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ORIGINAL RESEARCH

The resistance mechanism of Escherichia coli

induced by ampicillin in laboratory
This article was published in the following Dove Press journal:
Infection and Drug Resistance

Mengchen Li 1 Background: Multi-drug-resistant Escherichia coli poses a great threat to human health,
Qiaoli Liu 1 especially resistant to ampicillin (AMP), but the mechanism of drug resistance is not very clear.
Yanli Teng 1 Purpose: To understand the mechanism of resistance of E. coli to beta-lactam antibiotics by
Liuyang Ou 1 inducing drug resistance of sensitive bacteria in laboratory.
Methods: Clinical sensitive E. coli strain was induced into resistance strain by 1/2 minimum
Yuanlin Xi 1
inhibitive concentration (MIC) induced trails of AMP. The drug resistance spectrum was
Shuaiyin Chen 1, *
measured by modified K-B susceptibility test. Whole-genome sequencing analysis was used
Guangcai Duan 1,2, *
to analyze primary sensitive strain, and resequencing was used to analyze induced strains.
1
Department of Epidemiology, College of Protein tertiary structure encoded by the gene containing single nucleotide polymorphism
Public Health, Zhengzhou University,
Zhengzhou, Henan, People’s Republic of
(SNP) was analyzed by bioinformatics.
China; 2Henan Collaborative Innovation Results: After 315 hrs induced, the MIC value of E. coli 15743 reached to 256 µg/mL, 64
Center of Molecular Diagnosis and times higher than that of the sensitive bacteria. During the induction process, the bacterial
Laboratory Medicine, Xinxiang Medical
College, Xinxiang, Henan, People’s resistance process is divided into two stages. The rate of drug resistance occurs rapidly
Republic of China before reaching the critical concentration of 32 µg/mL, and then the resistance rate slows
down. Sequencing of the genome of resistant strain showed that E. coli 15743 drug-resistant
*These authors contributed equally to
this work strain with the MIC values of 32 and 256 µg/mL contained four and eight non-synonymous
SNPs, respectively. These non-synonymous SNPs were distributed in the genes of frdD, ftsI,
acrB, OmpD, marR, VgrG, and envZ.
Conclusion: These studies will improve our understanding of the molecular mechanism of
AMP resistance of E. coli, and may provide the basis for prevention and control of multi-
drug-resistant bacteria and generation of new antibiotics to treat E. coli infection.
Keywords: Escherichia coli, ampicillin, drug resistance

Introduction
Pathogenic Escherichia coli often causes diarrhea, sepsis, and other clinical symp-
toms, and is still one of the main intestinal pathogens affecting human and animal
health. Ampicillin (AMP), a semi-synthetic β-lactam antibiotics, is widely used to
treat of human and livestock E. coli infection, but recently its resistance rate has
increased.1–3 AMP works on the active replicating stage of bacteria, inhibiting the
synthesis of bacterial cell wall. Bacteria often resist such an antibiotics in the
Correspondence: Shuaiyin Chen;
Guangcai Duan following ways: encodes β-lactamase, changes the target protein in cell wall,
Department of Epidemiology, College of
Public Health, Zhengzhou University, reduces the permeability of outer membrane, and increases the expression of drug
No.100 Kexue Avenue, Zhengzhou, efflux pump. Antibacterial drugs are used by animals and then spread to the
Henan 450001, People’s Republic of China
Tel +86 1 352 340 8394 environment via excreta, which not only makes the environment polluted, but
Fax +86 6 778 1453 also brings great harm to human health and the sustainable development of the
Email sychen@zzu.edu.cn;
gcduan@yeah.net breeding industry.4,5

