Multidrug-resistant Pseudomonas aeruginosa from sputum of patients with cystic fibrosis demonstrates a high rate of susceptibility to ceftazidime-avibactam

Figures & data

Figure 1 Flowchart of subjects and isolate collection.

Note: A flowchart of CF individuals seen at the University of Texas Southwestern adult clinic from January 1, 2015, to December 31, 2015.
Abbreviations: AVI, avibactam; CAZ, ceftazidime; CF, cystic fibrosis; PsA, Pseudomonas aeruginosa; MDR, multidrug-resistant.

Table 1 Demographic information of patients for each P. aeruginosa isolate collected categorized by CAZ–AVI susceptibility

	CAZ-AVI sensitive (n=23)^a	CAZ-AVI resistant (n=9)^a	P-value
Age (years), mean ± sD	27.8±8.3	36.9±8.1	0.017^b
Sex			0.433^c
Male, n (%)	13 (52.6)	3 (33.3)
Female, n (%)	10 (47.4)	6 (66.7)
Race			0.509^c
Caucasian, n (%)	17 (73.6)	8 (88.9)
African American, n (%)	4 (15.7)	1 (11.1)
Hispanic, n (%)	2 (10.5)	0
Others	0	0
BMI, mean ± SD	21.6±2.7	19.1 ±5.4	0.049^b
ppFEV1 mean ± sD	39.2± 15.0	35.6±22.1	0.208^b
Genotype			0.056^c
F508del homozygous	12 (52.6)	1 (11.1)
F508del heterozygous	8 (47.3)	4 (44.4)
No F508del mutations	3 (15.7)	4 (44.4)

Notes: ^aBecause four subjects had 2 P. aeruginosa isolates some of which had differing sensitivity to CAZ–AVI, the subject’s demographics with two isolates was included twice.

b Unpaired t-test.

c chi-square test.

d Fisher’s exact test.

Abbreviations: AVI, avibactam; BMI, body mass index; CAZ, ceftazidime; P. aeruginosa, Pseudomonas aeruginosa; ppFEV₁,percent predicted forced expiratory volume in 1 second.

Figure 2 Antibiotic exposure of subjects prior to sputum collection.

Notes: Average number of days of subject exposure to listed antibiotic with standard error bars displayed in the 2 years prior to sputum sample collection. *P<0.05.
Abbreviations: AVI, avibactam; CAZ, ceftazidime.

Table 2 Antimicrobial susceptibility of 32 P. aeruginosa isolates from 28 patients with cystic fibrosis

Antimicrobial agent	MIC50 (g/mL)	MIC90 (g/mL)	MIC range (g/mL)	%S^a (CLSI/US-FDA)	%I or R^a (CLSI/US-FDA)
Ceftazidime-avibactam	4	64	0.5—> 128	71.9	28.1
Ceftazidime	64	>128	l6->128	0	l00
Aztreonam	64	>128	≤1-<128	3.1	96.9
Cefepime	>16	>16	8—> 16	0	l00
Meropenem	8	>16	≤0.12—> 16	34.3	65.7
Piperacillin/tazobactam	>128	>128	l6->128	3.1	96.9
Amikacin	32	>32	4->32	25.0	75.0
Gentamicin	>8	>8	2->8	12.5	87.5
Colistin	1	2	0.25-8	93.7	6.3
Levofloxacin	>4	>4	0.12—>4	21.9	78.1
Ciprofloxacin	>4	>4	0.06->4	25.0	75.0

Note: ^aUS-FDA breakpoints were applied for ceftazidime–avibactam, ceftazidime, and cefepime (≥8/≤16 µg/mL for S/R for all three drugs), and CLSI breakpoints were applied for the remaining drugs.

Abbreviations: FDA, Food and Drug Administration; CLSI, Clinical and Laboratory Standards Institute; R, resistant; I, intermediate; S, sensitive.

Figure 3 Characteristics of P. aeruginosa strain.

Notes: (A) Distribution of MDR, XDR, and PDR isolates found to be ceftazidime-avibactam sensitive versus resistant, P=0.0054. (B) CAZ–AVI susceptibility of P. aeruginosa isolates by morphotype, P=0.874.
Abbreviations: AVI, avibactam; CAZ, ceftazidime; MDR, multidrug-resistant; P. aeruginosa, Pseudomonas aeruginosa; PDR, pan-drug resistant; XDR, extensive drug resistant.

Figure 4 Ceftazidime–avibactam MBEC.

