SICs of CFZ and IMP Improve the Expression of Ampc and Improve Resistance to β-Lactam in EC Clinical Isolate
It was reported that cefoxitin and low concentration cefotaxime could improve AmpC expression [1]. To investigate whether other antibiotics can elicit the expression of AmpC, firstly, various antibiotics (including quinolones, β lactams and aminoglycosides) were tested for their minimum inhibitory concentration (MIC) against EC clinical isolate following the guideline outlined by the Clinical Laboratory Standard Institute (CLSI) [23]. The results are shown in Table S1. Later western blot assay was employed for determining whether or not the subinhibitory concentration (SIC, ≤1/4 MIC) of antibiotics induce AmpC expression. The findings (Fig. S1) demonstrate that CFZ, IMP, and cefoxitin have a strong induction impact on AmpC, whereas ceftriaxone, cefotaxime, ceftazidime, and cefepime have a modest induction effect. Other antibiotics, such as aminoglycosides and quinolones, showed no discernible effect on AmpC. Next, we explored the effect of induction of different concentrations CFZ and IMP on AmpC expression at mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and protein level by western blot, respectively. CFZ and IMP have a dosage impact on AmpC induction, according to our findings (Fig. 1A-C). Finally, the AmpC generated by CFZ and IMP was tested using a nitrocefin hydrolysis assay to see if it had good β-lactamase activity. The results show that CFZ and IMP increase AmpC β-lactamase activity in a dose-dependent manner when compared to the control group. (Fig. 1D).
To investigate the role of CFZ and IMP in resistance, the inhibition zone and MICs of aztreonam (ATM), ceftriaxone (CRO), ceftazidime (CAZ), piperacillin (PIP), piperacillin-tazobactam (TZP), and cefoperazone-sulbactam (SCF) against EC clinical isolate treated with or without SICs of CFZ and IMP were estimated by employing broth microdilution method and disk diffusion technique (Kirby-Bauer method) in accordance with the CLSI guideline [23], as indicated in Table 1 and Fig. S2A, inhibition zones of those antibiotics against EC treated with SICs of CFZ and IMP are decreased obviously compared with control and the MICs of TZP, PIP, CRO, SCF, CAZ, and ATM manifest a substantial increase by SICs of CFZ and IMP.
Table 1
MICs for the EC clinical isolate treated with CFZ and IMP
Antibiotics | MIC(µg/ml) |
control | CFZ(64µg/ml) | CFZ(256µg/ml) | IMP(0.0625µg/ml) | IMP(0.125µg/ml) |
PIP | 64 | 128 | 512 | 512 | 1024 |
TZP | 32 | 128 | 256 | 512 | 1024 |
ATM | 32 | 64 | 256 | 256 | 512 |
CRO | 16 | 32 | 64 | 256 | 512 |
CAZ | 16 | 64 | 128 | 128 | 256 |
SCF | 4 | 8 | 32 | 32 | 128 |
Abbreviations. MIC: minimum inhibitory concentration; CFZ: cefazolin; IMP: and imipenem. |
The Effects of Induction of CFZ and IMP on AmpC Expression and Resistance Were Abrogated in ΔnagZ
NagZ, existing in Gram-negative bacteria and involved in the peptidoglycan recycling pathway, is identified as an exo-N-acetyl-β-glucosaminidase. In some Gram-negative bacteria, NagZ inactivation has been reported to arrest and reverse the resistance to β-lactam antibiotics [24, 25]. As shown in Fig. S2A and Table 1, our data indicates the SICs of CFZ and IMP have the potential to improve the resistance of EC, so we speculated that NagZ may have an indispensable part in promoting resistance of EC. In an attempt to confirm the hypothesis, the knockout model of gene nagZ (ΔnagZ) was constructed by employing homologous recombination technology in EC clinical isolate (WT). As shown by RT-qPCR (Fig. 2A) and western blot (Fig. 2B), the nagZ gene was effectively knocked out. Next, we determined the effects of SICs of CFZ and IMP on the expression and activity of AmpC in ΔnagZ. The results, as shown in Fig. 2C-F, depict that the expression of AmpC was significantly downregulated, and the induction of AmpC by SICs of CFZ and IMP was completely abolished in ΔnagZ (Fig. 2C-E). At the same time, knocking down nagZ reduced AmpC's rising β-lactamase activity produced by CFZ and IMP SICs (Fig. 2F). Furthermore, antibiotic susceptibility tests revealed that EC resistance had been considerably reduced, while induction of CFZ and IMP had little impact on resistance in ΔnagZ (Table2 and Fig. S2B).
