Catheter-associated urinary tract infections (CAUTI) account for 70%∼80% of urinary tract infections (UTI) and can lead to adverse outcomes. Most CAUTIs are polymicrobial with resilient communities maintaining a consistent composition of species over time, despite antibiotic treatment and catheter replacement. However, the mechanisms promoting persistence are poorly understood. Here we examine how a chemical interaction between Gram-positive Enterococcus faecalis and Gram-negative Klebsiella pneumoniae can explain their high rate of co-occurrence on long-term indwelling urinary catheters. Sequence analyses of longitudinal isolates from several human patients co-infected with E. faecalis and K. pneumoniae revealed that despite frequent replacement, catheters became re-colonized with the same or a nearly identical consortium of strains throughout the study collection period. Using artificial urine medium (AUM), monoculture revealed that the K. pneumoniae isolates grew robustly and formed biofilm, while the E. faecalis isolates grew poorly and did not form biofilm. However, co-culture of paired isolates resulted in enhanced E. faecalis growth and biofilm, which could be reproduced by supplementing E. faecalis with cell-free K. pneumoniae conditioned AUM supernatant (KpAUMSup). Analyses using comparative transcriptomics, mutant strains and chemical inhibitors with cell culture and murine CAUTI models revealed that: i) KpAUMSup, but not AUM, stimulated expression of the E. faecalis Fsr quorum-sensing system; ii) Fsr was required for E. faecalis to respond to KpAUMSup; iii) E. faecalis cultured in KpAUMSup was more efficient in initiating CAUTI; and iv) Disruption of Fsr inhibited initiation of CAUTI. This interspecies signaling may help explain the high rate of co-colonization of these CAUTI pathogens and highlights new therapeutic strategies to treat polymicrobial CAUTI.

Culture with conditioned medium supernatant

E. faecalis growth and biofilm formation in AUM were also examined in cultures supplemented with K. pneumoniae or MRSA-1369 conditioned AUM supernatant. K. pneumoniae or MRSA1369 strains were first cultured in AUM in individual wells of a 96-well microplate for 24 hours, growth from wells was pooled, and then filtered (Millipore Sigma, SLGPR33RB) to remove bacterial cells (1-3). The resulting supernatants (SaAUMSup, KpAUMSup) were then lyophilized and used to supplement fresh E. faecalis AUM at a ratio of 1:1 for culture in microplate and catheter assays.

RNA Sequencing and comparative transcriptomic analysis

RNA sequencing (RNA-seq) and comparative transcriptomic analysis were used to compare: i) E. faecalis OG1RF grown in AUM in the presence or absence of KpAUMSup from TOP52 conditioned supernatant for 9 hrs to identify growth-associated differentially regulated genes, and ii) E. faecalis OG1RF grown in AUM supplemented with SaAUMSup from MRSA-1369 or TOP52 KpAUMSup  for 48 hrs to identify biofilm-associated differentially regulated genes. Briefly, E. faecalis OG1RF microplate cultures were grown as described above, multiple wells (200 µL/well × 50 wells = 10 mL total culture) were harvested, pooled, and subjected to centrifugation to collect bacterial pellets. RNA was extracted from bacterial pellets using the RNeasy Plus Mini Kit (Qiagen, 74134) with the quality of the purified RNA determined by spectroscopy (NanoDrop 2000, Thermo Fisher). Libraries for Illumina sequencing were prepared using the FastSelect RNA kit (Qiagen, 334222), according to the manufacturer’s protocol and sequences were determined using an Illumina NovaSeq 6000 and processed as previously described to generate RNA-seq reads for comparative transcriptomic analysis (4-12). Comparative transcriptomic analyses, including principal component analysis (PCA) for identifying transcriptomes differences and differential expression analysis for determining differentially expressed genes, were conducted as previously described (1, 4).

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