Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae 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 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