Pseudomonas aeruginosa Type III Secretion System Virulotypes and Their Association with Clinical Features of Cystic Fibrosis Patients

Figures & data

Table 1 Primers Sequences Used in the Molecular Detection of T3SS Related-Genes

Gene	Primers Sequence	Amplicons Size (bp)	Reference
exoS	F: 5ʹ-CGTCGTGTTCAAGCAGATGGTGCTG-3ʹ R: 5ʹ-CCGAACCGCTTCACCAGGC-3’	444	Finnan et al^Citation19
exoT	F: 5ʹ-CAATCATCTCAGCAGAACCC-3ʹ R: 5ʹ-TGTCGTAGAGGATCTCCTG-3’	1159
exoY	F: 5ʹ-TATCGACGGTCATCGTCAGGT-3ʹ R: 5ʹ-TTGATGCACTCGACCAGCAAG-3’	1035
exoU	F: 5ʹ- GGGAATACTTTCCGGGAAGTT-3ʹ R: 5ʹ-CGATCTCGCTGCTAATGTGTT-3’	428	Zhu et al^Citation25

Abbreviation: bp, base pairs.

Table 2 P. aeruginosa Infection Profile According to the Number of Exacerbations/Year, Age and Age at Diagnosis

Variable	Groups			p-value*
	Chronic Infection n=23	Intermittent Infection n=26	Infection-Free n=63
Number of exacerbations/year
Min. – Max.	0–4,0	0–2,0	0–4,0	0,0087^†
Median (DIQ)	0 (2,0)^a	0 (1,0)	0 (0)^a
P25/P75	0/2,0	0/1,0	0/0
CV%	127,5	141,2	241,9
Age (years)
Min. – Max.	2,0–63,0	1,0–38,0	0,2–70,0	0,0023^†
Median (DIQ)	21,0 (17,0)^a	9,0 (8,3)^a,b	15,0 (12,0)^b
P25/P75	9,0/26,0	7,0–15,3	11,0/23,0
CV%	73,5	66,1	73,3
Age at diagnosis (years)
Min. – Max.	0,3–62,0	0,1–30,0	0,1–64,0	0,0025^†
Median (DIQ)	12,0 (13,0)^a	3,0 (5,5)^a	6,0 (9,0)
P25/P75	6,0/19,0	0,8–6,3	2,0/11,0
CV%	101,6	135,4	133,0

Notes: *Kruskal-Wallis/Dunn test. DIQ, interquartile deviation; P25/P75, 25th and 75th percentiles; CV, coefficient of variation; ^†Statistically significant. Equal superscript letters indicate the groups that showed statistically significant differences from each other.

Table 3 Severity Category According to the Shwachman-Kulckycki Score Among the Studied Groups

Gravity	Groups			p-value*
	Chronic Infection n=23 (%)	Intermittent Infection n=26 (%)	Infection-Free n=63 (%)
Excellent	0 (0,0)^↓	12 (46,2)	30 (47,6)^↑	<0,0001^†
Good	05 (21,8)	09 (34,6)	23 (36,5)
Mild	12 (52,2)^↑	03 (11,5)	09 (12,3)^↓
Moderate	03 (13,0)	02 (7,7)	01 (1,6)^↓
Severe	03 (13,0)^↑	0 (0,0)	0 (0,0)^↓

Notes: *G-test of independence/Chi-Square Residue Analysis. ^†Statistically significant. ^↓Frequency lower than expected at random. ^↑Frequency higher than expected at random.

