Cecilia Jernberg

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

Antibiotic administration is known to cause short-term disturbances in the microbiota of the human gastrointestinal tract, but the potential long-term consequences have not been well studied. The aims of this study were to analyse the long-term impact of a 7-day clindamycin treatment on the faecal microbiota and to simultaneously monitor the ecological stability of the microbiota in a control group as a baseline for reference. Faecal samples from four clindamycin-exposed and four control subjects were collected at nine different time points over 2 years. Using a polyphasic approach, we observed highly significant disturbances in the bacterial community that persisted throughout the sampling period. In particular, a sharp decline in the clonal diversity of Bacteroides isolates, as assessed by repetitive sequence-based PCR (rep-PCR) and long-term persistence of highly resistant clones were found as a direct response to the antibiotic exposure. The Bacteroides community never returned to its original composition during the study period as assessed using the molecular fingerprinting technique, terminal restriction fragment length polymorphism (T-RFLP). Furthermore, using real-time PCR we found a dramatic and persistent increase in levels of specific resistance genes in DNA extracted from the faeces after clindamycin administration. The temporal variations in the microbiota of the control group were minor compared to the large and persistent shift seen in the exposed group. These results demonstrate that long after the selection pressure from a short antibiotic exposure has been removed, there are still persistent long term impacts on the human intestinal microbiota that remain for up to 2 years post-treatment.

Short-term antibiotic treatment has differing long-term impacts on the human throat and gut microbiome.

Antibiotic administration is the standard treatment for the bacterium Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer. However, the long-term consequences of this treatment on the human indigenous microbiota are relatively unexplored. Here we studied short- and long-term effects of clarithromycin and metronidazole treatment, a commonly used therapy regimen against H. pylori, on the indigenous microbiota in the throat and in the lower intestine. The bacterial compositions in samples collected over a four-year period were monitored by analyzing the 16S rRNA gene using 454-based pyrosequencing and terminal-restriction fragment length polymorphism (T-RFLP). While the microbial communities of untreated control subjects were relatively stable over time, dramatic shifts were observed one week after antibiotic treatment with reduced bacterial diversity in all treated subjects in both locations. While the microbiota of the different subjects responded uniquely to the antibiotic treatment some general trends could be observed; such as a dramatic decline in Actinobacteria in both throat and feces immediately after treatment. Although the diversity of the microbiota subsequently recovered to resemble the pre treatment states, the microbiota remained perturbed in some cases for up to four years post treatment. In addition, four years after treatment high levels of the macrolide resistance gene erm(B) were found, indicating that antibiotic resistance, once selected for, can persist for longer periods of time than previously recognized. This highlights the importance of a restrictive antibiotic usage in order to prevent subsequent treatment failure and potential spread of antibiotic resistance.

Long-term impacts of antibiotic exposure on the human intestinal microbiota

Although it is known that antibiotics have short-term impacts on the human microbiome, recent evidence demonstrates that the impacts of some antibiotics remain for extended periods of time. In addition, antibiotic-resistant strains can persist in the human host environment in the absence of selective pressure. Both molecular- and cultivation-based approaches have revealed ecological disturbances in the microbiota after antibiotic administration, in particular for specific members of the bacterial community that are susceptible or alternatively resistant to the antibiotic in question. A disturbing consequence of antibiotic treatment has been the long-term persistence of antibiotic resistance genes, for example in the human gut. These data warrant use of prudence in the administration of antibiotics that could aggravate the growing battle with emerging antibiotic-resistant pathogenic strains.

