Next Article in Journal
Oxygen Availability on the Application of Antimicrobial Photodynamic Therapy against Multi-Species Biofilms
Previous Article in Journal
A Systematic Meta-Analysis of Global Sarcocystis Infection in Sheep and Goats
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 12
Issue 7
10.3390/pathogens12070903
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Characterization of an Escherichia coli Isolate Coharboring the Virulence Gene astA and Tigecycline Resistance Gene tet(X4) from a Dead Piglet

by
Jianmei Wang
1,†,
Yuting Huang
1,2,†,
Chunjiu Guan
1,2,
Jie Li
3,
Hua Yang
1,
Guoping Zhao
4,
Canying Liu
2,
Jiangang Ma
1,* and
Biao Tang
1,*
1
State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products & Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China
2
School of Life Science and Engineering, Foshan University, Foshan 528225, China
3
College of Life Science, Liaocheng University, Liaocheng 252000, China
4
School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Pathogens 2023, 12(7), 903; https://doi.org/10.3390/pathogens12070903
Submission received: 14 April 2023 / Revised: 27 June 2023 / Accepted: 29 June 2023 / Published: 3 July 2023
(This article belongs to the Special Issue Update on Animal Pathogenic Escherichia coli)
Browse Figures
Versions Notes

Abstract

:
tet(X4) is the critical resistance gene for tigecycline degradation that has been continually reported in recent years. In particular, pathogenic bacteria carrying tet(X4) are a severe threat to human health. However, information describing Escherichia coli coharboring tet(X4) with virulence genes is limited. Here, we isolated an E. coli strain coharboring tet(X4) and the heat-stable toxin gene astA from a dead piglet. The strain named 812A1-131 belongs to ST10. The genome was sequenced using the Nanopore and Illumina platforms. The virulence genes astA and tet(X4) are located on the chromosome and in the IncHI1-type plasmid p812A1-tetX4-193K, respectively. The plasmid could be conjugatively transferred to recipient E. coli J53 with high frequency. In vivo experiments showed that strain 812A1-131 is pathogenic to Galleria mellonella and could colonize the intestines of mice. In summary, pathogenic E. coli could receive a plasmid harboring the tet(X4) gene, which can increase the difficulty of treatment. The prevalence and transmission mechanisms of pathogenic bacteria coharboring the tet(X4) gene need more attention.

1. Introduction

Antimicrobial resistance (AMR) has become a global public health problem. The emergence of multidrug-resistant (MDR) bacteria is threatening human health. Tigecycline is a third-generation tetracycline known as the last resort to treat MDR bacterial infections in hospitals [1]. Unfortunately, the tigecycline resistance gene tet(X) has emerged and spread widely in animal-derived bacteria, which may be accelerated by the overuse and misuse of tetracycline antibiotics in livestock and poultry [2]. Tigecycline-resistant bacteria harboring the tet(X4) gene have also been discovered in patients, limiting the antibiotic treatment and threatening human health [3].
The tet(X) gene family encoding flavin-dependent monooxygenase can degrade all tetracycline antibiotics, including tigecycline, omadacycline, and eravacycline. In recent years, plasmid-mediated tet(X4) gene transfer among animal-derived bacteria has caused wide public concern [4]. Notably, the IncX1, IncX3, IncHI1, and IncQ plasmids carrying the tet(X4) gene are most common and have been identified in Enterobacteriaceae [5,6,7]. Escherichia coli harboring tet(X4) have been isolated from multiple sources, including birds, retail meat, and the environment, and they have also been found in human clinical samples [8,9,10,11,12].
E. coli is a common opportunistic zoonotic pathogen. E. coli carrying virulence genes can cause human and animal diseases, such as gastroenteritis, cholecystitis, urinary tract infections (UTIs), and pneumonia in humans [13,14]. It can also cause severe diarrhea in pigs, chickens, cattle, and other animals, or even death, especially in newborn animals [15,16]. Diarrheagenic E. coli (DEC) can be classified into six groups according to their virulence genes: enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli (EHEC), shiga-toxin-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), and diffusely adherent E. coli (DAEC) [17]. The eae, stx, elt, estB, faeG, aggR, ipaH, and Afa-Dr adhesin genes are the main virulence genes in these pathogens [18,19]. The astA virulence gene encodes a heat-stable toxin that is widespread in pathogens, such as EAEC, ETEC, STEC, and EHEC [20]. E. coli carrying the astA virulence gene are associated with diarrhea and the enhanced pathogenicity of other virulence factors. Such strains can cause severe gastrointestinal disease in animals and lead to economic losses [21].
Multilocus sequence typing (MLST) is an important method for analyzing specific bacterial typing based on different housekeeping genes. MLST analysis could be helpful in understanding the processes of genomic evolution in diverse species and the characteristics of a specific clone [22]. E. coli ST types are extraordinarily diverse, including ST10, ST48, ST95, and so on [23]. E. coli ST10, as a non-host-restricted pathogenic bacterium, can spread in humans and animals [24]. ST10 strains are common among MDR E. coli and have always carried various virulence genes, including gad (glutamate decarboxylase), iss (increased serum survival), terC (tellurium ion resistance protein gene), and sitA (iron transport protein) [25,26]. It should be noted that mcr-1, tet(X4), and blaNDM-5 have been detected in ST10, which showed that ST10 strains are important carriers for ARGs [27]. Moreover, some ST10 strains belonging to the DEC have been discovered in diarrheal pigs and patients [4,28]. DEC ST10 carrying ARGs in sick animals and patients would increase the risk of antibiotic treatment failure.
In a routine antimicrobial resistance surveillance, we isolated an E. coli strain cocarrying the resistance gene tet(X4) and the virulence gene astA from a piglet of unknown cause of death. We tested the AMR of the strain and analyzed the genome characterization after whole-genome sequencing to elucidate the transmission mechanism of the tet(X4) gene and the virulence. The virulence of strain 812A1-131 was detected by in vivo experiments in Galleria mellonella and mice. This strain may pose a threat to other animals and humans.