Table 4 Spirometric Data of CF Patients According to Infection Status

Variable	Groups			p-value*
	Chronic Infection n=18	Intermittent Infection n=21	Infection-Free n=57
FEV1, %
Min. – Max.	18,0–124,0	30,0–121,0	31,0–136,0	<0,0001^†
Median (DIQ)	43,0 (51,0)^a,b	86,0 (32,0)^a	92,0 (24,3)^b
P25/P75	28,0/79,0	70,0/102,0	79,7/104,0
CV%	57,4	30,4	22,0
FVC, %
Min. – Max.	22,0–132,0	42,0–133,0	45,0–140,0	<0,0001^†
Median (DIQ)	54,0 (37,0)^a,b	102,0 (29,5)^a	102,0 (25,5)^b
P25/P75	44,0/81,0	80,5/110,0	87,0/112,5
CV%	46,4	28,4	20,1
FEV1/FVC, %
Min. – Max.	55,0–97,0	72,0–108,0	65,0–109,0	0,0660
Median (DIQ)	82,0 (25,0)	89,0 (25,0)	89,0 (11,0)
P25/P75	72,0/97,0	83,0/108,0	84,0/95,0
CV%	16,3	9,1	10,4
FEF25–75, %
Min. – Max.	10,0–120,0	13,0–155,0	11,0–156,0	0,0003^†
Median (DIQ)	30,0 (59,0)^a,b	86,0 (47,0)^a	86,0 (33,5)^b
P25/P75	16,0/75,0	63,0/110,0	67,0/100,5
CV%	81,4	45,8	34,7

Notes: *Kruskal-Wallis/Dunn test. FEV1: forced expiratory volume in the first second. FVC: forced vital capacity. FEF 25–75: forced expiratory flow. DIQ, interquartile deviation. P25/P75: 25th and 75th percentiles. CV, coefficient of variation. ^†Statistically significant. Equal superscript letters indicate the groups that showed statistically significant differences from each other.

Table 5 Association of T3SS Virulotypes with Infection Profile and Colony Morphology

Profile	Virulotype			p-value*
	exoS^+/exoU^+ n=30 (%)	exoS^+/exoU^− n=18 (%)	exoS^−/exoU^+ n=1 (%)
Groups
Chronic infection	9 (30,0)^↓	14 (77,8)^↑	0 (0,0)	0,0087^†
Intermittent infection	21 (70,0)^↑	4 (22,2)^↓	1 (100)
Morphology
Mucoid	7 (23,4)	8 (44,4)	0 (0,0)	0,8163
Non-mucoid	22 (73,3)	9 (50)	1 (100)
Both	1 (3,3)	1 (5,6)	0 (0,0)

Notes: *G-test of independence/Chi-Square Residue Analysis. ^†Statistically significant. ^↓Frequency lower than expected at random. ^↑Frequency higher than expected at random.

Table 6 Association of Shwachman-Kulckycki Score Categories with T3SS Genotypes and Infection-Free Patients

Variable	exoS^+/exoU^+ n=30	exoS^+/exoU^− n=18	Infection-Free n=63	p-value*
General activity
Min. – Max.	5,0–25,0	5,0–25,0	15,0–25,0	0,0232^†
Median (DIQ)	20 (5,0)	20,0 (7,7)^a	25,0 (5,0)^a
P25/P75	20,0/25,0	17,3/25,0	20,0/25,0
CV%	22,8	28,0	13,1
Physical examination
Min. – Max.	5,0–25,0	5,0–25,0	10,0–25,0	<0,0001^†
Median (DIQ)	20,0 (10,0)^a	15,0 (3,5)^b	20,0 (5,0)^a,b
P25/P75	15,0/25,0	15,0/18,5	20,0/25,0
CV%	30,2	28,5	18,0
Nutritional status
Min. – Max.	10,0–25,0	10,0–25,0	10,0–25,0	0,2394
Median (DIQ)	22,5 (6,2)	20,0 (10,0)	25,0 (5,0)
P25/P75	18,8/25,0	15,0/25,0	20,0/25,0
CV%	27,4	25,3	20,7
Radiological findings
Min. – Max.	5,0–20,0	5,0–20,0	10,0–25,0	<0,0001^†
Median (DIQ)	15,0 (10,0)^a	15,0 (8,0)^b	20,0 (5,0)^a,b
P25/P75	10,0/20,0	10,0/18,0	15,0/20,0
CV%	32,8	32,5	21,3

Notes: *Kruskal-Wallis/Dunn test. DIQ, interquartile deviation. P25/P75: 25th and 75th percentiles. CV, coefficient of variation. ^†Statistically significant. Equal superscript letters indicate the groups that showed statistically significant differences from each other.