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Whole-genome sequencing (WGS) has been shown to M-H liquid medium for amplification of bacteria. The
guide the prevention and control of bacterial resistance.6 above bacteria solution was cultured in M-H liquid med-
Single nucleotide polymorphism (SNP) mainly refers to ium containing 1/2MIC AMP, respectively, and the con-
the DNA sequence polymorphism caused by the variation centration of AMP was continuously increased during the
of a single nucleotide at the genomic level, and the rese- subculture process. When the concentration of antibiotics
quencing analysis to screen the different SNPs can more reached 16 μg/mL, 8 μg/mL was increased each time, and
directly study drug resistance. We simulated the process of each concentration was subcultured twice. When the value
clinical antibiotics in organisms by using the method of of the MIC change of a drug was greater than or equal to
AMP laboratory induction, and explored the relationship four times MIC before and after induction, it was consid-
between the degree of drug resistance and the mutation ered that the MIC change after induction had significant
site. Screening for non-synonymous single nucleotide significance.10 The culture medium of M-H broth without
polymorphism (non-SNP) between drug-resistant and sus- antibiotics was used as the control during the whole
ceptible strains to understand the role of non-SNP in drug- process.
resistant strains. The purpose of this study is to understand Multilocus sequence typing (MLST) of E. coli strains
the law and mechanism of drug resistance of E. coli, were classified by seven pairs of housekeeping genes con-
provide new targets for the development of new antibio- taining adk, fumC, gyrB, icd, mdh, purA, and recA.
tics, make the rational use of antibiotics, and solve the
multiple occurrence and treatment of multi-drug resistance Susceptibility testing
of E. coli in clinical practice. Kirby Bauer paper diffusion method was used to screen the
E. coli which was sensitive to eight kinds of antibiotics,
including aminoglycosides, penicillins, cephalosporins, tet-
Materials and methods
racycline, β-lactamase inhibitors, carbamates, sulfonamides,
Bacterial isolates and reagent and quinolones. The induced strains were repeated using
The E. coli strain used in this study (E. coli 15743) was
drug sensitivity test. The data interpretation was performed
isolated from a stool specimen from a patient at a hospital
in accordance with the Clinical and Laboratory Standards
in Suixian, Henan Province, China, in 2015. Characterization
Institute 2016 guidelines.11
of this strain by Kirby Bauer (K-B) paper diffusion method
First, we induced E. coli resistance to AMP, by culturing
showed that the strain was sensitive to eight classes of 20
E. coli with gradually increase the concentration of AMP
antibiotics. E. coli ATCC 25922 was used as a control for our
(2, 4, 8, 16, 32, 64, 128, and 256 μg/mL). After we obtained
study.
resistance strain, we compared the resistance spectrum of 20
M-H broth medium and M-H solid medium (Oxoid
antibiotics between the induced strain (E. coli 15743-256,
company, UK), Pharmaceutical sensitive paper (Hangzhou
induced at 256 μg/mL) and the original strain (E. coli
Binhe microbial company, Hangzhou, China), AMP stan-
15743) by performing drug sensitivity tests. The bacterial
dard products (Chinese drug identification Institute, Beijing,
suspension was spread onto an agar plate, with a small
China), DNA extraction kit (Shanghai Laifeng Biotech
circular pieces paper containing different antibiotics, and
company, Shanghai, China). Illumina Hiseq was done at
cultured at 37°C for 16–20 hrs. Antimicrobial ring diameter
Shanghai Lingen Biotechnology Co., Ltd.
was measured.
The E. coli used in the experiment was specifically
isolated for this study. The study was approved by the
WGS and resequencing analysis
Life Science Ethics Committee of Zhengzhou University,
The strains at MIC values of 32 and 256 were named as
and patient also signed written informed consent.
E. coli 15743-32 and E. coli 15743-256, respectively.
Whole-genome analysis was performed on the primary
Induction process sensitive strains, and resequencing was performed on
Minimum inhibitory concentration (MIC) was determined induced resistant strains. The results of resequencing
by microbroth dilution method.7–9 The strain of E. coli were compared with those of the original map.
(isolated from clinical and have MIC value) that is sensi- Screening non-SNPs that may affect protein function.
tive to AMP was cultured in MH solid medium, 37°C Sequencing was performed by Shanghai ling’en
culture after 18–24 hrs, pick a single colony in 8 mL Biotechnology Co. Ltd. (Shanghai, China). Illumina