Decreased gut microbiota diversity, delayed Bacteroidetes colonisation and reduced Th1 responses in infants delivered by Caesarean section

Objective The early intestinal microbiota exerts important stimuli for immune development, and a reduced microbial exposure as well as caesarean section (CS) has been associated with the development of allergic disease. Here we address how microbiota development in infants is affected by mode of delivery, and relate differences in colonisation patterns to the maturation of a balanced Th1/Th2 immune response. Design The postnatal intestinal colonisation pattern was investigated in 24 infants, born vaginally (15) or by CS (nine). The intestinal microbiota were characterised using pyrosequencing of 16S rRNA genes at 1 week and 1, 3, 6, 12 and 24 months after birth. Venous blood levels of Th1- and Th2-associated chemokines were measured at 6, 12 and 24 months. Results Infants born through CS had lower total microbiota diversity during the first 2 years of life. CS delivered infants also had a lower abundance and diversity of the Bacteroidetes phylum and were less often colonised with the Bacteroidetes phylum. Infants born through CS had significantly lower levels of the Th1-associated chemokines CXCL10 and CXCL11 in blood. Conclusions CS was associated with a lower total microbial diversity, delayed colonisation of the Bacteroidetes phylum and reduced Th1 responses during the first 2 years of life.

Monitoring of Antibiotic-Induced Alterations in the Human Intestinal Microflora and Detection of Probiotic Strains by Use of Terminal Restriction Fragment Length Polymorphism

ABSTRACT Terminal restriction fragment length polymorphism (T-RFLP) was investigated as a tool for monitoring the human intestinal microflora during antibiotic treatment and during ingestion of a probiotic product. Fecal samples from eight healthy volunteers were taken before, during, and after administration of clindamycin. During treatment, four subjects were given a probiotic, and four subjects were given a placebo. Changes in the microbial intestinal community composition and relative abundance of specific microbial populations in each subject were monitored by using viable counts and T-RFLP fingerprints. T-RFLP was also used to monitor specific bacterial populations that were either positively or negatively affected by clindamycin. Some dominant bacterial groups, such as Eubacterium spp., were easily monitored by T-RFLP, while they were hard to recover by cultivation. Furthermore, the two probiotic Lactobacillus strains were easily tracked by T-RFLP and were shown to be the dominant Lactobacillus community members in the intestinal microflora of subjects who received the probiotic.

Degradation of mixtures of phenolic compounds by Arthrobacter chlorophenolicus A6.

In this study the chlorophenol-degrading actinobacterium, Arthrobacter chlorophenolicus A6, was tested for its ability to grow on mixtures of phenolic compounds. During the experiments depletion of the compounds was monitored, as were cell growth and activity. Activity assays were based on bioluminescence output from a luciferase-tagged strain. When the cells were grown on a mixture of 4-chlorophenol, 4-nitrophenol and phenol, 4-chlorophenol degradation apparently was delayed until 4-nitrophenol was almost completely depleted. Phenol was degraded more slowly than the other compounds and not until 4-nitrophenol and 4-chlorophenol were depleted, despite this being the least toxic compound of the three. A similar order of degradation was observed in non-sterile soil slurries inoculated with A. chlorophenolicus. The kinetics of degradation of the substituted phenols suggest that the preferential order of their depletion could be due to their respective pKa values and that the dissociated phenolate ions are the substrates. A mutant strain (T99), with a disrupted hydroxyquinol dioxygenase gene in the previously described 4-chlorophenol degradation gene cluster, was also studied for its ability to grow on the different phenols. The mutant strain was able to grow on phenol, but not on either of the substituted phenols, suggesting a different catabolic pathway for the degradation of phenol by this microorganism.

Impact of 4-chlorophenol contamination and/or inoculation with the 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L, on soil bacterial community structure

