Andrea Wilcks

Selective pressure affects transfer and establishment of a Lactobacillus plantarum resistance plasmid in the gastrointestinal environment

OBJECTIVES AND METHODS A Lactobacillus plantarum strain recently isolated from French raw-milk cheese was tested for its ability to transfer a small plasmid pLFE1 harbouring the erythromycin resistance gene erm(B) to Enterococcus faecalis. Mating was studied in vitro and in different gastrointestinal environments using gnotobiotic rats as a simple in vivo model and streptomycin-treated mice as a more complex model. Transfer and establishment of transconjugants in the intestine were investigated with and without selective pressure. RESULTS Compared with the relatively low transfer frequency of approximately 5.7 x 10(-8) transconjugants/recipient obtained in vitro by filter mating, a surprisingly high number of transconjugants (10(-4) transconjugants/recipient) was observed in gnotobiotic rats even without antibiotic treatment. When erythromycin was administered, a transfer rate of approximately 100% was observed, i.e. the recipient population turned completely into transconjugants (3 x 10(9) cfu/g faeces). Additionally, the time to reach a stable transconjugant population level was much faster in the erythromycin-treated gnotobiotic rats (1 day) than in the untreated animals (4-5 days). Transconjugants persisted in the gut in relatively stable numbers at least 12 days after termination of antibiotic treatment. In the streptomycin-treated mice, no transfer was observed either with or without erythromycin treatment. CONCLUSIONS The overall results imply that the gastrointestinal tract may comprise a more favourable environment for antibiotic resistance transfer than conditions provided in vitro. However, the indigenous gut microbiota severely restricts transfer, thus minimizing the number of detectable transfer events. Treatment with erythromycin strongly favoured transfer and establishment of pLFE1.

Occurrence of natural Bacillus thuringiensis contaminants and residues of Bacillus thuringiensis-based insecticides on fresh fruits and vegetables

ABSTRACT A total of 128 Bacillus cereus-like strains isolated from fresh fruits and vegetables for sale in retail shops in Denmark were characterized. Of these strains, 39% (50/128) were classified as Bacillus thuringiensis on the basis of their content of cry genes determined by PCR or crystal proteins visualized by microscopy. Random amplified polymorphic DNA analysis and plasmid profiling indicated that 23 of the 50 B. thuringiensis strains were of the same subtype as B. thuringiensis strains used as commercial bioinsecticides. Fourteen isolates were indistinguishable from B. thuringiensis subsp. kurstaki HD1 present in the products Dipel, Biobit, and Foray, and nine isolates grouped with B. thuringiensis subsp. aizawai present in Turex. The commercial strains were primarily isolated from samples of tomatoes, cucumbers, and peppers. A multiplex PCR method was developed to simultaneously detect all three genes in the enterotoxin hemolysin BL (HBL) and the nonhemolytic enterotoxin (NHE), respectively. This revealed that the frequency of these enterotoxin genes was higher among the strains indistinguishable from the commercial strains than among the other B. thuringiensis and B. cereus-like strains isolated from fruits and vegetables. The same was seen for a third enterotoxin, CytK. In conclusion, the present study strongly indicates that residues of B. thuringiensis-based insecticides can be found on fresh fruits and vegetables and that these are potentially enterotoxigenic.

Effects of apples and specific apple components on the cecal environment of conventional rats: role of apple pectin

BackgroundOur study was part of the large European project ISAFRUIT aiming to reveal the biological explanations for the epidemiologically well-established health effects of fruits. The objective was to identify effects of apple and apple product consumption on the composition of the cecal microbial community in rats, as well as on a number of cecal parameters, which may be influenced by a changed microbiota.ResultsPrincipal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks), while no effects of apple juice, purée or pomace on microbial composition in cecum were observed. Administration of either 0.33 or 3.3% apple pectin in the diet resulted in considerable changes in the DGGE profiles.A 2-fold increase in the activity of beta-glucuronidase was observed in animals fed with pectin (7% in the diet) for four weeks, as compared to control animals (P < 0.01). Additionally, the level of butyrate measured in these pectin-fed animal was more than double of the corresponding level in control animals (P < 0.01). Sequencing revealed that DGGE bands, which were suppressed in pectin-fed rats, represented Gram-negative anaerobic rods belonging to the phylum Bacteroidetes, whereas bands that became more prominent represented mainly Gram-positive anaerobic rods belonging to the phylum Firmicutes, and specific species belonging to the Clostridium Cluster XIVa.Quantitative real-time PCR confirmed a lower amount of given Bacteroidetes species in the pectin-fed rats as well as in the apple-fed rats in the four-week study (P < 0.05). Additionally, a more than four-fold increase in the amount of Clostridium coccoides (belonging to Cluster XIVa), as well as of genes encoding butyryl-coenzyme A CoA transferase, which is involved in butyrate production, was detected by quantitative PCR in fecal samples from the pectin-fed animals.ConclusionsOur findings show that consumption of apple pectin (7% in the diet) increases the population of butyrate- and β-glucuronidase producing Clostridiales, and decreases the population of specific species within the Bacteroidetes group in the rat gut. Similar changes were not caused by consumption of whole apples, apple juice, purée or pomace.

