Table 1
Specificity tests with the newly designed primer set
Taxon
|
Strain
|
ReactionA
|
Pseudomonas aeruginosa
|
ATCC 10145T
|
+
|
Pseudomonas aeruginosa
|
ATCC 9027
|
+
|
Pseudomonas aeruginosa
|
ATCC 15442
|
+
|
Pseudomonas aeruginosa
|
JCM 2776
|
+
|
Pseudomonas aeruginosa
|
JCM 5961
|
+
|
Pseudomonas aeruginosa
|
DSM 6195
|
+
|
Pseudomonas alcaligenes
|
ATCC 14909T
|
-
|
Pseudomonas fluorescens
|
ATCC 13525T
|
-
|
Pseudomonas fluorescens
|
ATCC 15916
|
-
|
Pseudomonas brenneri
|
ATCC 49642
|
-
|
Pseudomonas luteola
|
ATCC 43273T
|
-
|
Pseudomonas oryzihabitans
|
JCM 2952T
|
-
|
Pseudomonas oryzihabitans
|
JCM 3843
|
-
|
Pseudomonas pseudoalcaligenes
|
JCM 5968T
|
-
|
Pseudomonas putida
|
ATCC 12633T
|
-
|
Pseudomonas putida
|
ATCC 49128
|
-
|
Pseudomonas stutzeri
|
ATCC 17588T
|
-
|
Pseudomonas tolaasii
|
ATCC 33618T
|
-
|
Bacteroides vulgatus
|
ATCC 8482T
|
-
|
Bifidobacterium adolescentis
|
ATCC 15703T
|
-
|
Blautia producta
|
JCM 1471T
|
-
|
Clostridium difficile
|
DSM 1296T
|
-
|
Clostridium perfringens
|
JCM 1290T
|
-
|
Collinsella aerofaciens
|
DSM 3979T
|
-
|
Enterococcus faecalis
|
ATCC 19433T
|
-
|
Escherichia coli
|
ATCC 11775T
|
-
|
Faecalibacterium prausnitzii
|
ATCC 27768T
|
-
|
Prevotella melaninogenica
|
ATCC 25845T
|
-
|
Lactobacillus brevis
|
ATCC 14869T
|
-
|
Lactobacillus casei
|
ATCC 334T
|
-
|
Lactobacillus fermentum
|
ATCC 14931T
|
-
|
Lactobacillus fructivorans
|
ATCC 8288T
|
-
|
Lactobacillus gasseri
|
DSM 20243T
|
-
|
Lactobacillus plantarum
|
ATCC 14917T
|
-
|
Lactobacillus reuteri
|
JCM 1112T
|
-
|
Lactobacillus ruminis
|
JCM 1152T
|
-
|
Lactobacillus sakei
|
ATCC 15521T
|
-
|
Staphylococcus aureus
|
ATCC 12600T
|
-
|
Streptococcus mutans
|
ATCC 27175T
|
-
|
A The specificity of the qRT-PCR assay for target bacteria performed with the s-Pa-F/s-Pa-R primer set was investigated by using RNA extracts corresponding to 105 cells from each strain described.
+: when it was more than that of 104 standard cells; -: when it was less than that of 101 standard cells.
|
23S rRNA gene sequencing of P. aeruginosa and related strains.
