Martin Iain Bahl

Establishment of Intestinal Microbiota during Early Life: a Longitudinal, Explorative Study of a Large Cohort of Danish Infants

ABSTRACT Fecal samples were obtained from a cohort of 330 healthy Danish infants at 9, 18, and 36 months after birth, enabling characterization of interbacterial relationships by use of quantitative PCR targeting 31 selected bacterial 16S rRNA gene targets representing different phylogenetic levels. Nutritional parameters and measures of growth and body composition were determined and investigated in relation to the observed development in microbiota composition. We found that significant changes in the gut microbiota occurred, particularly from age 9 to 18 months, when cessation of breastfeeding and introduction of a complementary feeding induce replacement of a microbiota characterized by lactobacilli, bifidobacteria, and Enterobacteriaceae with a microbiota dominated by Clostridium spp. and Bacteroides spp. Classification of samples by a proxy enterotype based on the relative levels of Bacteroides spp. and Prevotella spp. showed that enterotype establishment occurs between 9 and 36 months. Thirty percent of the individuals shifted enterotype between 18 and 36 months. The composition of the microbiota was most pronouncedly influenced by the time of cessation of breastfeeding. From 9 to 18 months, a positive correlation was observed between the increase in body mass index and the increase of the short-chain-fatty-acid-producing clostridia, the Clostridum leptum group, and Eubacterium hallii. Considering previously established positive associations between rapid infant weight gain, early breastfeeding discontinuation, and later-life obesity, the corresponding microbial findings seen here warrant attention.

Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains.

The conjugative plasmid pOLA52, which confers resistance to olaquindox and other antimicrobial agents through a multidrug efflux pump, was investigated for its ability to promote biofilm formation in Escherichia coli. Screening of a transposon-mutagenized pOLA52 clone library revealed several biofilm-deficient mutants, which all mapped within a putative operon with high homology to the mrkABCDF operon of Klebsiella pneumoniae, where these genes are responsible for type 3 fimbriae expression, attachment to surfaces and biofilm formation. Biofilm formation in microtitre plates and in urinary catheters of clones containing pOLA52 with a disrupted putative mrk operon was reduced by more than 100-fold and 2-fold, respectively, compared to mutants with an intact mrk operon. The conjugative transfer rate of pOLA52 was also significantly lower when the mrk operon was disrupted. Through reverse transcriptase analysis, it was demonstrated that the genes contained in the putative mrk operon were linked and likely to be expressed as a single operon. Immunoblotting with type 3 fimbriae (MrkA)-specific antibodies further verified expression of type 3 fimbriae. When transferred to other, potentially pathogenic, members of the family Enterobacteriaceae, including Klebsiella pneumoniae, Salmonella Typhimurium, Kluyvera sp. and Enterobacter aerogenes, pOLA52 facilitated increased biofilm formation. pOLA52 is believed to represent the first example of a conjugative plasmid encoding type 3 fimbriae, resulting in enhanced conjugation frequencies and biofilm formation of the plasmid-harbouring strain.

All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant

The presence and diversity of IncP-1 plasmids in the influent of a Danish wastewater treatment plant was studied by PCR amplification of the trfA gene in community DNA followed by sequencing. Three sets of PCR primers were designed to amplify a 281bp fragment of trfA from all currently sequenced IncP-1 plasmids. A neighbor-joining tree, based on a multiple alignment of 72 obtained sequences together with homologous sequences of previously published IncP-1 plasmids, revealed that all established subgroups of IncP-1 plasmids, alpha, beta, gamma and delta, were present in the wastewater treatment plant influent. Also sequences representing the recently described fifth subgroup, the epsilon subgroup, were detected in the wastewater. Thus, these results confirm the presence of at least five phylogenetically distinct subgroups of IncP-1 plasmids and represent the first time that sequences associated with plasmids of all of these five subgroups have been detected in a single setting. Additionally, the results confirm that wastewater constitutes a reservoir for the conjugative IncP-1 plasmids, which often harbor multiple antibiotic resistance genes.

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

Aims:u2002 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.

Persistence Mechanisms of Conjugative Plasmids

Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid-free cells. Finally, various molecular adaptations of plasmids to better match the genetic background of their bacterial host cell will be described.

Acclimation of subsurface microbial communities to mercury

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.

Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry

A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry. To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells. Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype. The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations. This could be useful in future investigations of plasmid stability and population selection in bacterial communities.

Construction and use of flow cytometry optimized plasmid-sensor strains.

Determining the stability of plasmids in bacterial populations is traditionally performed by isolating a large number of clones followed by screening for the presence of plasmids by replica transfer to plasmid-selective agar plates. This is often a laborious task, especially when the intrinsic stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid-harboring cells in a bacterial population facilitates a very high throughput of cells and thus provides excellent sensitivity and statistics toward detecting even very low levels of plasmid instability.