Conjugative gene transfer in the gastrointestinal environment.

Publisher Summary This chapter focuses on knowledge on conjugative transfer of antibiotic resistance genes in the animal gut, particularly, on the impact of bacterial transfer physiology and intestinal environment on the kinetics of transfer and establishment of new genetic traits within the gut microbial population. The justified concern about the increasing emergence of antibiotic resistant bacteria focuses about transfer and establishment of resistance genes in the gut. Dissemination of resistance genes in the gut bacterial population can happen either through horizontal transfer, as reviewed, or through vertical transfer. The different mechanisms for exchange of genetic information among intestinal bacteria such as plasmid transfer from gram‐negative bacteria, plasmid transfer from gram-positive bacteria, and conjugative transposition are presented in this chapter. The transfer frequency of conjugative transposons is a product of the frequency of transposons excision from the chromosome and of the transfer event itself, these transposons are transferred in vitro at a much lower frequency than is seen for most conjugative plasmids. Environmental factors also can affect the frequency and outcome of conjugative gene transfer. Impact of the gut environment on conjugative transfer is also discussed in this chapter. Another factor that affects the efficiency of conjugative transfer is the physiological state of the cells involved in the mating. For potent gene transfer mechanisms such as conjugative plasmids, colonization and establishment in the gut is not a prerequisite for genetic exchange.

Gram-negative bacteria account for main differences between faecal microbiota from patients with ulcerative colitis and healthy controls

Detailed knowledge about the composition of the intestinal microbiota may be critical to unravel the pathogenesis of ulcerative colitis (UC), a human chronic inflammatory bowel disease, since the intestinal microbes are expected to influence some of the key mechanisms involved in the inflammatory process of the gut mucosa. The aim of this study was to investigate the faecal microbiota in patients either with UC in remission (n=6) or with active disease (n=6), and in healthy controls (n=6). The composition of Gram-negative bacteria and Gram-positive bacteria was examined. Antigenic structures of Gram-negative bacteria such as lipopolysaccharides have been related to the inflammatory responses and pathogenesis of inflammatory bowel disease. Dice cluster analysis and principal component analysis of faecal microbiota profiles obtained by denaturing gradient gel electrophoresis and quantitative PCR, respectively, revealed that the composition of faecal bacteria from UC patients with active disease differed from the healthy controls and that this difference should be ascribed to Gram-negative bacteria. The analysis did not show any clear grouping of UC patients in remission. Even with the relatively low number of subjects in each group, we were able to detect a statistically significant underrepresentation of Lactobacillus spp. and Akkermansia muciniphila in UC patients with clinically active disease compared to the healthy controls. In line with previous communications, we have shown that the microbiota in UC patients with active disease differ from that in healthy controls. Our findings indicate that alterations in the composition of the Gram-negative bacterial population, as well as reduced numbers of lactobacilli and A. muciniphila may play a role in UC.

Intra- and Interspecies Conjugal Transfer of Tn916-Like Elements from Lactococcus lactis In Vitro and In Vivo

