Prevalence of Quorum Sensing and Virulence Factor Genes Among Pseudomonas aeruginosa Isolated from Patients Suffering from Different Infections and Their Association with Antimicrobial Resistance

Figures & data

Table 1 The Primers Used in This Study and Their Sequences

Primer Type	Primer Sequence (5’-3’)	Amplicon Size (bp)	Annealing Temp.	Reference
lasB-F	GGAATGAACGAAGCGTTCTC	300	55	[59]
lasB-R	GGTCCAGTAGTAGCGGTTGG
toxA-F	GGTAACCAGCTCAGCCACAT	352	50	[59]
toxA-R	TGATGTCCAGGTCATGCTTC
aprA-F	ACCCTGTCCTATTCGTTCC	140	65	[59]
aprA-R	GATTGCAGCGACAACTTGG
algD-F	ATGCGAATCAGCATCTTTGGT	1311	55	[59]
algD-R	CTACCAGCAGATGCCCTCGGC
plcH-F	GAAGCCATGGGCTACTTCAA	307	55	[59]
plcH-R	AGAGTGACGAGGAGCGGTAG
ExoS-F	CTTGAAGGGACTCGACAAGG	504	58	[60]
ExoS-R	TTCAGGTCCGCGTAGTGAAT
LasI-F	CGCACATCTGGGAACTCA	605	65	[61]
LasI-R	CGGCACGACGATCATCATCT
LasR-F	CTGTGGATGCTCAAGGACTAC	725	65	[61]
LasR-R	AACTGGTCTTGCCGATGG
rhII-F	GTAGCGGGTTTGCGGATG	155	65	[61]
rhII-R	CGGCATCAGGTCTTCATCG
rhIR-F	GCCAGCGTCTTGTTCGG	133	60	[61]
rhIR-R	CGGTCTGCCTGAGCCATC

Figure 1 Antibiotic resistance profile of P. aeruginosa according to the type of sample.

Abbreviations: CIP, ciprofloxacin; AK, amikacin; CPM, cefepime; NX, norfloxacin; IPM, imipenem; GEN, gentamicin, TOB, tobramycin; ATM, aztreonam; PIT, piperacillin-tazobactam; CL, colistin.

Table 2 Distribution of MDR Resistant P. aeruginosa Among Isolates Among Different Types of Infection

Sample Type	MDR		Non-MDR		P value
	No.	%*	No.	%*
U	10	66.7	5	33.3	0.767
W	21	60	14	40	0.644
ER	20	62.5	12	37.5	0.924
SP	19	70.4	8	29.6	0.383
ST	9	56.3	7	43.7	0.537
Total	79	63.2**	46	36.8**

Notes: Type of clinical specimen; U: urine specimen; W: wound exudate; ER: ear discharge; SP: sputum; ST: stool. *P. aeruginosa isolates related to the total number of isolates from the same source. **The total number of P. aeruginosa isolates.

Table 3 Prevalence of MDR Isolates Among the Different Types of Infections

Type of Samples	MDR Isolates (N=79)
	No.	%*
Urine	10	12.6
Wound	21	26.5
Ear discharge	20	25.3
Sputum	19	24.05
Stool	9	11.3

Note: *Percents were correlated to the total number of MDR isolates.

Abbreviation: MDR, Multi-drug resistant.

Table 4 Distribution of Quorum Sensing and Virulence Factors Regulating Genes in P. aeruginosa Isolated from Different Clinical Specimens

Tested Genes		Type of Sample %*					Total (%**)	P value
		U	W	ER	SP	ST
QS genes	rhlI	46.7	74.3	71.9	70.4	75	69.6	0.355
	rhlR	100	100	93.8	92.6	81.3	94.4	0.078
	lasI	53.3	91.4	96.9	74.1	68.8	81.6	0.001***
	lasR	60	88.6	84.4	85.2	62.5	80	0.055
Virulence factors genes	lasB	86.7	88.6	90.6	92.6	87.5	89.6	0.969
	aprA	73.3	85.7	87.5	88.9	87.5	85.6	0.697
	exoS	80	82.9	90.6	85.2	75	84	0.690
	algD	66.7	80	84.4	81.5	81.3	80	0.717
	toxA	66.7	80	78.1	77.8	75	76.8	0.888
	plcH	60	77.1	75	81.5	75	75.2	0.644

Notes: *P. aeruginosa isolates related to the total number of isolates from the same source. **The total number of P. aeruginosa isolates, ***P-value was significant if it was ≤0.05.

