Molecular Mechanisms Mediating Ceftazidime/Avibactam Resistance Amongst Carbapenem-Resistant Klebsiella pneumoniae Isolates from Cancer Patients

Figures & data

Table 1 Antimicrobial Susceptibility Profile of the Test CRKP Isolates

Antimicrobials	CRKP Isolates (n= 47)
	Susceptible		Intermediate		Resistant
	Number	%	Number	%	Number	%
Ceftazidime/avibactam	35	74.5	0	0.00	12	25.5
Amikacin	14	29.8	0	0.00	33	70.2
Levofloxacin	9	19.1	0	0.00	38	80.9
Cefepime	8	17.0	1	2.1	38	80.9
Gentamicin	6	12.8	1	2.1	40	85.1
Piperacillin/tazobactam	2	4.3	1	2.1	44	93.6
Ciprofloxacin	2	4.3	3	6.4	42	89.3
Ceftriaxone	1	2.1	0	0.00	46	97.9
Sulfamethoxazole/trimethoprim	1	2.1	0	0.00	46	97.9
Ceftazidime	0	0.0	0	0.00	47	100.0
Cefotaxime	0	0.0	0	0.00	47	100.0
Ampicillin/sulbactam	0	0.0	0	0.00	47	100.0
Aztreonam	0	0.0	0	0.00	47	100.0

Abbreviation: CRKP, carbapenem-resistant Klebsiella pneumoniae.

Figure 1 Antibiotic resistance profile of the test Klebsiella pneumoniae isolates. Out of 134 Klebsiella pneumoniae isolates, 35.07% and 30.6% demonstrated resistance to imipenem and meropenem, respectively. High resistance profiles were observed for most of the investigated β-lactams, including the third generation cephalosporins CTX (98.51%), CAZ (96.27%), and CRO (94.03%). Resistance rates to fluoroquinolones were recorded at 87.31% for CIP and 85.07% for LEV.

Abbreviations: CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; SAM, ampicillin/sulbactam; ATM, aztreonam; TZP, piperacillin/tazobactam; CIP, ciprofloxacin; LEV, levofloxacin; CN, gentamicin; FEP, cefepime; SXT, sulfamethoxazole/trimethoprim; AK, amikacin; IMP, imipenem; MEM, meropenem; CZA, ceftazidime/avibactam.

Table 2 Risk Factors and Outcome of Ceftazidime/Avibactam Resistance Amongst the Study Cohort

Risk Factors	CZA-S/CRKP (n= 35)	CZA-R/CRKP (n= 12)	OR (95% CI)	P-value
Age, years (± SD)^a	45.5 ± 2.2 (24–76)	65 ± 2.32 (51–81)	–	0.12
Gender
Male	20 (57.1)	9 (75.0)	2.25 (0.52–9.77)	0.27
Female	15 (42.8)	3 (25.0)
Underlying malignancy
Solid organ tumors	26 (74.29)	8 (66.67)	1.44 (0.34–5.97)	0.61
Hematologic	9 (25.71)	4 (33.33)
Comorbidity
Pulmonary disease	10 (28.57)	1 (8.33)	0.22 (0.02–2.0)	0.15
Chronic liver disease	6 (17.14)	5 (41.67)	3.45 (0.81–14.65)	0.08
Diabetes mellitus	4 (11.43)	1 (8.33)	0.71 (0.07–7.01)	0.76
Cardiac disease	1 (2.86)	0 (0.0)	1.35 (1.14–1.60)	0.55
Chronic kidney disease	1 (2.86)	3 (25.0)	11.33 (1.04–122.38)	0.018*
IUC	16 (45.71)	7 (58.33)	1.66 (0.44–6.26)	0.45
PVC	13 (37.14)	5 (41.67)	1.21 (0.31–4.60)	0.78
CVC	12 (34.29)	4 (33.33)	0.95 (0.23–3.84)	0.95
MV	1 (2.9)	5 (41.7)	24.29 (2.45–241.26)	0.003*
Surgical intervention	5 (14.29)	0 (0.0)	1.4 (1.15–1.69)	0.17
Prior carbapenem use	10 (28.57)	5 (41.67)	1.78 (0.45–6.97)	0.40
ICU admission	6 (17.14)	7 (58.33)	6.76 (1.59–28.72)	0.006*
LOS	2 (5.71)	7 (58.33)	23.1 (3.7–144.21)	0.001*
Neutropenia	21 (60.0)	8 (66.67)	1.33 (0.33–5.28)	0.68
30-day mortality	0 (0.0)	8 (66.67)	0.10 (0.04–0.26)	0.001*

