<abstract xmlns="http://www.w3.org/1999/xhtml"><p>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 <italic>Enterobacteriaceae</italic> are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive <italic>Enterobacteriaceae</italic> 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 <italic>Klebsiella pneumoniae</italic> strains harbored <italic>bla</italic><sub>SHV-1</sub> only, and four harbored <italic>bla</italic><sub>SHV-1</sub> and <italic>bla</italic><sub>TEM-1B</sub>. Two <italic>Escherichia coli</italic> strains carried <italic>bla</italic><sub>TEM-1B</sub> only, while two <italic>Klebsiella oxytoca</italic> isolates harbored <italic>bla</italic><sub>OXY-1-3</sub> and <italic>bla</italic><sub>OXY-1-1</sub>, 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 <italic>K. pneumoniae</italic>. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one <italic>K. oxytoca</italic> strain, and no OmpK gene was found in the other. No Omp mutations were found in <italic>E. coli</italic> 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 <italic>Enterobacteriaceae</italic>.</p>
<p><table-wrap id="j_pjm-2022-023_tab_001" position="float">
<label></label><caption><p>Antibiotic susceptibility tests</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th align="left" rowspan="2">Antibiotics</th>
<th align="center" rowspan="2">Breakpoint, (μg/ml)</th>
<th align="center" colspan="7"><italic>Klebsiella pneumoniae</italic><hr/></th>
<th align="center" colspan="2"><italic>Escherichia cou</italic><hr/></th>
<th align="center" colspan="2">Klebriehd axyoca<hr/></th>
</tr>
<tr>
<th align="center">E1</th>
<th align="center">E3</th>
<th align="center">E4</th>
<th align="center">E7</th>
<th align="center">E9</th>
<th align="center">E10</th>
<th align="center">E11</th>
<th align="center">E6</th>
<th align="center">E8</th>
<th align="center">E2</th>
<th align="center">E5</th>
</tr></thead>
<tbody>
<tr><td align="left"><bold>CRO</bold></td>
<td align="center"><bold>≤1≥4</bold></td>
<td align="center"><bold>≤0.5</bold></td>
<td align="center"><bold>≤0.5</bold></td>
<td align="center"><bold>≤0.5</bold></td>
<td align="center"><bold>≤0.5 1</bold></td>
<td align="center"></td>
<td align="center"><bold>≤0.5</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>≤0.5</bold></td>
<td align="center"><bold>≤0.5</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>1</bold></td>
</tr>
<tr><td align="left"><bold>CAZ</bold></td>
<td align="center"><bold>4 ≥16</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>4</bold></td>
<td align="center"><bold>4</bold></td>
<td align="center"><bold>4</bold></td>
<td align="center"><bold>4</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>4</bold></td>
<td align="center"><bold>1 1</bold></td>
<td align="center"></td>
</tr>
<tr><td align="left"><bold>FEP</bold></td>
<td align="center"><bold>≤2 216 1</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>0.5</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>1 1</bold></td>
<td align="center"></td>
</tr>
<tr><td align="left"><bold>AMC</bold></td>
<td align="center"><bold>≤8 ≥32</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>≥128</bold></td>
</tr>
<tr><td align="left"><bold>CSL</bold></td>
<td align="center"><bold>≤16 ≥64</bold></td>
<td align="center"><bold>64</bold></td>
<td align="center"><bold>64</bold></td>
<td align="center"><bold>64</bold></td>
<td align="center"><bold>64</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>128</bold></td>
<td align="center"><bold>≥128</bold></td>
<td align="center"><bold>64</bold></td>
<td align="center"><bold>128</bold></td>
<td align="center"><bold>128</bold></td>
<td align="center"><bold>≥128</bold></td>
</tr>
<tr><td align="left"><bold>TZP</bold></td>
<td align="center"><bold>≤16 ≥128</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>2256</bold></td>
<td align="center"><bold>2256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
<td align="center"><bold>≥256</bold></td>
</tr>
<tr><td align="left"><bold>FPT</bold></td>
<td align="center"><bold>≤2 ≥16</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>0.5</bold></td>
<td align="center"><bold>0.06</bold></td>
<td align="center"><bold>0.125</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>0.125</bold></td>
<td align="center"><bold>0.25</bold></td>
</tr>
<tr><td align="left"><bold>FPZ</bold></td>
<td align="center"><bold>≤2 216</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>0.06</bold></td>
<td align="center"><bold>0.125</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>0.25 1</bold></td>
<td align="center"></td>
<td align="center"><bold>0.125</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>0.125</bold></td>
<td align="center"><bold>0.125</bold></td>
</tr>
<tr><td align="left"><bold>CZA</bold></td>
<td align="center"><bold>≤8 216 1</bold></td>
<td align="center"></td>
<td align="center"><bold>0.5</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>0.25</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>0.5</bold></td>
<td align="center"><bold>0.5</bold></td>
<td align="center"><bold>0.5</bold></td>
<td align="center"><bold>0.25</bold></td>
</tr>
<tr><td align="left"><bold>CZT</bold></td>
<td align="center"><bold>≤2 28</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>0.5 1</bold></td>