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Hiseq combined with third-generation sequencing technol- Statistical analysis

ogy was used to complete genomic sequencing of the SPSS17.0 was used for simple linear regression analysis
strains in this project. and the regression equation was tested. Size of a test was
0.05 (α=0.05).
RT-PCR
Remove the reverse transcribed DNA from the 4°C freezer Results
and prepare the desired concentration of the reagent Drug susceptibility test results
according to the instructions. Turn on the ABI Fast7500 Our data showed that E. coli 15743 was sensitive to 20
instrument, set 95°C for 30 s, react for 40 cycles, 95°C for different antibiotics. After induction, E. coli 15743-256 was
3 s, 60°C for 30 s, and dissolve the curve for 95°C for 15 resistant to AMP, piperacillin, cefuroxime, cefazolin, cefox-
s, 60°C for 60 s, and 95°C for 15 s. Add the sample to the itin, AMP/sulbactam, amoxicillin/clavulanic acid, piperacil-
8-row EP tube, three replicate wells per sample, and lin/tazobactam, and aztreonam, but still sensitive to
remove the bubbles by centrifugation. The average CT remaining 11 antibiotics (Table 1, Note that Intermediaries
value of each sample was recorded after the reaction was were also defined as drug resistance). Our results indicated
completed. The relative expression level of the gene of that original sensitive E. coli was not only induced resistant
interest was calculated using 2−ΔΔCT. (ΔCT = CT value of to AMP, but also resistant to a variety of other antibiotics
the target gene-CT value of the internal reference gene. and became multi-drug resistant during induction.
ΔΔCT = experimental sample ΔCT - control group ΔCT.)
The occurrence of drug resistance
Bioinformatics analysis (determined by MIC value) during induction
SWISS-MODEL software was used to analyze the To study kinetics of drug resistance, we cultured E. coli
amino acid sequence of the protein encoded before with increasing concentration of AMP for different periods
and after gene mutation, and predict protein tertiary and measured MIC at each concentration as indicated in
structure.12,13 Table 2. Regression analysis was performed on the MIC

Table 1 Antibacterial ring diameter of Escherichia coli

Antibiotics 15743 15743-256 CLSI criterion (mm)

R I S

Gentamicin 20 18 ≤12 13–14 ≥15

Tobramycin 19 19 ≤12 13–14 ≥15
Ampicillin 19 7 ≤13 14–16 ≥17
Piperacillin 26 18 ≤17 18–20 ≥21
Cefepime 31 22 ≤14 15–17 ≥18
Cefuroxime 21 8 ≤14 15–17 ≥18
Ceftazidime 23 16 ≤14 15–17 ≥18
Cefoperazone 30 22 ≤15 16–20 ≥21
Cefazolin 25 8 ≤19 20–22 ≥23
Cefoxitin 24 8 ≤14 15–17 ≥18
Tetracycline 20 15 ≤11 12–14 ≥15
Ampicillin/sulbactam 24 8 ≤11 12–14 ≥15
Amoxicillin/clavulanic acid 22 8 ≤13 14–17 ≥18
Peracillin/tazobartan 24 16 ≤17 18–20 ≥21
Meropenem 35 27 ≤19 20–22 ≥23
Imipenem 32 25 ≤19 20–22 ≥23
Aztreonam 28 16 ≤17 18–20 ≥21
Sulfamethoxazole 26 19 ≤10 11–15 ≥16
Evofloxacin 27 26 ≤13 14–16 ≥17
Ciprofloxacin 29 25 ≤15 16–20 ≥21
Abbreviations: R, resistance; I, intermediaries; S, sensitivity.