Notes: Data are expressed as numbers and percentages unless otherwise indicated; ^aSignificance was tested using the independent samples t-test; *P < 0.05 (statistically significant).

Abbreviations: CZA-S/CRKP, ceftazidime/avibactam-susceptible/carbapenem-resistant Klebsiella pneumoniae; CZA-R/CRKP, ceftazidime/avibactam-resistant/carbapenem-resistant Klebsiella pneumoniae; SD, standard deviation; OR, odds ratio; CI, confidence interval; IUC, indwelling urinary catheter; PVC, peripheral venous catheter; CVC, central venous catheter; MV, mechanical ventilation; ICU, intensive care unit; LOS, length of hospital stay.

Table 3 Multivariate Analysis for Predictors of Ceftazidime/Avibactam Resistance Amongst the Study Cohort

Risk Factors	CZA-S/CRKP (n= 35)	CZA-R/CRKP (n= 12)	OR (95% CI)	P-value
Chronic kidney disease	1 (2.86)	3 (25.0)	0.24 (0.006–10.09)	0.46
MV	1 (2.9)	5 (41.7)	0.08 (0.004–1.39)	0.08
ICU admission	6 (17.14)	7 (58.33)	0.84 (0.07–10.85)	0.89
LOS	2 (5.71)	7 (58.33)	0.10 (0.01–0.86)	0.03*

Notes: Data are expressed as numbers and percentages; *P < 0.05 (statistically significant).

Abbreviations: CZA-S/CRKP, ceftazidime/avibactam-susceptible/carbapenem-resistant Klebsiella pneumoniae; CZA-R/CRKP, ceftazidime/avibactam-resistant/carbapenem-resistant Klebsiella pneumoniae; OR, odds ratio; CI, confidence interval; MV, mechanical ventilation; ICU, intensive care unit; LOS, length of hospital stay.

Table 4 Phenotypic and Genotypic Characterization of the Investigated CRKP Isolates (N= 47)