We newly sequenced the 23S rRNA genes of 6 strains of P. aeruginosa (P. aeruginosa ATCC 10145T [accession number: LC515958], P. aeruginosa ATCC 9027 [accession number: LC515959], P. aeruginosa ATCC 15442 [accession number: LC515960], P. aeruginosa JCM 2776 [accession number: LC515961], P. aeruginosa JCM 5961 [accession number: LC515962], and P. aeruginosa DSM 6195 [accession number: LC515963]) and 8 species related to P. aeruginosa (P. alcaligenes ATCC 14909T [accession number: LC515970], P. fluorescens ATCC 13525T [accession number: LC515971], P. luteola ATCC 43273T [accession number: LC515966], P. oryzihabitans JCM 2952T [accession number: LC515967], P. pseudoalcaligenes JCM 5968T [accession number: LC515964], P. putida ATCC 12633T [accession number: LC515968], P. stutzeri ATCC17588T [accession number: LC515965], and P. tolaasii ATCC 33618T [accession number: LC515969]) using the primer sets designed in this study for 23S rRNA sequencing. qRT-PCR was conducted in a one-step reaction using a Qiagen OneStep RT-PCR kit (QIAGEN, Hilden, Germany), and the reaction mixture was incubated at 50 °C for 30 min for reverse transcription. The continuous amplification program consisted of one cycle at 95 °C for 15 min, followed by 45 cycles at 94 °C for 20 s, followed by 20 s at the annealing temperature of each primer, and 72 °C for a duration according to the respective size of each amplicon. Each primer set used in the amplification is described in Additional File 2: Supplementary Table 2. Then, the amplified DNA fragment was sequenced, and the full-length 23S rRNA gene sequence was determined by a primer walking method. Sequencing was performed with a 3500 Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA) after the reaction using a BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) and purification.
Development of 23S rRNA gene-targeted primers specific to P. aeruginosa.
Multiple alignment of the 6 P. aeruginosa strains and 8 related species was performed with the CLUSTAL_X program [25] using the 23S rRNA gene sequences obtained from the previous experiment. After comparison of the sequences in silico, target sites for P. aeruginosa species-specific detection were identified, and a primer set, s-Pa-F (5ʹ-GTC TTT TAG ATG ACG AAG TGG-3ʹ) and s-Pa-R (5ʹ-TGG TAT CTT CGA CCA GCC AGA-3ʹ), was newly constructed. The product size and annealing temperature were 234 bp and 60 °C, respectively. The specificity of the designed primer pair was confirmed by submitting the sequences to the BLAST program of the National Center for Biotechnology Information (NCBI) (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Total RNA extraction.
For RNA stabilization, 2 volumes of RNAprotect Bacteria Reagent (QIAGEN, Hilden, Germany) were added to samples of each bacterial strain. After being kept for 10 min at room temperature, the bacterial suspensions were centrifuged at 13,000 × g for 10 min. The supernatant was discarded, and the pellet was stored at -80 °C until used for RNA extraction. RNA extraction was performed using a previously described method [21]. Briefly, each thawed sample was resuspended in a solution containing 346.5 µl of RLT buffer (QIAGEN, Hilden, Germany), 3.5 µl of β-mercaptoethanol (Sigma-Aldrich Co., St. Louis, MO, USA), and 100 µl of Tris-EDTA buffer (Wako Pure Chemical Industries, Osaka, Japan). Glass beads (300 mg; diameter, 0.1 mm) (TOMY Seiko, Tokyo, Japan) were added to the suspension, and the mixture was vortexed vigorously for 5 min with a ShakeMaster Auto machine (BioMedical Science Inc., Tokyo, Japan). Then, 500 µl of water-saturated phenol (Wako Pure Chemical Industries, Osaka, Japan) was added to the mixture, which was incubated at 60 °C for 10 min. Next, 100 µl of chloroform-isoamyl alcohol (24:1) was added to the mixture. After centrifugation of the mixture at 13,000 × g for 10 min at 4 °C, the supernatant (470 µl) was collected, and an equal volume of chloroform-isoamyl alcohol was added to it. After centrifugation at 12,000 × g and 4 °C for 5 min, the supernatant (400 µl) was collected and subjected to isopropanol precipitation. Finally, the nucleic acid fraction was suspended in nuclease-free water (Ambion Inc., Waltham, MA, USA).
qRT-PCR.