Notes: Dose-dependent response of ceftazidime–avibactam on eradication of a Pseudomonas aeruginosa biofilm generated from a representative strain with an MIC of 1 µg/mL to ceftazidime–avibactam. *P<0.05.
Abbreviation: MBEC, minimum biofilm eradication concentration.

Table 3 Expression analysis of OprD, AmpC, and efflux pumps from nine resistant isolates

	MIC	OprDloss^a	Fold change in gene expression compared to control:
			AmpC^b	MexCD-OprJ^b	MexXY-OprM^b	MexAB-OprM^b	MexEF-OprD^b
1	64	+	3702.7++	29.8++	0.9	1.9	0.03
2	64	+	2141++	1.9	1.7	1.5	0.06
3	64	+	1.9	2.8	0.64	0.87	0.05
4	16	+	25.4++	0.047	1.2	3.0	0.01
5	128	+	I8.I++	0.28	I.I	0.88	0.12
6	>128	+	677.8++	I4.9++	0.39	2.0	0.04
7	16	-	0.9	0.03	0.67	1.8	0.02
8	128	+	28.0^++	0.46	8.8^+	5.3^+	0.01
9	64	-	1.7	1.7	5.3^+	6.5^+	0.02

Notes: ^aOprD loss: +, protein not detected; -, protein was detected.

b AmpC and efflux pumps’ interpretation: ⁺⁺, ≥10-fold change = elevated expression; ⁺, >5-fold change but <10-fold change = moderate expression.

Table S1 Antimicrobial susceptibility of the 32 isolates

	CAZ	FEP	ATM	TZP	MEM	LVX	CIP	AMK	GEN	CST	CAZ-AVI
1	64	>16	32	>128	<0.12	1	0.5	32	>8	2	2
2	32	16	16	>128	0.5	1	0.5	16	8	2	1
3	128	>16	>128	>128	>16	>4	4	16	>8	1	8
4	32	>16	32	64	>16	4	2	16	4	0.5	2
5	128	>16	128	>128	4	>4	>4	>32	>8	2	8
6	16	16	16	128	2	>4	4	32	>8	2	1
7	64	>16	32	>128	0.5	4	2	16	4	2	2
8	16	16	16	64	0.25	>4	4	32	>8	2	2
9	16	8	16	128	8	>4	2	>32	>8	0.5	2
10	>128	>16	128	>128	4	>4	>4	>32	>8	1	4
11	>128	>16	128	128	0.25	4	1	32	>8	0.5	2
12	64	>16	64	>128	16	>4	>4	>32	>8	2	4
13	>128	>16	32	>128	<0.12	1	1	16	8	1	8
14	64	>16	32	>128	1	2	1	>32	>8	0.5	2
15	128	>16	64	>128	16	>4	4	>32	>8	4	4
16	16	8	8	16	0.5	4	2	32	>8	8	1
17	>128	>16	>128	>128	>16	>4	2	>32	>8	0.5	8
18	16	8	16	64	1	0.25	0.06	8	2	2	2
19	64	>16	64	64	2	2	1	32	>8	0.25	4
20	64	>16	>128	>128	8	>4	>4	4	2	1	2
21	64	>16	>128	32	8	>4	<4	16	8	0.5	2
22	16	>16	128	32	16	>4	4	32	>8	2	8
23	>128	>16	16	>128	16	>4	2	>32	>8	0.5	8
24	>128	>16	64	>128	8	4	4	32	>8	0.25	64
25	>128	>16	128	>128	>16	>4	>4	>32	>8	0.5	64
26	>128	>16	>128	>128	0.5	0.12	0.12	>32	>8	1	16
27	64	>16	128	32	>16	>4	>4	32	>8	2	64
28	>128	>16	128	>128	>16	>4	>4	32	>8	0.5	64
29	>128	>16	64	>128	16	>4	>4	>32	>8	1	16
30	>128	>16	>128	>128	8	>4	>4	>32	>8	0.5	>128
31	>128	>16	128	>128	>16	>4	>4	>32	>8	0.5	128
32	64	>16	16	64	4	>4	4	32	>8	0.25	16

Abbreviations: AMK, amikacin; ATM, aztreonam; CAZ–AVI, ceftazidime–avibactam; CAZ, ceftazidime; CIP, ciprofloxacin; CST, colistin; FEP, cefepime; GEN, gentamicin; LVX, levofloxacin; MEM, meropenem; TZP, piperacillin–tazobactam.

Figure S1 Outer membrane protein (OprD) expression.

Notes: Outer membrane protein (OprD) expression by Western blot of the nine ceftazidime–avibactam-resistant Pseudomonas aeruginosa strains. PAO1 was used as a positive control.