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Table 2 The MIC value of E. coli 15743 over time and induced MLST results
concentration
To demonstrate that the induced strain (E. coli 15743-256)
Induced concentration (µg/mL) Time (hrs) MIC (µg/mL) was indeed derived from the original strain (E. coli 15743),
2 5 4 we performed MLST of above two strains. Genomic DNA
4 21 8 was extracted by bacterial DNA extraction kit, PCR ampli-
8 39 16 fied, and sequenced by Sangon Biotech (Shanghai) Co.,
16 63 32 Ltd. Blast searching of NCBI database indicated that these
32 81 48
two strains have identical, MLST type, adk-13, fumC-363,
64 113 64
128 187 128
gyrB-302, icd-97, mdh-17, purA-94, and recA-93. Our data
256 315 256 indicate that the induction process was not contaminated,
and the resistant strain E. coli 15743-256 was derived from
the sensitive strain E. coli 15743.
value and induction time using SPSS 17.0. The regression
equation was y=1.0435lnx−0.7316. The fitting effect of the Whole-genome analysis
equation was evaluated, R2=0.9605, P<0.05. The MIC E. coli 15743 contained 4408 genes, 22 rRNA, and 85
value reaching 32 µg/mL is the critical value, and the tRNA. The gene density was 0.945 kb, the GC content was
MIC value increased faster before reaching 32 µg/mL 51.7%, the gene percentage was 88.3%, the intergenic
than after (Table 2). region length was 545,151, the intergenic region GC con-
Meanwhile, the part with MIC value less than or equal tent was 42.6%, and the intergenic region accounted for
to 32 µg/mL was selected for regression analysis, and the 11.7% of the genome. The characteristics of E. coli 15743
regression equation was y=0.0358x+1.2812. The fitting genomes are summarized in Figure 2. E. coli 15743 did
effect of the equation was evaluated, R2=0.991, P<0.05. not contain plasmids.
The MIC value of E. coli 15743 increased with the increase The genome map of the strain includes distribution of
of induction concentration and induction time (Figure 1). genes on the chains of justice and antisense, functional
classification of Clusters of Orthologous Groups of pro-
teins (COG), GC content, genome island, and homologous
genes, which can fully display the features of the genome.

COG
The functional classification of COG of E. coli 15743
showed that most genes were related to amino acid trans-
port and metabolism, carbohydrate transport and metabo-
lism, energy production and conversion, general function
prediction only, inorganic ion transport and metabolism,
and cell envelope biogenesis (Figure 3).

Non-SNPs
To determine whether there was a change in the E. coli
genome after induction of the original strain, we per-
formed a genome-wide sequencing of the induced resis-
tant strains (E. coli 15743-32 and E. coli 15743-256)
and analyzed the number of mutations and the site of
the mutation.
Compared to the original E. coli strain (E. coli 15743),
there were nine non-SNPs in two induced drug-resistant
strains, including three shared non-SNPs, which were pre-
sent in the genes orf00819, orf01200, and orf02235. Other
Figure 1 The change of MIC value over time.
Abbreviation: MIC, minimum inhibitiveconcentration. non-SNPs were present in the genes orf01916, orf00490,

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Figure 2 The genomic map of E. coli 15743.

Notes: The outermost circle of the circle map is the genome-sized logo, each scale is 0.1 Mp. The second and third circles are CDS on the positive and negative chains, and
the different colors indicate different COG classifications of the CDS. The fourth circle is rRNA or tRNA. The fifth circle is the GC content, and the outward red part
indicates that the GC content in the region is higher than the whole-genome average GC content. The higher the peak value indicates the greater the difference from the
average GC content, and the inward blue portion indicates that the GC content in the region is low. For the whole-genome average GC content, a higher peak indicates a
greater difference from the average GC content. The innermost circle is the GC skew value. The specific algorithm is G−C or G+C. When the value is positive in the
biological sense, the positive chain tends to transcribe CDS. When it is negative, the negative chain tends to transcribe CDS.
Abbreviation: COG, Clusters of Orthologous Groupsof proteins.