qRT-PCR was performed using a previously described method [21]. Briefly, in the case of bacterial cultures or blood samples, qRT-PCR was conducted in a one-step reaction using a Qiagen OneStep RT-PCR kit (QIAGEN, Hilden, Germany). The 20 µl reaction mixture was composed of 1 × Qiagen OneStep RT-PCR buffer, 0.5 × Q-solution buffer, 400 µM of each deoxynucleoside triphosphate, 1:100,000 dilution of SYBR green I (Lonza, Basel, Switzerland), 1 µl of Qiagen OneStep RT-PCR enzyme mixture, 0.6 µM of each specific primer, and 2 µl of template RNA. Amplification and detection were performed in 96-well optical plates (WATSON, Tokyo, Japan) using a 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA). In the case of fecal samples, qRT-PCR was performed with a Qiagen OneStep RT-PCR kit. The 10 µl reaction mixture was composed of 1 × Qiagen OneStep RT-PCR buffer, 0.5 × Q-solution buffer, 400 µM of each deoxynucleoside triphosphate, 1:100,000 dilution of SYBR green I, 0.4 µl of Qiagen OneStep RT-PCR enzyme mixture, 0.6 µM of each specific primer, and 5 µl of template RNA. Amplification and detection were performed in 384-well optical plates (Life Technologies, Carlsbad, CA, USA) with a QuantStudio™ 12K Flex Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA). The reaction mixture was incubated at 50 °C for 30 min for reverse transcription. In either case, the continuous amplification program consisted of one cycle at 95 °C for 15 min and 45 cycles at 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 35 s. A melting curve was obtained to distinguish targeted PCR products from non-targeted ones by slow heating at temperatures from 60 to 95 °C at a rate of 0.2 °C/s with continuous fluorescence collection. Standard curves for P. aeruginosa ATCC 10145T were generated by using the threshold cycle (CT) values and the corresponding cell counts, which were determined microscopically with DAPI staining as described elsewhere. CT values in the linear range of the assay were applied to the analytical curve generated in the same experiment to obtain the corresponding bacterial count in each nucleic acid sample; this count was converted to the count per sample.
DNA extraction and qPCR.
Fresh cultures of each bacterial strain (1 ml) were centrifuged at 13,000 × g for 10 min. Then, the supernatant (800 µl) was discarded and the pellet was stored at -80 °C until used for DNA extraction. The bacterial suspension (200 µl) was subjected to DNA extraction. RNA extraction and qPCR were performed using a previously described method [26]. Briefly, each thawed sample was mixed with 250 µl of extraction buffer (100 mM Tris-HCl, 40 mM EDTA; pH 9.0) and 50 µl of 10% sodium dodecyl sulfate. Glass beads (300 mg; diameter, 0.1 mm) and 500 µl of Tris-EDTA (TE)-saturated phenol were added to the suspension, and the mixture was subjected to vigorous vortexing for 10 min on a ShakeMaster Auto apparatus (Bio Medical Science Inc., Tokyo, Japan). After phenol-chloroform purification and isopropanol precipitation, the nucleic acid fraction was suspended in 100 µl of nuclease-free water. qPCR was carried out using a Qiagen OneStep RT-PCR kit (QIAGEN, Hilden, Germany). Each reaction mixture (20 µl) contained the same components as those for qRT-PCR, except for the replacement of 2 µl template RNA with the same amount of template DNA. The reaction mixture was incubated at 95 °C for 15 min and 45 cycles at 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 35 s. The subsequent procedures were the same as those for qRT-PCR.
Determination of primer specificity.
Total RNA fractions extracted from the bacterial cells of each strain (shown in Table 1) at a dosorresponding to 105 cells were assessed by qRT-PCR using the primer set of s-Pa-F and s-Pa-R. Using the standard curve for P. aeruginosa as described above, we judged the amplified signal to be positive (+) when it was more than that of 104 standard cells and negative (−) when it was less than that of 101 standard cells. The amplified signal was also defined as negative (−) when the corresponding melting curve had a peak different from that of the standard strain.
Determination of qRT-PCR sensitivity.
Total RNA and DNA fractions of the 6 strains of P. aeruginosa were extracted from culture samples, and bacterial counts were determined microscopically with DAPI staining as described elsewhere. Serial RNA and DNA dilutions corresponding to bacterial counts ranging from 10− 2 to 104 cells were assessed by qRT-PCR and qPCR assays, respectively. The range of RNA and DNA concentrations at which there was linearity with the CT value was confirmed. Results are expressed as the mean and standard deviation of the results from triplicate samples.
Quantification of P. aeruginosa spiked to human blood by qRT-PCR and culture methods.