The 4-chlorophenol-degrading strain, Arthrobacter chlorophenolicus A6L (chromosomally tagged with the firefly luciferase gene, luc) was inoculated into 4-chlorophenol-contaminated soil to assess the impact of bioaugmentation with a biodegrading strain on the indigenous microbiota. Simultaneously, the impact of 4-chlorophenol alone, or inoculation with A. chlorophenolicus into non-contaminated soil, was addressed. Using terminal restriction fragment length polymorphism (T-RFLP) several significant changes were detected in community fingerprint patterns obtained from soil microcosms treated under the different conditions. The relative abundances of some populations, as judged by the relative intensity of terminal restriction fragments, were significantly impacted by either 4-chlorophenol, A. chlorophenolicus inoculation, or by a combination of both inoculation and 4-chlorophenol contamination. Some populations were significantly stimulated and others were significantly repressed when compared to control soil with no additions. For several peaks, the positive or negative impact imposed by the treatments increased over the 13-day incubation period. Some members of the bacterial community were specifically sensitive to A. chlorophenolicus inoculation or to 4-chlorophenol contamination, whereas other populations remained relatively unaffected by any of the treatments. The A. chlorophenolicus inoculum was also monitored by T-RFLP and was found to have a significantly higher relative abundance in soil contaminated with 4-chlorophenol. These results were substantiated by a high correlation to luciferase activity measurements and the number of colony forming units of the inoculum. Therefore, the A. chlorophenolicus A6L population was positively stimulated by the presence of the 4-chlorophenol substrate (180 microg g(-1) soil) that it catabolized during the first 8 days of the incubation period as a carbon and energy source. Together, these results demonstrate that specific populations in the soil bacterial community rapidly fluctuated in response to specific disturbances and the resulting shifts in the community may therefore represent an adjustment in community structure favoring those populations best capable of responding to novel stress scenarios.

Restored fitness leads to long-term persistence of resistant Bacteroides strains in the human intestine

Acquired antibiotic resistance typically confers a cost to the bacteria, but these costs can be reduced by genetic compensation over time. The fitness of two Bacteroides thetaiotaomicron clones consecutively isolated in vivo was studied using an in vitro pair-wise competition method. The isolates derived from faecal samples of two clindamycin-exposed healthy volunteers and the two B. thetaiotaomicron clone types could be followed up to 18 months in these two subjects. The two clones were originally susceptible to clindamycin and lacked erm genes; however, after 7 days of clindamycin administration they carried the erm (erythromycin methylase)(G) or (F) gene, respectively, and expressed phenotypic clindamycin resistance. The initial cost of acquired resistance was high as seen in the in vitro pair-wise competition experiments. At 2 weeks post-administration, no growth disadvantage was detected for isolates of either of the two clones in the in vitro experiments and this regained fitness remained for isolates collected up to 18 months. Competition analysis of an in vitro isolated erm(G) positive transconjugant also demonstrated an initial reduction of fitness that was restored over time. The results indicate that the biological cost associated with a resistance gene can rapidly be compensated during in vivo growth. Thus, once the resistant clone has gained its resistance determinant it will be difficult to eliminate.

Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori

A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

Adaptive mutations and replacements of virulence traits in the Escherichia coli O104:H4 outbreak population.

The sequencing of highly virulent Escherichia coli O104:H4 strains isolated during the outbreak of bloody diarrhea and hemolytic uremic syndrome in Europe in 2011 revealed a genome that contained a Shiga toxin encoding prophage and a plasmid encoding enteroaggregative fimbriae. Here, we present the draft genome sequence of a strain isolated in Sweden from a patient who had travelled to Tunisia in 2010 (E112/10) and was found to differ from the outbreak strains by only 38 SNPs in non-repetitive regions, 16 of which were mapped to the branch to the outbreak strain. We identified putatively adaptive mutations in genes for transporters, outer surface proteins and enzymes involved in the metabolism of carbohydrates. A comparative analysis with other historical strains showed that E112/10 contained Shiga toxin prophage genes of the same genotype as the outbreak strain, while these genes have been replaced by a different genotype in two otherwise very closely related strains isolated in the Republic of Georgia in 2009. We also present the genome sequences of two enteroaggregative E. coli strains affiliated with phylogroup A (C43/90 and C48/93) that contain the agg genes for the AAF/I-type fimbriae characteristic of the outbreak population. Interestingly, C43/90 also contained a tet/mer antibiotic resistance island that was nearly identical in sequence to that of the outbreak strain, while the corresponding island in the Georgian strains was most similar to E. coli strains of other serotypes. We conclude that the pan-genome of the outbreak population is shared with strains of the A phylogroup and that its evolutionary history is littered with gene replacement events, including most recently independent acquisitions of antibiotic resistance genes in the outbreak strains and its nearest neighbors. The results are summarized in a refined evolutionary model for the emergence of the O104:H4 outbreak population.