Abbreviation: QS genes, Quorum sensing genes.

Table 5 Distribution of Virulence Factor Genes Among MDR and Non-MDR P. aeruginosa Isolates

		MDR		Non-MDR		P value
		No.	%*	No.	%**
QS genes	rhlI	65	82.3	22	47.8	<0.001***
	rhlR	79	100	39	84.9	<0.001***
	lasI	70	88.6	32	69.6	0.008***
	lasR	74	93.7	26	56.5	<0.001***
Virulence factors genes	lasB	78	98.7	34	73.9	<0.001***
	aprA	76	96.2	31	67.4	<0.001***
	exoS	73	92.4	32	69.6	0.001***
	algD	69	87.3	31	67.4	0.007***
	toxA	68	86.1	28	60.9	0.001***
	plcH	67	84.8	27	58.7	0.001***

Notes: *The total number of MDR P. aeruginosa isolates (79). **The total number of Non-MDR P. aeruginosa isolates (46), ***P-value was significant if it was ≤0.05.

Table 6 Resistance Pattern of P. aeruginosa Among Biofilm Producers and Non-Producer

Antimicrobial Agent	Biofilm Producers No. (%)*	Biofilm Non Producers No. (%)**	P value
Ciprofloxacin	81 (73.6)	3 (20)	<0.001***
Amikacin	47 (42.7)	1 (6.7)	<0.001***
Cefepime	110 (100)	6 (40)	<0.001***
Norfloxacin	70 (63.6)	2 (13.3)	<0.001***
Imipenem	15 (13.6)	0 (0)	0.127
Gentamicin	34 (30.9)	2 (13.3)	<0.001***
Tobramycin	21 (19.1)	0 (0)	0.179
Aztreonam	60 (54.5)	3 (29)	0.012***
Piperacillin-tazobactam	19 (17.3)	1 (6.7)	0.089
Colistin	73 (66.4)	2 (13.3)	<0.001***

Notes: *Percents were correlated to total biofilm producers (110). **Percents were related to total biofilm non producers (15). ***P-value was significant if it was ≤0.05.

Figure 2 Correlation between biofilm formation and type of resistant P. aeruginosa isolates (P > 0.001).

Abbreviation: MDR, Multi-drug resistant.

Table 7 Distribution of Virulence Genes Among P. aeruginosa Clinical Isolates Collected from Different Sources

No. of Virulence Genes Found in Each Isolate	Type of Samples %*					Total (n=125) N (%**)	P value
	Urine	Wound Exudate	Ear Discharge	Sputum	Stool
0	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0 (0)	0.893
1	0 (0)	1 (2.9)	0 (0)	0 (0)	1 (6.3)	2 (1.6)
2	1 (6.7)	1 (2.9)	1 (3.1)	0 (0)	2 (12.5)	5 (4)
3	2 (13.3)	2 (5.7)	3 (9.4)	1 (3.7)	1 (6.3)	9 (7.2)
4	2 (13.3)	1 (2.9)	2 (6.3)	1 (3.7)	2 (12.5)	8 (6.4)
5	4 (26.7)	5 (14.3)	2 (6.3)	4 (14.8)	2 (12.5)	17 (13.6)
6	3 (20)	6 (17.1)	8 (25)	5 (18.5)	3 (18.8)	25 (20)
7	2 (13.3)	4 (11.4)	4 (12.5)	5 (18.5)	1 (6.3)	16 (12.8)
8	1 (6.7)	7 (20)	4 (12.5)	6 (22.2)	3 (18.8)	21 (16.8)
9	0 (0)	5 (14.3)	6 (18.8)	3 (11.1)	1 (6.3)	15 (12)
10	0 (0)	3 (8.6)	2 (6.3)	2 (7.4)	0 (0)	7 (5.6)

Notes: *Percentages were correlated to the total number of P. aeruginosa isolates from each type of sample. **Percentages were correlated to the total number of P. aeruginosa isolates. P-value was significant if it was ≤0.05.