Commercially available human blood type A (Kohjin Bio Co., Saitama, Japan) was used in this study to determine the detection limit of qRT-PCR and to compare the bacterial counts determined by qRT-PCR and the culture method. P. aeruginosa ATCC 10145T prepared as described above was serially diluted with 1% Tween 20 (Wako Pure Chemical Industries, Osaka, Japan) in trypticase soy broth (TSB, Beckton Dickinson Co., Franklin Lakes, NJ, USA) and then spiked to make final concentrations ranging from 100 to 104 cells/ml of blood. For qRT-PCR assays, 1 ml of the blood sample was added to 2 ml of RNAprotect bacterial reagent (QIAGEN, Hilden, Germany). After being kept for 10 min at room temperature and centrifuged at 15, 000 × g for 10 min, the pellet was stored at -80 °C until used for RNA extraction. Then, RNA fractions extracted from the mixture were assessed by qRT-PCR assay. The obtained CT values were applied to the standard curve generated with the RNA dilution series for P. aeruginosa ATCC 10145T to determine the qRT-PCR counts with the primer set of s-Pa-F and s-Pa-R. For the culture method, to avoid the effect of reduced colony-forming capacity by the presence of antimicrobial substances in human blood such as lysozyme and complement proteins, the P. aeruginosa-spiked human blood sample was immediately cultured on NCC agar plates. One ml of each appropriate dilution series of blood sample was cultured on 10 culture plates (100 µl each), at 37 °C for 24 h. The CFU counts of P. aeruginosa in 1 ml of blood was calculated by summed up the results of 10 culture plates.
Quantification of P. aeruginosa added to human feces by qRT-PCR and culture methods.
Fecal samples collected from three healthy adult volunteers who had been confirmed in advance by qRT-PCR not to include P. aeruginosa in their indigenous intestinal populations were used in this study. Each fecal sample was weighed and suspended in 9 volumes of TSB. P. aeruginosa ATCC 10145T prepared as described above was serially diluted with 1% Tween 20 in TSB and then spiked at final concentrations ranging from 102 to 107 cells/g of feces. In preparation for total RNA extraction, 400 µl of each fecal homogenate was added to 800 µl of RNA later (Ambion Inc., Waltham, MA, USA). After being kept for 10 min at room temperature, 120 µl of the mixture was added to 1 ml of Dulbecco’s PBS (-) (Nissui Pharmaceutical Co., Tokyo, Japan) Then, after centrifugation of the mixture at 15,000 × g for 10 min, the pellet was stored at -80 °C until used for RNA extraction. RNA fractions extracted from the pellet were assessed by qRT-PCR assays. The CT values obtained were applied to the standard curve generated with the RNA dilution series for P. aeruginosa ATCC 10145T to determine the qRT-PCR counts with the primer set of s-Pa-F and s-Pa-R. For the culture method, 100 µl of each appropriate dilution series of fecal homogenate was cultured on NCC agar plates at 37 °C for 24 h.
Monitoring of MDRP exposed to antibiotics by three different methods.
P. aeruginosa ATCC BAA-2108™ adjusted to a final concentration of 106 CFU/ml was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA), 1% MEM nonessential amino acids (Gibco, Waltham, MA, USA), 5 µg/ml colistin sulfate salt (Sigma-Aldrich Co., St. Louis, MO, USA), and 100 µg/ml doripenem hydrate (SHIONOGI & CO., LTD., Osaka, Japan) at 37 °C with shaking (140 rpm). After the start of culture, 10 ml of the culture solution was periodically sampled and washed by centrifugation at 13,000 × g for 10 min, and the supernatant was discarded. The precipitated pellets were resuspended in 10 ml of antibiotic-free and serum-free DMEM. Then, the culture solution was distributed for RNA extraction (1 ml), DNA extraction (1 ml), culturing for CFU counts (1 ml), cell infection (2 ml), and acquisition of fluorescent images (3 ml). For RNA extraction, 1 ml of the dispersed culture solution was added to 2 ml of RNA later. After being kept for 10 min at room temperature and centrifuged at 15,000 × g for 10 min, the pellet was stored at -80 °C until used for RNA extraction. RNA extraction and qRT-PCR with the primer set s-Pa-F/s-Pa-R were performed as described above. Also, for DNA extraction, 1 ml of the dispersed culture solution was centrifuged at 15,000 × g for 10 min, and the pellet was stored at -80 °C until used for DNA extraction. DNA extraction and qPCR with the primer set s-Pa-F/s-Pa-R were performed as described above. For the culture method, 1 ml of the appropriate dilution series was cultured on TSA plates at 37 °C for 24 h.
