Antibiotics promote intestinal growth of carbapenem-resistant Enterobacteriaceae by enriching nutrients and depleting microbial metabolites

The intestine is the primary colonisation site for carbapenem-resistant Enterobacteriaceae (CRE) and serves as a reservoir of CRE that cause invasive infections (e.g. bloodstream infections). Broad-spectrum antibiotics disrupt colonisation resistance mediated by the gut microbiota, promoting the expansion of CRE within the intestine. Here, we show that antibiotic-induced reduction of gut microbial populations leads to an enrichment of nutrients and depletion of inhibitory metabolites, which enhances CRE growth. Antibiotics decrease the abundance of gut commensals (including Bifidobacteriaceae and Bacteroidales) in ex vivo cultures of human faecal microbiota; this is accompanied by depletion of microbial metabolites and enrichment of nutrients. We measure the nutrient utilisation abilities, nutrient preferences, and metabolite inhibition susceptibilities of several CRE strains. We find that CRE can use the nutrients (enriched after antibiotic treatment) as carbon and nitrogen sources for growth. These nutrients also increase in faeces from antibiotic-treated mice and decrease following intestinal colonisation with carbapenem-resistant Escherichia coli. Furthermore, certain microbial metabolites (depleted upon antibiotic treatment) inhibit CRE growth. Our results show that killing gut commensals with antibiotics facilitates CRE colonisation by enriching nutrients and depleting inhibitory microbial metabolites.

For the whole genome sequencing data from the CRE patient isolates, raw reads were trimmed to remove sequences and low-quality bases with Trimmomatic v0.39. Bacterial species from assembled genomes was confirmed using Kraken2 v2.0.8-beta and its full bacterial database. Draft genomes were generated de novo using SPAdes v3.15.2. Assembly statistics were checked using QUAST v5.0.2. Acquired antimicrobial resistance genes were detected from draft genomes using ABRicate v1.0.1 with the ResFinder database. Multi locus sequence types (MLST) were determined from draft genomes using MLST v2.19.0. The following MLST databases were used to determine MLST: E. hormaechei -Enterobacter cloacae MLST scheme, E. coli -Achtman's E. coli MLST, K. pneumoniae -Klebsiella pneumoniae MLST scheme.
For the whole genome sequencing data from the CRE patient isolates, the Kraken bacterial database is available at http://ccb.jhu.edu/software/kraken2/, the ResFinder database is available at https://cge.food.dtu.dk/services/ResFinder/, and the MLST databases are available at https://pubmlst.org and https:// bigsdb.pasteur.fr/klebsiella/. For the 16S rRNA gene sequencing data the SILVA bacterial database version 138.1 is available at https://www.arb-silva.de/. For the 1H-NMR spectroscopy data the compound database in the Chenomx NMR Suite v9.02 is available at https://www.chenomx.com/.
Male and female participants were used equally for the study as there is no biological reason to favour one sex over the other. Findings apply to both sexes and both sexes were included in this study. Sex was determined based on self-reporting. In the first faecal culture experiment, 6 males and 5 females provided faecal donations to seed the faecal cultures. In the second faecal culture experiment, 2 males and 3 females provided faecal donations to seed the faecal cultures. For the donors that provided faecal samples to measure the concentration of specific microbial metabolites, 5 males and 7 females provided faecal samples.
Donors were between 18-65 years old and had not received antibiotic treatment in the 6+ months prior to donation.
Healthy donors were approached and asked if they would be interested in participating in this study. Informed consent was obtained from participants with information provided in the form of an Information Sheet detailing the research objectives and the samples needed. Samples were collected after the participant had agreed to participate in the study, had read the Information Sheet, and had signed the Informed Consent form.
As healthy donors were approached and asked to participate in our study (rather than through open advertisement to recruit donors), this study was not affected by self-selection bias. There is nothing in the donor recruitment process that would favour a particular sex, ethnicity, or other characteristic over others and participation was entirely voluntary.

March 2021
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Power calculations were used to determine sample size using the appropriate statistical test (e.g. paired or unpaired t-test), an alpha of 0.05, a power of 0.80, and an effect size calculated from an appropriate existing data set.
For the mouse dataset, one NMR spectrum (from day 2) had more than 50% of integrated peaks identified as an outlier or extreme outlier (using the identify_outliers function within the rstatix library within R) and was excluded. As paired t-tests were conducted, samples from this mouse were also excluded at the other time points (as paired t-test can only use cases that have non-missing values).
One sample was excluded from the TZP-treated faecal culture for E. coli ST410 outlined in Figure 1c as this was an extreme significant outlier (as calculated using the Grubbs' test in GraphPad).
Experiments were repeated the number of times indicated in the figure legends. All attempts at replication were successful.
Mice were randomly allocated into different groups.
For the human faecal culture experiments, each donor faecal sample was homogenised and split to seed the antibiotic-naïve group and each antibiotic-treated group. This means that each donor was allocated into all of the treatment groups.
For the human faecal samples used to determine a minimum, average, and maximum concentration of each metabolite, there were no treatment groups.
Investigators were not blinded for in vitro assays or 1H-NMR preparation because the same investigator was doing group allocation during data collection. Blinding was not used for animal experiments because the investigators needed to know the treatment groups in order to administer the interventions. 16S rRNA gene sequencing library preparation was blinded.
Eight-to ten-week-old female wild-type C57BL/6 mice were purchased from Envigo (Huntingdon, UK) and acclimatised for 1 week prior to the start of the experiment. Each test group contained 5 mice that were housed 5 per cage (in individually ventilated cages). Mice were provided autoclaved food (RM1, Special Diet Services, Essex, UK), water (provided ad libitum), and bedding (Aspen chip 2 bedding, NEPCO, Warrensburg, New York). Mice were maintained at 20-22°C and 45-65% humidity in a 12-hour light and 12-hour dark cycle (all interventions were performed during the light cycle).