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Figure 3 The functional classification of COG of E. coli 15743.

Abbreviation: COG, Clusters of Orthologous Groups of proteins.

orf03479, orf04094. Three non-SNPs mutations occurred Our data showed that there were four non-SNPs in E. coli
in the orf03479 gene, and only one SNP mutation occurred 15743-32, which were on four genes. There were eight non-
in each of the remaining genes. Three non-SNPs were in SNPs in E. coli 15743-256, spread across six genes. The
genes that encode cell membrane proteins. Three were in functional classification of COG showed that most genes
genes with unknown functions. One was related to the were related to amino acid transport and metabolism, carbo-
transport and metabolism of inorganic ion, one was related hydrate transport and metabolism, energy production and
to transcription, and one was related to signal transduction conversion, general function prediction only, inorganic ion
mechanisms (Table 3). transport and metabolism, and cell envelope biogenesis.

Table 3 The non-SNPs analysis results of E. coli 15743-32 and E. coli 15743-256
Location Gene-id Position Base Codon Aa COG Gene

E. coli 15743-32 orf00819 899,435 G→A GTT→ATT Val→Ile M ftsI

orf01200 1,302,112 C→T TCG→TTG Ser→Leu P acrB
orf01916 2,031,602 C→T GCC→GTC Ala→Val M OmpD
orf02235 2,368,737 C→A GCA→GAA Ala→Glu K marR

E. coli 15743-256 orf00490 541,538 T→A GTC→GAC Val→Asp M frdD

orf00819 899,435 G→A GTT→ATT Val→Ile M ftsI
orf01200 1,302,112 C→T TCG→TTG Ser→Leu P acrB
orf02235 2,368,737 C→A GCA→GAA Ala→Glu K marR
orf03479 3,681,194 G→C AGG→AGC Arg→Ser S VgrG
orf03479 3,681,453 C→T CTC→TTC Leu→Phe S VgrG
orf03479 3,681,480 C→G CGG→GGG Arg→Gly S VgrG
orf04094 4,314,203 C→A CGT→AGT Arg→Ser T envZ
Abbreviations: Ala, alanine; Asp, aspartic acid; Glu, glutamic acid; Ile, isoleucine; Ser, serine; Val, valine; Thr, threonine; Lys, lysine; HIS, histidine; Gln, glutamine; Asn,
asparagine; C, energy production and conversion; M, cell wall or envelope biogenesis; S, unknown function.

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RT-PCR Before the mutation of orf04094, 4cti.1.B was selected

Whole-genome re-sequencing E. coli 15743-32 and E. coli as reference template protein (Figure 6A). The model
15743-256, fluorescent real-time quantitative PCR detec- range of residual infrastructure was 184–436, the sequence
tion of consensus genes. The genes in which non-SNPs similarity was 0.56, and the template coverage was 0.59.
occur were screened, and E. coli 15743-32 and E. coli After the mutation of orf04094, 3ib7.1.A was selected as
15743-256 had three identical genes (orf00819, orf01200, reference template protein (Figure 6B). The model range
orf02235), and the expression levels of these genes in each of residual infrastructure was 10–262, the sequence simi-
generation strain are shown in Figure 4A–C, respectively. larity was 0.33, and the template coverage was 0.91.
RT-PCR showed that the orf01200, orf00819, orf02235
genes showed high expression in resistant strains (E. coli Discussion
15743-32, E. coli 15743-64, E. coli 15743-128, E. coli Regression analysis of MIC and induction time showed
15743-256). that the MIC value of the strain increased with the increase
of exogenous antibiotic pressure and the induction time.
Protein structure prediction Liu et al, showed that during the induction of E. coli
The tertiary structure changes only in proteins encoded by resistance by imipenem, the MIC value increased with
genes orf01200 and orf04094, and the predicted results are time.14 Even when the induced concentration reached
shown in Figures 5 and 6. 128 times the MIC value of the primary strain, the induc-
Before the mutation of orf01200, 2hrt.1.A was tion was continued, and the MIC value continued to
selected as reference template protein (Figure 5A). The increase with induction. Consistent with the results of
model range of residual infrastructure was 2-1033, the this study, the MIC value of E. coli increased with time
sequence similarity was 0.59, and the template coverage and induced concentration. It shows that if the dose is not
was 1.00. After the mutation of orf01200, 1iwg.1.A was limited, the resistance of the strain will become more and
selected as reference template protein (Figure 5B). The more serious.
model range of residual infrastructure was 7-1036, the AMP was induced to E. coli 15743 for 63 hrs (MIC
sequence similarity was 0.59, and the template coverage reached 32 µg/mL), and the MIC value was eight times
was 1.00. that of the susceptible strain. Prior to this, the MIC value

