This study was a single-center prospective observational study to investigate perioperative changes in the gut microbiota associated with cardiac surgery and the association between gut flora and postoperative pseudopsia or insomnia. The study was approved by the institutional ethics committee and conforms to the ethical norms and standards in the Declaration of Helsinki.
In this study, 21 adult patients who underwent elective cardiac surgery with cardiopulmonary bypass were enrolled. Participants provided written informed consent. Exclusion criteria included another major illness except for preoperative hypertension, diabetes mellitus, or dyslipidemia; selective cerebral perfusion; deep hypothermic circulatory arrest; aortic surgery; and preoperative administration of antibiotics. Preoperative and perioperative patient characteristics were collected from medical records.
Postoperative delirium was assessed using the CAM-ICU scale (https://www.icudelirium.org/medical-professionals/delirium/monitoring-delirium-in-the-icu) during the week after extubation, which has been used most frequently., Furthermore, postoperative pseudopsia and insomnia were investigated simultaneously. They are not included in the CAM-ICU, but are included in the Diagnostic and Statistical Manual of Mental Disorders, 5th edition. Symptoms of delirium were also included. Pseudopsia and insomnia were assessed by Intensive Care Unit (ICU) physicians. The insomnia group included patients who had insomnia for more than 2 days after surgery to exclude the effects of general anesthesia. The pseudopsia group contained patients who had pseudopsia on more than one occasion.
All patients underwent induction of general anesthesia with 0.5–1 mg/kg of midazolam, 2–10 μg/kg of fentanyl, and 0.6–1 mg/kg of rocuronium. Anesthesia was maintained with 4–6 mg/kg/h of propofol, 30 μg/kg/h of remifentanil, and 0.4–0.5 mg/kg/h of rocuronium. Sevoflurane was sometimes added in order to control blood pressure for short periods of time. Local anesthesia was not always used. All patients received 3 g/day of cefazolin on postoperative day (POD) 0 and 1.
Fecal Sample Collection
Patients collected their own fecal samples at three time points. The first sample, which served as the control, was harvested a few days before surgery. The second sample was from the first or second bowel movement after surgery. In general, the first postoperative sample was harvested but some patients were critically ill and the second sample was harvested. The last sample was harvested sometime between (POD) 6 and 8. Patients placed the fecal samples directly into two tubes (approximately 1.0 g/tube). One tube contained 2 mL RNAlater (an RNA stabilization solution; Ambion, Austin, TX, USA), and the other was empty. The samples with RNAlater were placed in a refrigerator at 4°C for analysis of the fecal microbiota. The other samples were placed in a freezer at -80°C within 30 minutes of excretion for analysis of fecal organic acid concentrations and pH. Samples were transported to the Yakult Central Institute at -20°C for analysis. The patient’s identity, clinical information, and study group were unknown to the technicians performing the analysis.
Gut Microbiota Analysis
To quantify the bacteria present in the samples, we extracted total RNA fractions from the fecal samples using previously described methods.[14-17] We examined gut microbiota composition using 16S and 23S rRNA targeted quantitative Reverse Transcription PCR (qRT-PCR) using the Yakult Intestinal Flora-SCAN analysis system (YIF-SCAN®; Yakult Honsha, Tokyo, Japan). Three serial dilutions of each extracted RNA sample were used for rRNA-targeted qRT-PCR. Threshold cycle values in the linear range of the assay were applied to the standard curve to obtain the corresponding bacterial cell count for each fecal or blood sample. In this study, predominant anaerobes present in the human intestine (Clostridium coccoides group, Clostridium leptum subgroup, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, and Prevotella) and intestinal subdominant populations (Clostridium difficile, Clostridium perfringens, Lactobacillus, Enterobacteriaceae, Enterococcus, Streptococcus, Staphylococcus, and Pseudomonas) were examined. The specificity of the qRT-PCR assay using group-specific, genus-specific, and species-specific primers was determined as described previously.[14, 15, 17, 18], 
Short-chain Fatty Acids Concentration (SCFAs) and pH Measurement
Concentrations of fecal SCFAs were measured as described previously  with slight modifications. Briefly, the frozen samples were homogenized in four-fold volumes of 0.15 mol/l perchloric acid and allowed to stand at 4°C for 12 hours. The suspension was subjected to centrifugation at 20,400 x g at 4°C for 10 minutes. The resultant supernatant was passed through a filter with a pore size of 0.45 μm (Millipore Japan, Tokyo, Japan). The sample was analyzed for organic acids using a high-performance liquid chromatography system (Waters 432 Conductivity Detector, Waters, Milford, MA, USA). Organic acid concentrations were calculated with the use of external standards and expressed as µmol/g of wet feces. The lower limits for fecal organic acid concentrations using this procedure were 0.075 µmol/g for succinic acid, 0.2 µmol/g for lactic acid, 0.05 µmol/g for formic acid, 0.4 µmol/g for acetic acid, 0.5 µmol/g for propionic acid, 0.55 µmol/g for butyric acid, 0.8 µmol/g for isovaleric acid, and 0.65 µmol/g for valeric acid. Fecal pH was measured by directly inserting the glass electrode of a D-51 pH meter (Horiba Seisakusho, Tokyo, Japan) into a sample of homogenized feces.
Postoperative microbiota counts and fecal organic acid concentrations were compared with preoperative control values using the paired t-test. If microbiota counts were lower than the limit of detection, the count of the sample was treated as half of the limit of detection value. Moreover, microbiota counts and fecal organic acid concentrations were compared between patients with or without pseudopsia and insomnia using the unpaired t-test. Analyses were performed using Stata/SE, version 16 (StataCorp, College Station, TX, USA).