Table 8 Resistance Pattern of Highly Virulent Biofilm Producing P. aeruginosa Harboring ≥6 Virulence Genes

No. of Virulence Genes	No. of Isolates	QS Genes				Virulence Genes						Antibiotics
		lasI	lasR	rhlI	rhlR	lasB	aprA	algD	toxA	exoS	plcH	CIP	AK	CPM	NX	IPM	GEN	TOB	ATM	PIT	CL
Group 1 (6 genes)	7	+	+	+		+		+		+		+		+					+		+
	6	+		+	+		+		+	+		+	+	+	+
	5		+		+	+		+	+		+	+		+			+				+
	3	+	+		+		+			+	+			+	+						+
	2		+	+		+	+	+	+			+						+	+
	2	+		+	+	+			+		+		+			+					+
Group 2 (7 genes)	5		+	+	+	+	+	+		+		+		+			+
	4	+		+		+		+	+	+	+			+	+						+
	4	+	+		+	+	+	+		+		+		+					+
	2			+	+	+	+	+	+		+		+	+			+
	1	+	+		+	+	+			+	+	+				+					+
Group 3 (8 genes)	6	+	+	+	+	+	+	+		+		+		+	+				+		+
	5	+	+	+	+	+	+	+			+	+		+	+					+
	3	+		+		+	+	+	+	+	+		+	+	+		+		+
	3	+	+			+	+	+	+	+	+	+	+					+	+		+
	2		+	+	+	+		+	+	+	+		+	+		+	+		+		+
	1	+	+	+	+	+	+	+		+	+	+		+	+			+	+		+
	1	+	+	+	+	+	+	+	+	+		+		+		+		+	+	+
Group 4 (9 genes)	5	+	+	+	+	+	+	+		+	+	+	+	+	+		+		+
	5	+	+	+	+	+	+	+	+		+	+		+	+		+	+	+		+
	3	+	+		+	+	+	+	+	+	+	+		+	+	+		+	+		+
	2	+	+	+		+	+	+	+	+	+	+	+	+		+		+	+	+
	2	+	+	+	+	+	+	+	+	+		+	+	+	+	+	+		+	+
Group 5 (10 genes)	7	+	+	+	+	+	+	+	+	+	+	+	+	+	+		+		+	+	+

Abbreviations: CIP, ciprofloxacin; AK, amikacin; CPM, cefepime; NX, norfloxacin; IPM, imipenem; GEN, gentamicin; TOB, tobramycin; ATM, aztreonam; PIT, piperacillin-tazobactam; CL, colistin.

Table 9 Correlation Between Different Groups of Virulent Genes of Highly Virulent Biofilm Producing P. aeruginosa and Number of Resistant Antibiotics

	Group I	Group II	Group III	Group IV	Group V	P value
	N=25	N=16	N=21	N=17	N=7
No of resistant antibiotics	3.7±0.5	3±0	5±0.7	6.8±0.6	8±0	<0.001*

Notes: Group I: strains which were positive for 6 genes, Group II: strains which were positive for 7 genes, Group III: strains which were positive for 8 genes, Group IV: strains which were positive for 9 genes and Group V: strains which were positive for all tested genes. *P-value was significant if it was ≤0.05.

Table 10 Correlation Between Number of Genes and Number of Antibiotics

	Number of Antibiotics
	r	P value
Number of genes	0.815	<0.001*

Notes: Spearman correlation (r), *P-value was significant if it was ≤ 0.05.

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