Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation
Authors: Yang Fengzhen , Zhao Qi , Wang Lipeng , Wu Jinying , Jiang Lihua , Sheng Li , Zhang Leyan , Xue Zhaoping , Yi Maoli 
Published: June 2022

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