Strain no.	Source	Carbapenemase Type	CZA Zone Diameter (mm)	MIC (µg/mL)	CLSI Criteria	ompK35	ompK36
1.	Sputum	blaKPC	23	4	S	P	P
2.	Sputum	blaKPC	28	0.5	S	P	P
3.	Blood	blaKPC	25	1	S	P	P
4.	Sputum	blaKPC	23	1	S	P	P
5.	Blood	blaKPC	31	0.25	S	P	P
6.	Blood	blaKPC	22	0.5	S	N	N
7.	Urine	blaOXA-48	18	128	R	N	N
8.	Sputum	blaKPC/ blaIMP	22	0.5	S	P	P
9.	Blood	blaKPC	26	0.25	S	P	P
10.	Urine	blaOXA-48	28	1	S	P	P
11.	Blood	blaNDM-1	24	4	S	P	P
12.	ETA	blaOXA-48	25	4	S	P	P
13.	Blood	blaKPC	25	0.25	S	P	P
14.	Blood	blaKPC	28	0.25	S	P	P
15.	Blood	blaIMP	12	64	R	P	P
16.	Blood	blaNDM-1	27	0.5	S	P	P
17.	Sputum	blaOXA-48	31	0.5	S	P	P
18.	Urine	blaOXA-48	27	0.25	S	P	P
19.	Blood	blaKPC	23	1	S	P	P
20.	Blood	blaOXA-48/blaNDM-1	25	1	S	P	P
21.	Blood	blaKPC	13	128	R	N	N
22.	Blood	blaNDM-1	30	1	S	P	P
23.	Urine	blaKPC	24	2	S	P	P
24.	Blood	blaOXA-48/blaNDM-1	28	0.25	S	P	P
25.	Blood	blaOXA-48	31	0.25	S	P	P
26.	Blood	blaOXA-48/blaNDM-1	14	64	R	N	N
27.	Blood	blaOXA-48/blaNDM-1	27	0.5	S	P	P
28.	W. swab	blaOXA-48	33	0.25	S	P	P
29.	Blood	blaKPC	15	32	R	N	N
30.	Blood	blaKPC	11	128	R	N	N
31.	Blood	blaOXA-48	19	16	R	P	P
32.	Urine	blaKPC	26	4	S	P	P
33.	Urine	blaNDM-1	11	64	R	P	P
34.	Urine	blaKPC/blaNDM-1	22	1	S	N	N
35.	Blood	blaOXA-48/blaNDM-1	27	2	S	P	P
36.	W. swab	blaOXA-48	33	4	S	P	P
37.	Blood	blaKPC/blaIMP	25	2	S	P	P
38.	Sputum	blaNDM-1	13	64	R	P	P
39.	Blood	blaKPC	14	64	R	N	N
40.	Blood	blaKPC	11	64	R	P	P
41.	W. swab	blaOXA-48	30	0.25	S	P	P
42.	ETA	blaKPC	19	32	R	N	N
43.	Sputum	blaKPC/blaNDM-1	22	0.5	S	N	N
44.	Sputum	blaOXA-48/blaNDM-1	24	0.5	S	P	P
45.	Sputum	blaKPC	27	1	S	P	P
46.	ETA	blaKPC	25	4	S	P	+
47.	W. swab	blaOXA-48	31	2	S	P	P

Note: Inhibition zone diameters and MIC breakpoints of CZA were interpreted in accordance with the guidelines of the CLSI, 2020.

Abbreviations: CRKP, carbapenem-resistant Klebsiella pneumoniae; CZA, ceftazidime/avibactam; MIC, minimum inhibitory concentration; CLSI, Clinical and Laboratory Standards Institute; W. swab, wound swab; ETA, endotracheal aspirate; KPC, Klebsiella pneumoniae carbapenemase; NDM, New Delhi metallo-β-lactamase; OXA-48, oxacillinase-48; IMP, imipenemase; OmpK35, outer membrane porin of Klebsiella pneumoniae 35; OmpK36, outer membrane porin of Klebsiella pneumoniae 36; S, susceptible; R, resistant; P, positive; N, negative.

Figure 2 (A and B) Gel electrophoresis of multiplex polymerase chain reaction (PCR) amplified products for carbapenemase-encoding and outer membrane porin genes amongst ceftazidime/avibactam-resistant isolates. (A) bla_NDM−1 (82 bp), bla_IMP (120 bp), bla_OXA-48 (177 bp), and bla_KPC (798 bp). Lane M: DNA Ladder (1000 bp); Lane 1: positive result for both bla_OXA-48 and bla_NDM−1; Lanes 2–4: positive result for bla_NDM-1; Lanes 5–10: positive result for bla_KPC; Lane 8: positive result for both bla_KPC and bla_NDM−1; Lane 11: positive result for both bla_KPC and bla_IMP; and Lane 12: negative control. (B) ompK35 (778 bp), and ompK36 (588 bp). Lane M: DNA Ladder (1000 bp); Lanes 1, 2, 3, 5, and 7: negative result for both ompK35 and ompK36; Lanes 4 and 6: positive result for both ompK35, and ompK36; Lane 8: negative control for both ompK35 and ompK36; Lane 9: Klebsiella pneumoniae (ATCC 13883) positive control that harbors both ompK35, and ompK36.