Figure 4 Results of mRNA expression in different generations of strains.

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Figure 5 The tertiary structure of the protein encoded by orf01200 gene.

Notes: (A) Before the mutation; (B) after the mutation.

Figure 6 The tertiary structure of the protein encoded by orf04094 gene.

Notes: (A) Before the mutation; (B), after the mutation.

increased rapidly, whereas when induced to a MIC value also to piperacillin, cefuroxime, cefazolin, cefoxitin, AMP/
of 32 µg/mL, the induction continued and the growth rate sulbactam, amoxicillin/clavulanic acid, piperacillin/tazo-
of the MIC value decreased. Considering bacterial resis- bactam, and aztreonam were also resistant. It is considered
tance can occur shortly before reaching the drug resistance that during the induction of E. coli by AMP, the expression
threshold (MIC value of 32 µg/mL). After reaching the system of AcrAB-TolC is activated, or more than one of
critical value, the bacteria may be lazy and grow slowly, the multiple efflux pump systems is activated, and there
but the MIC value continues to increase. It is also believed are other resistance mechanisms other than the efflux
that this strain activates certain resistance mechanisms and mechanism.
changes the drug resistance status of the bacteria. The molecular mechanism of bacterial resistance is still
Zhang et al, showed that chloramphenicol induced unclear. In order to investigate the specific molecular
sensitive Shigella to the drug-resistant state, and its drug mechanism of E. coli resistance to AMP, bacterial WGS
resistance spectrum would change.10 As a result, Shigella analysis was performed. The sequencing results were com-
was not only resistant to chloramphenicol, but also resis- pared with the reference sequence, and 20 SNPs were
tant to other types of antibiotics. Consistent with the screened from the sequence of E. coli 15743-32, 4 of
results of this study, the drug resistance spectrum of which were non-synonymous SNPs. Twenty-six SNPs
E. coli was amplified after induction. The results showed were screened from the E. coli 15743-256 strain, eight of
that E. coli 15743-256 was not only resistant to AMP, but which were non-synonymous SNPs. Xiang et al, showed