ABSTRACT Tetracycline-resistant Lactococcus lactis strains originally isolated from Polish raw milk were analyzed for the ability to transfer their antibiotic resistance genes in vitro, using filter mating experiments, and in vivo, using germfree rats. Four of six analyzed L. lactis isolates were able to transfer tetracycline resistance determinants in vitro to L. lactis Bu2-60, at frequencies ranging from 10−5 to 10−7 transconjugants per recipient. Three of these four strains could also transfer resistance in vitro to Enterococcus faecalis JH2-2, whereas no transfer to Bacillus subtilis YBE01, Pseudomonas putida KT2442, Agrobacterium tumefaciens UBAPF2, or Escherichia coli JE2571 was observed. Rats were initially inoculated with the recipient E. faecalis strain JH2-2, and after a week, the L. lactis IBB477 and IBB487 donor strains were introduced. The first transconjugants were detected in fecal samples 3 days after introduction of the donors. A subtherapeutic concentration of tetracycline did not have any significant effect on the number of transconjugants, but transconjugants were observed earlier in animals dosed with this antibiotic. Molecular analysis of in vivo transconjugants containing the tet(M) gene showed that this gene was identical to tet(M) localized on the conjugative transposon Tn916. Primer-specific PCR confirmed that the Tn916 transposon was complete in all analyzed transconjugants and donors. This is the first study showing in vivo transfer of a Tn916-like antibiotic resistance transposon from L. lactis to E. faecalis. These data suggest that in certain cases food lactococci might be involved in the spread of antibiotic resistance genes to other lactic acid bacteria.

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

Aims:  To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats.

A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species

Optimal conditions and a standardized method for conjugation between two model lactococcal strains, Lactococcus lactis SH4174 (pAMbeta1-containing, erythromycin resistant donor) and L. lactis Bu2-60 (plasmid-free, erythromycin sensitive recipient), were developed and tested in a inter-laboratory experiments involving five laboratories from different countries. The ultimate goal of the study was to assess the microbial potential of antibiotic resistance transfer among Lactic Acid Bacteria (LAB). The influence of culture age (various OD values) and ratios of donor and recipient cultures as well as filter, solid and liquid mating techniques, were examined in order to optimize the conjugation protocol. In the result of these studies, we concluded that the donor-to-recipient ratio appear to be important; the most efficient technique for conjugation was filter mating and the optimal conditions for gene transfer were observed when late logarithmic cultures of both donor and recipient were used. Comparison of conjugal transfer frequencies between five partner laboratories showed that results are sufficiently intra-laboratory repeatable and inter-laboratory comparable. This is the first study of this kind, in which a standardized protocol of conjugal mating for testing antibiotic resistance dissemination among LAB was established and validated.

Analysis of the intestinal microbiota of oligosaccharide fed mice exhibiting reduced resistance to Salmonella infection.

Certain indigestible carbohydrates, known as prebiotics, are claimed to be beneficial for gut health through a selective stimulation of certain gut microbes including bifidobacteria. However, stimulation of such microbes does not necessarily imply a preventive effect against pathogen infection. We recently demonstrated a reduced resistance to Salmonella infection in mice fed diets containing fructo-oligosaccharides (FOS) or xylo-oligosaccharides (XOS). In the present study, faecal and caecal samples from the same mice were analysed in order to study microbial changes potentially explaining the observed effects on the pathogenesis of Salmonella. Denaturing gradient gel electrophoresis revealed that the microbiota in faecal samples from mice fed FOS or XOS were different from faecal samples collected before the feeding trial as well as from faecal profiles generated from control animals. This difference was not seen for caecal profiles. Further analysis of faecal samples by real-time PCR demonstrated a significant increase in the Bacteroidetes phylum, the Bacteroides fragilis group and in Bifidobacterium spp. in mice fed FOS or XOS. The observed bifidogenic effect was more pronounced for XOS than for FOS. The Firmicutes phylum and the Clostridium coccoides group were reduced by both FOS and XOS. Surprisingly, no significant differences were detected between faecal samples collected before and after pathogen challenge in any of the groups. Furthermore, no effect of diets on caecal concentrations of short-chain fatty acids was recorded. In conclusion, diets supplemented with FOS or XOS induced a number of microbial changes in the faecal microbiota of mice. The observed effects of XOS were qualitatively similar to those of FOS, but the most prominent bifidogenic effect was seen for XOS. An increased level of bifidobacteria is thus not in itself preventive against Salmonella infection, since the same XOS or FOS-fed mice were previously reported to be more severely affected by Salmonella than control animals.

Metabolic footprint of Lactobacillus acidophilus NCFM at different pH

Lactobacillus acidophilus NCFM is a well known microorganism from the genomic and probiotic point of view. In order to analyze the potential interactions of NCFM with the surrounding environment, in vitro tests with the metabolic footprinting approach were performed. It was found that NCFM increased the concentration of lactic acid, succinic acid, adenine and arginine in the medium. The metabolism of NCFM did not change significantly between pH 5 and 7, suggesting that other environmental factors than pH might have bigger impact on its colonization throughout the gastrointestinal tract.