Infection of Caco-2 cells with MDRP exposed to antibiotics.
Caco-2 cells (86010202) obtained from Public Health England (London, UK) were grown in DMEM supplemented with 10% FBS, 1% MEM nonessential amino acids, 1% penicillin-streptomycin (10,000 units/ml penicillin, 10,000 µg/ml streptomycin) (Gibco, Waltham, MA, USA) at 37 °C in the presence of humidified 5% CO2 in air. Cells were plated on 6-well plates (Costar, Corning, NY, USA) at a density of 3.0 × 105 cells/well and incubated for 24 h for the infection assays. After incubation, the cells were washed three times with D-PBS (Thermo Fisher Scientific Inc., Waltham, MA, USA) to remove penicillin G and streptomycin contained in the cell culture. For cell infection, 2 ml of MDRP suspension treated with antibiotics and washed as described above was applied to the cells, and cells were incubated at 37 °C with 5% CO2 in air for 6 h. To compare with a reference value of uninfected cells, fresh antibiotic-free culture medium was also applied to the cells as a control, and then the cells were incubated at 37 °C with 5% CO2 in air for 6 h.
Quantification of inflammatory response in infected Caco-2 cells.
The bacterial proinflammatory effect was assessed by measuring the transcription level of IL-8 and IL-8 secretion in infected Caco-2 cells. After a 6-hour incubation, 2 ml of the cell culture medium was sampled to measure IL-8 production. The medium was centrifuged at 2,300 g for 5 min, and the supernatant was stored at -80 °C until used for determinations. Then, total RNA was extracted from infected Caco-2 cells using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA). qRT-PCR was performed in 96-well optical plates with a 7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, MA, USA) using a Qiagen OneStep RT-PCR kit (QIAGEN, Hilden, Germany), and the reaction mixture was incubated at 95 °C for 15 min and 45 cycles at 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 35 s. The following primers were used: IL-8, 5’-AGGACAAGAGCCAGGAAGAA-3’ (forward primer) and 5’-CAGAGCTGCAGAAATCAGGA-3’ (reverse primer) [27]; GAPDH, 5’-GGAGCGAGATCCCTCCAAAAT-3’ (forward primer) and 5’-GGCTGTTGTCATACTTCTCATGG-3’ (reverse primer) [28]. Standard curves were obtained for each primer set with serial dilutions of RNA. The RNA used for standard RNA solutions was extracted from Caco-2 cells infected with P. aeruginosa following culturing for 6 h on medium containing antibiotics. The relative amounts of IL-8-encoding transcripts were calculated and normalized to that of GAPDH, and the relative gene expression values were compared to those of uninfected cells (treated with DMEM not containing antimicrobials; n = 4). IL-8 levels in cell culture supernatants were assayed using an ELISA Quantikine kit (BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol.
Observation and acquisition of fluorescent images of MDRP.
Fluorescence in situ hybridization (FISH) analyses were performed as described previously with minor modifications [29]. Briefly, the bacterial suspension for acquisition of fluorescent images, which was sampled at 0, 48, and 336 h after cultivation, was centrifuged at 13, 000 × g for 10 min and condensed to 100 times concentration. The suspension was fixed with 3 volumes of 4% paraformaldehyde and left at 4 °C for 16 h. Ten microliters of the fixed sample, after being condensed to 4 times the concentration by centrifugation, was smeared on a MAS-coated slide glass (Matsunami Glass Ind., Ltd., Osaka, Japan), which was then hybridized with the TAMRA-labeled 16S rRNA probe Eub338 (5’-GCTGCCTCCCGTAGGAGT-3’). After washing once more with distilled water, the cover glass was mounted with mounting medium containing DAPI (VECTASHIELD with DAPI, Vector Laboratories, Burlingame, CA, USA). An automated Leica DM6000 B research microscope was used for fluorescence observation and image acquisition. All fluorescent images obtained were analyzed (HiGauss and flattening) using image analysis software (Image-Pro Plus v. 5.1, Media Cybernetics, Inc., Rockville, MD).
qRT-PCR quantification of P. aeruginosa and monitoring of drug-resistant P. aeruginosa in clinical samples.
