Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation

Abstract

Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial activity. CSL and TZP-nonsusceptible Enterobacteriaceae are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive Enterobacteriaceae strains, which were resistant to CSL and TZP yet susceptible to CRO, were collected from January to December 2020. Antibiotic susceptibility tests and whole-genome sequencing were conducted to elucidate the mechanism for this rare phenotype. Antibiotic susceptibility tests showed that all isolates were amoxicillin/clavulanic-acid resistant and sensitive to ceftazidime, cefepime, cefepime/tazobactam, cefepime/zidebactam, ceftazidime/avibactam, and ceftolozane/tazobactam. Whole-genome sequencing revealed three of seven Klebsiella pneumoniae strains harbored bla _SHV-1 only, and four harbored bla _SHV-1 and bla _TEM-1B. Two Escherichia coli strains carried bla _TEM-1B only, while two Klebsiella oxytoca isolates harbored bla _OXY-1-3 and bla _OXY-1-1, respectively. No mutation in the β-lactamase gene and promoter sequence was found. Outer membrane protein (Omp) gene detection revealed that numerous missense mutations of OmpK36 and OmpK37 were found in all strains of K. pneumoniae. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one K. oxytoca strain, and no OmpK gene was found in the other. No Omp mutations were found in E. coli isolates. These results indicated that narrow spectrum β-lactamases, TEM-1, SHV-1, and OXY-1, alone or in combination with Omp mutation, contributed to the resistance to CSL and TZP in CRO-susceptible Enterobacteriaceae.

Antibiotic susceptibility tests

Antibiotics	Breakpoint, (μg/ml)	Klebsiella pneumoniae							Escherichia cou		Klebriehd axyoca
		E1	E3	E4	E7	E9	E10	E11	E6	E8	E2	E5
CRO	≤1≥4	≤0.5	≤0.5	≤0.5	≤0.5 1		≤0.5	1	≤0.5	≤0.5	1	1
CAZ	4 ≥16	1	2	1	4	4	4	4	2	4	1 1
FEP	≤2 216 1	1		0.25	1	2	2	2	0.5	2	1 1
AMC	≤8 ≥32	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128	≥128
CSL	≤16 ≥64	64	64	64	64	≥128	128	≥128	64	128	128	≥128
TZP	≤16 ≥128	≥256	≥256	≥256	≥256	2256	2256	≥256	≥256	≥256	≥256	≥256
FPT	≤2 ≥16	1	0.5	0.06	0.125	2	1	2	0.25	1	0.125	0.25
FPZ	≤2 216	0.25	0.25	0.06	0.125	0.25	0.25 1		0.125	0.25	0.125	0.125
CZA	≤8 216 1		0.5	0.25	0.25	1	0.25	1	0.5	0.5	0.5	0.25
CZT	≤2 28	2	1	0.5 1		2	2	2 1		1	2	2

CROceftriaxone, CAZceftazidime, FEPcefepime, AMC:amoxicillin clavulanic-acid, CSLcefoperazone/sulbactam, TZP:piperadllin/tazobactam, FPT:cefepime tazobactam, FPZ:cefepime/zidebactam, CZA:ceftazidime/avibactam, CZTceftolozane/tazobactam

Gene sequencing results

Number	Strain	ST	p-Lactamase gene	Promoter sequence mutation	Omp mutation
El	Kpn	45	blaSHV-1, blaTEM-lB	none	OmpK36, OmpK3 7
E3	Kpn	45	blaSHV-1, blaTEM-lB	none	OmpK36. OmpK3 7
E4	Kpn	2854	blaSHV-1	none	OmpK36, OmpK3 7
E7	Kpn	2358	blaSHV-1 - blaTEM-lB	none	OmpK36, OmpK3 7
E9	Kpn	2358	blaSHV-1. blaTEM-lB	none	OmpK36. OmpK3 7
E10	Kpn 18	9	blaSHV-1	none	OmpK36. OmpK3 7
Ell	Kpn	45	blaSHV-1	none	OmpK36, OmpK3 7
E6	Eco	88	blaTEM-lB	none	none
ES	Eco	409	blaTEM-1B	none	none
E2	Kox	194	blaOXY-1-3	none	OmpK36 mutations. OmpK35 and OmpK 37 deficiency
E5	Kox 11		blaOXY-1-1	none	no OmpK (OmpK3 5, OmpK36 and OmpK37) gene found