Third generation cephalosporins and piperacillin/tazobactam have distinct impacts on the microbiota of critically ill patients

Effective implementation of antibiotic stewardship, especially in critical care, is limited by a lack of direct comparative investigations on how different antibiotics impact the microbiota and antibiotic resistance rates. We investigated the impact of two commonly used antibiotics, third-generation cephalosporins (3GC) and piperacillin/tazobactam (TZP) on the endotracheal, perineal and faecal microbiota of intensive care patients in Australia. Patients exposed to either 3GC, TZP, or no β-lactams (control group) were sampled over time and 16S rRNA amplicon sequencing was performed to examine microbiota diversity and composition. While neither treatment significantly affected diversity, numerous changes to microbiota composition were associated with each treatment. The shifts in microbiota composition associated with 3GC exposure differed from those observed with TZP, consistent with previous reports in animal models. This included a significant increase in Enterobacteriaceae and Enterococcaceae abundance in endotracheal and perineal microbiota for those administered 3GC compared to the control group. Culture-based analyses did not identify any significant changes in the prevalence of specific pathogenic or antibiotic-resistant bacteria. Exposure to clinical antibiotics has previously been linked to reduced microbiota diversity and increased antimicrobial resistance, but our results indicate that these effects may not be immediately apparent after short-term real-world exposures.


Figure S3
Family level taxonomic composition of the (A) endotracheal, (B) perineal and (C) faecal microbiota samples collected after 48 hours of 3GC or TZP administration or admission to the ICU, (3GC-third-generation cephalosporins, TZP-piperacillin/tazobactam, control-no β-lactams). Data is shown for each patient categorised based on treatment group. OTUs that were not assigned to a family are categorised as "Family unassigned". Bacterial families with a relative abundance less than 3 % in all three treatments for each body site are grouped as "Other". The relative abundance of the 16S rRNA gene amplicons in the families were determined using QIIME and graphed using GraphPad Prism (version 9, GraphPad Software, USA, www.graphpad.com).

Figure S4
Bacterial families in the (A) endotracheal, (B) perineal, and (C) faecal microbiota showing significantly different abundances between the 3GC and TZP groups after 48 hours of antibiotic exposure (3GC-third-generation cephalosporins and TZP-piperacillin/ tazobactam). The histograms show the linear discriminant analysis (LDA) scores computed for each bacterial family. LEfSe analyses were performed with the following parameters, Kruskal-Wallis test among classes (P < 0.05), Wilcoxon test between classes (P < 0.01) and the threshold on the logarithmetic LDA score for discriminative features > 2.0.

Table S1
Number of samples collected per treatment group on each day.* (Provided as an Excel file) *(A) A summary of the number of samples collected for each treatment group within and after 48 hours of ICU admission. Details on patient identifiers and the number of (B) endotracheal, (C) perineal and (D) faecal microbiota samples collected on each day are provided.

Table S2
Bacterial families that were found to be significantly differentially abundant in the 3GC and TZP groups compared to the control group within the first 48 hours of antibiotic administration.* (Provided as an Excel file) *Data was obtained based on LEfSe analyses between 3GC vs control group and TZP vs control group in the (A) endotracheal (B) perineal and (C) faecal microbiota. LEfSe analyses were performed with the following parameters: Kruskal-Wallis test among classes (P < 0.05), Wilcoxon test between classes (P < 0.01) and the threshold on the logarithmetic LDA score for discriminative features > 2.0. 3GC-third-generation cephalosporins, TZP-piperacillin/tazobactam, control-no β-lactams.

Table S3
The relative abundance of bacterial OTUs that were found to be significantly differentially abundant between the three treatments after 48 hours of admission.* (Provided as an Excel file) *Data was obtained based on LEfSe analyses between (A) 3GC vs control group in the endotracheal microbiota, (B) TZP vs control group in the endotracheal microbiota, (C) 3GC vs control group in the perineal microbiota, (D) TZP vs control in the perineal microbiota, (E) 3GC vs control in the faecal microbiota and (F) TZP vs control in the faecal microbiota. LEfSe analyses were performed with the following parameters: Kruskal-Wallis test among classes (P < 0.05), Wilcoxon test between classes (P < 0.01) and the threshold on the logarithmetic LDA score for discriminative features > 2.0. 3GC-third-generation cephalosporins, TZP-piperacillin/tazobactam, control-no β-lactams.