<td align="center"></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>2 1</bold></td>
<td align="center"></td>
<td align="center"><bold>1</bold></td>
<td align="center"><bold>2</bold></td>
<td align="center"><bold>2</bold></td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn-group>
<fn id="j_pjm-2022-023_tfn_101"><p>CROceftriaxone, CAZceftazidime, FEPcefepime, AMC:amoxicillin clavulanic-acid, CSLcefoperazone/sulbactam, TZP:piperadllin/tazobactam, FPT:cefepime tazobactam, FPZ:cefepime/zidebactam, CZA:ceftazidime/avibactam, CZTceftolozane/tazobactam</p></fn>
</fn-group>
</table-wrap-foot>
</table-wrap>
</p>
<p><table-wrap id="j_pjm-2022-023_tab_002" position="float">
<label></label><caption><p>Gene sequencing results</p></caption>
<table frame="hsides" rules="groups">
<thead><tr>
<th align="left">Number</th>
<th align="center">Strain</th>
<th align="center">ST</th>
<th align="center">p-Lactamase gene</th>
<th align="center">Promoter sequence mutation</th>
<th align="center">Omp mutation</th>
</tr></thead>
<tbody>
<tr><td align="left"><bold>El</bold></td>
<td align="center"><bold>Kpn</bold></td>
<td align="center"><bold>45</bold></td>
<td align="center"><bold>blaSHV-1, blaTEM-lB</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36, OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>E3</bold></td>
<td align="center"><bold>Kpn</bold></td>
<td align="center"><bold>45</bold></td>
<td align="center"><bold>blaSHV-1, blaTEM-lB</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36. OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>E4</bold></td>
<td align="center"><bold>Kpn</bold></td>
<td align="center"><bold>2854</bold></td>
<td align="center"><bold>blaSHV-1</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36, OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>E7</bold></td>
<td align="center"><bold>Kpn</bold></td>
<td align="center"><bold>2358</bold></td>
<td align="center"><bold>blaSHV-1 - blaTEM-lB</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36, OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>E9</bold></td>
<td align="center"><bold>Kpn</bold></td>
<td align="center"><bold>2358</bold></td>
<td align="center"><bold>blaSHV-1. blaTEM-lB</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36. OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>E10</bold></td>
<td align="center"><bold>Kpn 18</bold></td>
<td align="center"><bold>9</bold></td>
<td align="center"><bold>blaSHV-1</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36. OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>Ell</bold></td>
<td align="center"><bold>Kpn</bold></td>
<td align="center"><bold>45</bold></td>
<td align="center"><bold>blaSHV-1</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36, OmpK3 7</bold></td>
</tr>
<tr><td align="left"><bold>E6</bold></td>
<td align="center"><bold>Eco</bold></td>
<td align="center"><bold>88</bold></td>
<td align="center"><bold>blaTEM-lB</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>none</bold></td>
</tr>
<tr><td align="left"><bold>ES</bold></td>
<td align="center"><bold>Eco</bold></td>
<td align="center"><bold>409</bold></td>
<td align="center"><bold>blaTEM-1B</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>none</bold></td>
</tr>
<tr><td align="left"><bold>E2</bold></td>
<td align="center"><bold>Kox</bold></td>
<td align="center"><bold>194</bold></td>
<td align="center"><bold>blaOXY-1-3</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>OmpK36 mutations. OmpK35 and OmpK 37 deficiency</bold></td>
</tr>
<tr><td align="left"><bold>E5</bold></td>
<td align="center"><bold>Kox 11</bold></td>
<td align="center"></td>
<td align="center"><bold>blaOXY-1-1</bold></td>
<td align="center"><bold>none</bold></td>
<td align="center"><bold>no OmpK (OmpK3 5, OmpK36 and OmpK37) gene found</bold></td>
</tr>
</tbody>
</table>
</table-wrap>
</p>
</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 blaSHV-1 only, and four harbored blaSHV-1 and blaTEM-1B. Two Escherichia coli strains carried blaTEM-1B only, while two Klebsiella oxytoca isolates harbored blaOXY-1-3 and blaOXY-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

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

Polish Journal of Microbiology

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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

Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial...

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

Article Category: Original Paper

Publicado en línea: 19 jun 2022

Páginas: 251 - 256

Recibido: 08 mar 2022

Aceptado: 30 abr 2022

DOI: https://doi.org/10.33073/pjm-2022-023

Palabras clave
cefoperazone/sulbactam, piperacillin/tazobactam, TEM, SHV, OXY

© 2022 Fengzhen Yang et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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

Article Category: Original Paper

Publicado en línea: 19 jun 2022

Páginas: 251 - 256

Recibido: 08 mar 2022

Aceptado: 30 abr 2022

DOI: https://doi.org/10.33073/pjm-2022-023

Palabras clavecefoperazone/sulbactam, piperacillin/tazobactam, TEM, SHV, OXY

© 2022 Fengzhen Yang et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Palabras clave
cefoperazone/sulbactam, piperacillin/tazobactam, TEM, SHV, OXY