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that the resistance level of mutant strains was higher than bacteria.25–27 Since most of the effluent system transports
that of non-mutant strains, and there was a quantitative substrates widely, and many active effluent systems can
reaction between point mutations and bacterial resistance exist in the same bacteria, this system can lead to bacterial
levels, and multiple gene mutations could enhance the resistance to various antibacterial drugs with completely
resistance of bacteria to antibiotics.15 Consistent with the different structures, namely multiple resistance. In Marlen
results of this study, the number of mutant genes in E. coli Adler’s study, mutations in the ftsI gene alone did not
15743-32 was less than E. coli 15743-256, indicating that increase antibiotic resistance, whereas ftsI and envZ gene
the number of mutations may be related to the degree of mutations increased the MIC of antibiotics multiple times.
drug resistance, and the more mutation sites, the higher the Cohen et al, demonstrated that the function of the inhibi-
degree of drug resistance. tory protein encoded by the mutated MarR gene would be
After sequencing, the non-SNPs screened in this reduced, and the effect of the bacteria on the multiple
experiment were distributed in the genes of orf00490, resistance of antibiotics was small when the MarR muta-
orf00819, orf01916, orf01200, orf02235, orf03479, and tion was only detected.28 Merric et al, found that E. coli
orf04094. Among them, genes orf00490, orf00819, and showed only low levels of multi-drug resistance when the
orf01916 are involved in cell wall synthesis. The annota- MarR gene was mutated.29 The results of this study
tions in KEGG are fumarate reductase subunit D (frdD), showed that multiple genes were simultaneously mutated
cell division protein ftsI (penicillin-binding protein 3) and and E. coli resistance to AMP increased.
porin outer membrane protein OmpD, respectively. Studies Gene orf03479 is annotated as valine glycine repeat G
have shown that the frd gene encodes a FRD enzyme to (VgrG) protein in KEGG. The Type VI Secretion System
catalyze the conversion between fumarate reductase and (T6SS) is a phage-related system that exists in many
succinate dehydrogenase.16 It has also been found that bacterial pathogens, such as E. coli, Pseudomonas aerugi-
amplification of the frdD gene using a plasmid vector nosa, and Burkholderia cenocepacia. The effector factors
can increase the yield of succinic acid.17,18 In combination can be secreted to the extracellular of bacteria, and the
with this study, it is considered that the frdD gene is protein secretion system is closely related to virulence of
involved in certain metabolic pathways, perhaps asso- pathogenic bacteria. Wang Jianfeng et al, showed that
ciated with AMP resistance. In E. coli, the main targets VgrG gene mutation affects the toxicity and drug resis-
of β-lactam antibiotics are PBP1 (maintaining cell mor- tance of bacteria, but the function of glutamate valine
phology), PBP2 (maintaining E. coli tension and rod repeat protein is still unclear.30 This study considers that
shape), and PBP3 (related to bacterial division). PBP3 is the VgrG gene may be associated with AMP resistance,
a core component of cell division proteins that catalyze the and its mechanism needs further investigation.
cross-linking of cell wall peptidoglycans during cell In summary, the COG function of these mutant genes is
division.19–22 Studies have shown that down-regulation related to the origin of cell membranes, transport and
of OmpD protein and OmpD gene expression in bacterial metabolism of inorganic ions, transcription and signal
biofilms leads to decreased cell membrane permeability transduction mechanisms. Studies have shown that under
and increased resistance to antibiotics.23,24 Consistent antibiotic stress, bacteria can take both active defense and
with the results of this study, the OmpD gene mutation passive defense to ensure their survival.31 In passive
initiates a mechanism of bacterial resistance to β-lactam defense, bacteria make itself dormant, reduce the vitality
antibiotics, and the decrease in E. coli cell membrane of life and block the combination of antibiotics and target
permeability is one of the reasons for the increased resis- to reduce the killing effect of antibiotics. In active defense,
tance to AMP. It is considered that these changes in the they increase the activity of efflux pump to increase the
function of proteins encoded by genes involved in cell efflux of antibiotics and reduce the accumulation of anti-
wall synthesis affect the resistance of bacteria to AMP. biotics in bacteria, thereby reducing the killing effect of
Genes orf04094, orf01200, orf02235 are annotated in antibiotics on bacteria. This study suggests that the resis-
KEGG as osmotic pressure sensor histidine kinase (envZ), tance of E. coli to AMP is a combination of active defense
multi-drug efflux pump gene (acrB), and multi-drug resis- systems and passive defense systems. Drug resistance can
tance protein involved in transcriptional regulation occur shortly before the bacterial MIC value reaches the
(marR). In recent years, the active efflux mechanism is drug resistance threshold. Genes frdD, ftsI, acrB, OmpD,
the main reason for the multiple drug resistance of marR, VgrG, and envZ are associated with AMP

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