Table 7 Association of Spirometric Data with T3SS Genotypes and Infection-Free Patients

Spirometric Data	Virulotype		Infection-Free n=57	p-value*
	exoS^+/exoU^+ n=24	exoS^+/exoU^− n=14
FEV1, %
Min. – Max.	18,0–124,0	18,0–121,0	31,0–136,0	0,0005^†
Median (DIQ)	82,5 (54,3)	49,0 (51,0)^a	92,0 (24,3)^a
P25/P75	44,8/99,00	28,0–79,0	79,7/104,0
CV%	39,2	56,6	22,0
FVC, %
Min. – Max.	22,0–132,0	22,0–133,0	45,0–140,0	0,0007^†
Median (DIQ)	92,0 (51,8)	54,0 (43,0)^a	102,0 (25,5)^a
P25/P75	56,5/108,3	47,0–90,0	87,0/112,5
CV%	35,5	46,6	20,1
FEV1/FVC, %
Min. – Max.	55,0–108,0	57,0–99,0	65,0–109,0	0,1473
Median (DIQ)	87,0 (11,0)	83,0 (19,0)	89,0 (11,0)
P25/P75	82,0/93,0	72,0–91,0	84,0/95,0
CV%	12,0	15,6	10,4
FEF25–75, %
Min. – Max.	10,0–143,0	10,0–150,0	11,0–156,0	0,0067^†
Median (DIQ)	81,5 (65,3)	30,0 (66,0)^a	86,0 (33,5)^a
P25/P75	30,0–95,8	21,0–87,0	67,0/100,5
CV%	53,9	83,1	34,7

Notes: *Kruskal-Wallis/Dunn test. FEV1: forced expiratory volume in the first second. FVC: forced vital capacity. FEF 25–75: forced expiratory flow. DIQ, interquartile deviation. P25/P75: 25th and 75th percentiles. CV, coefficient of variation. ^†Statistically significant. Equal superscript letters indicate the groups that showed statistically significant differences from each other.

Table 8 Association of Spirometric Data and Shwachman-Kulckycki Score of Patients with P. Aeruginosa Chronic Infection with T3SS Genotypes and Infection-Free Patients

Variable	Virulotypes of Chronically Infected Patients		Infection-Free n=57	p-value*
	exoS^+/exoU^+ n= 07	exoS^+/exoU^− n=12
FEV1, %
Min. – Max.	18,0–124,0	18,0–100,0	31,0–136,0	<0,0001^†
Median (DIQ)	43,0 (54,0)	44,0 (40,2)^a	92,0 (24,3)^a
P25/P75	29,0/83,0	27,3/67,5	79,7/104,0
CV%	64,5	51,9	22,0
FVC, %
Min. – Max.	22,0–132,0	22,0–104,0	45,0–140,0	<0,0001^†
Median (DIQ)	78,0 (48,0)^a	52,0 (32,0)^s	102,0 (25,5)^a,b
P25/P75	38,0/86,0	44,8/76,8	87,0/112,5
CV%	53,3	41,0	20,1
FEV1/FVC, %
Min. – Max.	55,0–97,0	57,0–96,0	65,0–109,0	0,0669
Median (DIQ)	82,0 (17,0)	82,5 (20,0)	89,0 (11,0)
P25/P75	77,0/94,0	70,5/90,5	84,0/95,0
CV%	17,3	16,4	10,4
FEF25–75, %
Min. – Max.	10,0–120,0	10,0–88,0	11,0–156,0	0,0004^†
Median (DIQ)	20,0 (80,0)	30,0 (48,2)^a	86,0 (33,5)^a
P25/P75	16,0/96,0	12,8/61,0	67,0/100,5
CV%	90,3	73,5	34,7
Overall Severity Score	n=09	n=14	n=63
Min. – Max.	25,0–85,0	25,0–85,0	50,0–100,0	<0,0001^†
Median (DIQ)	65,0 (25,0)^a	65,0 (11,8)^b	85,0 (15,0)^a,b
P25/P75	47,5/72,5	59,5/71,3	75,0/90,0
CV%	29,7	24,6	13,5

Notes: *Kruskal-Wallis/Dunn test. DIQ, interquartile deviation. P25/P75: 25th and 75th percentiles. CV, coefficient of variation. ^†Statistically significant. Equal superscript letters indicate the groups that showed statistically significant differences from each other.

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