-84a.     

Determining the source of Bacillus cereus and Bacillus licheniformis isolated from raw milk, pasteurized milk and yoghurt

Aims:  Strain-specific detection of Bacillus cereus and Bacillus licheniformi s in raw and pasteurized milk, and yoghurt during processing.
Methods and Results:  Randomly selected isolates of Bacillus spp. were subjected to PCR analysis, where single primer targeting to the repetitive sequence Box elements was used to fingerprint the species. The isolates were separated into six different fingerprint patterns. The results show that isolates clustered together at about the 57% similarity level with two main groups at the 82% and 83% similarity levels, respectively. Contamination with identical strains both of B. cereus and B . licheniformis in raw and pasteurized milk was found as well as contaminated with different strains (in the case of raw milk and yoghurt/pasteurized milk and yoghurt). Several BOX types traced in processed milk samples were not discovered in the original raw milk.
Conclusions:  BOX-PCR fingerprinting is useful for characterizing Bacillus populations in a dairy environment. It can be used to confirm environmental contamination, eventually clonal transfer of Bacillus strains during the technological processing of milk.
Significance and Impact of the Study:  Despite the limited number of strains analysed, the two Bacillus species yielded adequately detectable banding profiles, permitting differentiation of bacteria at the strain level and showing their diversity throughout dairy processing.     

Transfer and properties of some natural and suicide replicons in Pasteurella multocida

We tested the transfer of several plasmids and transposons from Escherichia coli to Pasteurella multocida by filter mating. Two plasmids, pRKTV5 (pRK2013::Tn7) and pUW964 (pRKTV5::Tn5), were derived from pRK2013--a narrow-host-range plasmid with the broad-host-range IncP conjugation genes. Most P. multocida transconjugants obtained with pRKTV5 had Tn7 insertions in the chromosome but some had insertions of the whole plasmid. By contrast, all the transconjugants obtained with pUW964 had insertions of this plasmid or a deleted variant. pUW964 mediated low-frequency transfer of Tn7 or chromosomal markers between P. multocida strains. Broad-host-range IncP plasmid RP4 (RK2) did not yield selectable transconjugants in P. multocida but two plasmids derived by Tn5 insertion into a kanamycin-sensitive derivative of RP4 did yield transconjugants. pSUP1011, a narrow-host-range p15A replicon with the RP4 mob region allowing mobilization by the IncP conjugation genes also yielded transconjugants while several other plasmids tested did not transfer markers to P. multocida.     

Transfer of conjugative plasmid pAMβ1 from Lactococcus lactis to mouse intestinal bacteria

Conjugal transfer of plasmid pAMβ1 from Lactococcus lactis to intestinal bacteria of BALB/c mice was studied. Plasmid transfer was observed to Enterococcus faecalis in vitro by a filter mating method with transfer frequencies of 2.3 × 10⁻³ and with lower frequencies to other species. In vivo , using gastric intubation with the pAMβ1-bearing Lactococcus lactis as donor and Ent. faecalis as recipient, a few transconjugants were detected from faecal Ent. faecalis . However, when these mice were given erythromycin through drinking water, a large number of conjugated Ent. faecalis were detected in faeces. Plasmid transfer to Ent. faecalis occurred at high frequency, 1.2 × 10⁻³, in mice whose anus was artificially closed after gastric intubation with pAMβ1-bearing Lactococcus lactis . These results demonstrate clearly that pAMβ1 transfer occurs between Gram-positive bacteria in the gut of mice harbouring many species of bacteria.     

Growth of bifidobacteria and clostridia on human and cow milk saccharides

For healthy infants, which were born normally and fully breastfed, the dominant component of the intestinal microflora are bifidobacteria. However, infants born by caesarean section possess clostridia as a dominant intestinal bacterial group. The aim of the present study was to determine whether bifidobacteria and clostridia are able to grow on human milk oligosaccharides (HMOs) and other carbon sources - lactose, cow milk (CM) and human milk (HM). Both bifidobacteria and clostridia grew on lactose and in CM. Bifidobacteria grew in HM and on HMOs. In contrast, 3 out of 5 strains of clostridia were not able to grow in HM. No clostridial strain was able to utilise HMOs. While both bifidobacterial strains were resistant to lysozyme, 4 out of 5 strains of clostridia were lysozyme-susceptible. It seems that HMOs together with lysozyme may act as prebiotic-bifidogenic compounds inhibiting intestinal clostridia.