Table 2
The effect of nagZ on MICs for six anbiotics
Antibiotics | MIC(µg/ml) |
WT | ΔnagZ | ΔnagZ+CFZ (4 µg/ml) | ΔnagZ+IMP (0.0625µg/ml) | ΔnagZ+NagZ | ΔnagZ+NagZ+ CFZ(256µg/ml) | ΔnagZ+NagZ+ IMP(0.125µg/ml) |
PIP | 64 | 2 | 2 | 1 | 32 | 256 | 512 |
TZP | 32 | 2 | 1 | 0.5 | 32 | 128 | 512 |
ATM | 32 | 1 | 1 | 0.5 | 16 | 256 | 512 |
CRO | 16 | 0.5 | 0.5 | 0.25 | 32 | 64 | 1024 |
CAZ | 16 | 0.5 | 0.5 | 0.25 | 16 | 64 | 128 |
SCF | 4 | 0.25 | 0.125 | 0.0625 | 4 | 64 | 64 |
Abbreviations. ΔnagZ: nagZ-knockout Enterobacter cloacae; ΔnagZ+CFZ: ΔnagZ treated with CFZ; ΔnagZ+IMP: ΔnagZ treated with IMP; ΔnagZ+NagZ+CFZ: ΔnagZ+NagZ treated with CFZ; ΔnagZ+NagZ+IMP: ΔnagZ+NagZ treated with IMP. |
Ectopic Expression of Nagz Rescues Induction Effect of CFZ and IMP on AmpC Expression and Resistance In Δ nagz
To investigate whether NagZ complementation rescues the expression of ampC and enhances resistance in ΔnagZ treated with or without SICs of CFZ and IMP, nagZ coding sequence (CDS) was cloned into the vector of pBAD33cm-rp4 (pBAD33-nagZ, nagZ overexpression vector). Later, the pBAD33-nagZ and the vector of pBAD33cm-rp4 (pBAD33, as control vector) were transformed into ΔnagZ by electroporator. RT-qPCR and western blot analyses were employed for detecting the availability of the pBAD33-nagZ vector (Fig. 3A, B). Next, the ampC expression was ascertained using western blot and RT-qPCR, the outcome showed that ampC levels of mRNA (Fig. 3C) and protein (Fig. 3D-E) were rescued by NagZ complementation in ΔnagZ in response to SICs of CFZ and IMP. Furthermore, NagZ was investigated in terms of its influence on the AmpC β-lactamase activity, and the result indicates that reduced activity of β- lactamase resulting from the elimination of nagZ was reversed by NagZ complementing in ΔnagZ (Fig. 3F). Additional confirmation of the significance of NagZ in resistance of ΔnagZ was acquired by measuring the inhibition zones and MICs of TZP, PIP, CRO, ATM, CAZ, and SCF. The findings demonstrate that NagZ overexpression may greatly reduce the inhibition zone and that SICs of CFZ or IMP can further reduce the inhibition zone (Fig. S2C). Consistent with the inhibition zone, NagZ complementation and SICs of CFZ or IMP can increase the MICs obviously (Table 2). To put it briefly, ampC expression and its β-lactamase activity are promoted by NagZ, which thereby enhances resistance in ΔnagZ.
CFZ and IMP Promote AmpC Expression Through the NagZ-AmpR-AmpC Pathway
Peptides from peptidoglycan degradation are transported by AmpG permease into the cytoplasm. In the cytoplasm, GlcNAc-1,6‐anhydroMurNAc‐peptides detach GlcNAc moiety with the aid of NagZ and forms 1,6‐anhydroMurNAc‐peptides (anhMurNAc) [24, 26]. Under normal physiological growth, AmpD cleaves anhMurNAc to generate free peptides and then synthesizes UDP-pentapeptides, which suppresses AmpR activity and represses AmpC transcription [27–29]. However, in the presence of inducers (such as β-lactams), AmpD cannot cleave the high amounts of anhMurNAc effectively. The accumulating anhMurNAc activates AmpR and increases AmpC transcription, which is also the main mechanistic step responsible for developing resistance to most β -lactams in Pseudomonas aeruginosa [30, 31]. Besides, several studies have shown that AmpR regulates the expression of a multitude of genes and is thus a global transcription factor (the genes regulated by AmpR include oxyR, rsmA, rpoS, phoP, and grpE) in Pseudomonas aeruginosa [30, 32]. Therefore, we hypothesize that, like Pseudomonas aeruginosa, there is a pathway in Enterobacter cloacae and that the induction of AmpC by SICs of CFZ and IMP is dependent on AmpR. To confirm our hypothesis, Pseudomonas aeruginosa and Enterobacter cloacae were both analyzed for their NagZ and AmpR protein sequence conservations. The AmpR sequences of the two species were identified (Fig. 4A) and the conservation of the NagZ sequence was as high as 67% (Fig. 4B). In addition, a high homology is additionally observed in the -35bp-0bp region (generally considered as transcriptional parameter zone of binding) for ampC among Pseudomonas aeruginosa and Enterobacter cloacae (Fig. 4C).
In an attempt to confirm if the induction of CFZ and IMP to AmpC is dependent on NagZ-mediated AmpR activation, we detected the effect of CFZ and IMP upon the expression of AmpR target genes in wild type EC and ΔnagZ. The outcome implies that CFZ and IMP are able to promote the AmpR target genes expression (for instance oxyR, rsmA, grpE, rpoS, and phoP) in wild type EC (Fig. 5A), while in the ΔnagZ strain, CFZ and IMP did not affect the expressions of AmpR target genes (Fig. 5B). In conclusion, these findings demonstrate that CFZ and IMP increase AmpC expression and resistance in Enterobacter cloacae in a NagZ-dependent manner.