For the analysis of clinical samples, we used total RNA isolated from fecal samples collected in a previous study [30], which were stored at -80 °C until used in this study. In the previous study, fecal samples were acquired from 65 patients who were more than 16 years old and had been placed on a ventilator within 3 days after admission to the ICU, and who were diagnosed as having sepsis in the Department of Traumatology and Acute Critical Medicine, Osaka University Medical School, and Osaka General Medical Center during the period from November 2011 to September 2016. If sepsis occurred, patients were initially treated empirically for the underlying clinical syndrome and then according to the results of antibiotic susceptibility testing of the bacterial isolate causing the sepsis. Antibiotics were administered under the same policy during the entire study period. Also, we collected fecal samples from 44 healthy Japanese adults and prepared total RNA in the same manner as for the clinical samples, as described above. Then, three serial dilutions of each extracted RNA sample were used for qRT-PCR, and the CT values obtained were applied to the standard curve generated with the RNA dilution series for P. aeruginosa ATCC 10145T to determine the qRT-PCR counts with the primer set of s-Pa-F and s-Pa-R. For monitoring of drug-resistant P. aeruginosa in fecal samples from 4 ICU patients, qRT-PCR was used to check for the presence of resistance genes using the following specific primers: blaIMP, 5’-GGAATAGAGTGGCTTAAYTCTC-3’ (forward primer) and 5’-GGTTTAAYAAAACAACCACC-3’ (reverse primer) [31]; ampC, 5’-GGGCTGGCCTCGAAAGAGGAC-3’ (forward primer) and 5’- GCACCGAGTCGGGGAACTGCA-3’ (reverse primer) [32]; and mexA, 5’-AACCCGAACAACGAGCTG-3’ (forward primer) and 5’-ATGGCCTTCTGCTTGACG-3’ (reverse primer) [33], in combination with the primer set of s-Pa-F and s-Pa-R. Each different reaction mixture was incubated at 95 °C for 15 min and 45 cycles at 94 °C for 20 s, 60 °C for 20 s, and 72 °C for 35 s. To confirm the validity of the qRT-PCR amplification, sequence analysis of the RT-PCR products was performed [34].
Fecal microbiota analysis.
The microbiota compositions of 4 ICU patients were analyzed using the YIF-SCAN® version of a 16S and 23S rRNA-targeted qRT-PCR system using specific primers for the 6 most prevalent obligate anaerobic bacterial groups (Clostridium coccoides group, C. leptum subgroup, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, and Prevotella) and 5 facultative anaerobic and aerobic bacterial groups (Lactobacillus, Enterobacteriaceae, Enterococcus, and Staphylococcus] as described previously [21, 22]. A standard curve was generated with qRT-PCR using the CT value, i.e., the cycle number when the threshold fluorescence was reached, and the corresponding cell count was determined microscopically with DAPI staining for a dilution series of the standard strains as described elsewhere. To determine the types of bacteria present in the samples, three serial dilutions of an extracted RNA sample were used for qRT-PCR, and the CT values in the linear range of the assay were applied to the standard curve to obtain the corresponding bacterial cell counts in each nucleic acid sample. These data were then used to determine the number of bacteria per sample.
Statistical analysis.
Statistical analyses were performed using IBM SPSS Statistics Desktop version 22.0 software (IBM Japan Ltd., Tokyo, Japan). Fisher's exact test was used to compare the detection rate of P. aeruginosa between 2 groups. The Mann-Whitney U test (2-tailed) was used to compare the average P. aeruginosa counts between 2 groups. Pearson’s correlation coefficient was used to analyze the relationship between the P. aeruginosa count quantified with each measurement method and the cytokine levels of the infected Caco-2 cells. P values < 0.05 were considered statistically significant. Spearman’s rank correlation coefficient was used to analyze the association between the number of P. aeruginosa and fecal occupation rate of representative microorganisms.