2. Materials and Methods

2.1. Pathogen Detection

An anal swab of a dead pig (of unknown cause of death) at 18 days of age was sent from a farm. The RNA in the feces was extracted according to the instructions for the Column Stool RNAOUT kit (Yaji biological Co., Ltd., Shanghai, China). The RNA was immediately subjected to reverse-transcription PCR (Vazyme Biotech Co., Ltd., Nanjing, China) using the primer Oligo dT. Then, the RT-PCR amplified procedure and the detection primers for porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), and porcine rotavirus (RV) were used as described in a previous study [29].

2.2. Bacterial Isolation and Identification

The swab was put into a sterile tube containing 5 mL BPW (Hangzhou Microbial Reagent Co., Ltd., Hangzhou, China) to enrich the bacteria and incubated at 37 °C at 200 rpm for 12–18 h. The bacterial solution was scribed onto MacConkey agar with a disposable inoculating loop and statically incubated at 37 °C for 12–18 h. A single colony on the McConkey agar plate was inoculated on eosin methylene blue (EMB) agar and Luria–Bertani (LB) medium and incubated overnight at 37 °C. The culture mediums were purchased from Beijing Land Bridge Technology Co. Ltd., Beijing, CHN. The strain was identified by PCR using the primers described in a previous study [30]. The stain was named 812A1-131 and preserved in a cryovial containing 1 mL of 25% glycerol at −80 °C.

2.3. Antimicrobial Susceptibility Testing

The broth dilution method was used to determine the minimum inhibitory concentration (MIC) of strain 812A1-131 according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. E. coli ATCC25922 was used as a quality control, as previously described [31]. A total of 13 antibiotics were selected for antimicrobial susceptibility testing, including ampicillin (AMP), amoxicillin/clavulanic acid (A/C), cefotaxime (CTX), meropenem (MEM), amikacin (AMK), gentamicin (GEM), colistin (COL), ceftiofur (CEF), ciprofloxacin (CIP), trimethoprim/sulfamethoxazole (SXT), tetracycline (TET), tigecycline (TIG), and florfenicol (FFC). The antibiotics were purchased from Biofosun, Fosun Diagnostics, Shanghai, China.
In short, the concentrated antibiotic solutions were diluted, and 100 μL volumes were added to 96-well plates by double dilution according to the CLSI instructions. Then, 2–5 single colonies of E. coli were scraped from a fresh LB agar plate and adjusted to 0.5 McFarland standard inoculum (1.5 × 108 CFU/mL) using saline solution. The suspension was added to MH nutrient broth at a ratio of 1:200, and then 100 μL of diluent was added to each well. The 96-well plate containing the antibiotics and bacterial suspension was placed in an incubator at a constant temperature of 37 °C for 16–18 h.
The MIC values of tigecycline were verified by the agar dilution method and E-test assay (Tang et al., 2022). E. coli ATCC 25922 was used as a quality control. Briefly, serial dilutions of bacterial suspensions (10−1~10−6) were inoculated onto LB agar plates with different concentrations of tigecycline and subsequently incubated at 37 °C for 12 h [32].

2.4. Whole-Genome Sequencing (WGS) and Bioinformatics Analysis

Whole-genome sequencing is a valuable way to analyze ARGs and virulence factors and has been used in the diagnosis of animal pathogens [33,34,35,36]. The genome of the tigecycline-resistant strain was extracted using the Generay DNA kit (Generay, Shanghai, China). WGS was performed using the Illumina HiSeq and Nanopore sequencing platforms. Unicycler v.0.4.3 was used for assembly, and RAST (https://rast.nmpdr.org/ (accessed on 17 January 2023)) was used for gene annotation [37]. The Center for Genomic Epidemiology (https://cge.cbs.dtu.dk//services/ (accessed on 17 January 2023)) was used to analyze ARGs with ResFinder 4.1 [38]. PlasmidFinder 2.1 and VirulenceFinder 2.0 were used to predict plasmid replicon and virulence genes [39]. The serotype was predicted by SerotypeFinder 2.0 (https://cge.cbs.dtu.dk/services/SerotypeFinder/ (accessed on 17 January 2023)) [40]. BacWGSTdb was used to observe the geographical distribution of closely related plasmids and, based on the cgMLST strategy to generate a phylogenetic tree, analyze the relationship between resistance genes of similar strains [41]. The plasmids were visualized using BRIG [42].

2.5. Conjugative Transfer

The sodium-azide-resistant E. coli strain J53 was used as the recipient, and the tet(X4)-positive strain 812A1-131 was used as the donor. E. coli J53 is sensitive to tigecycline, as demonstrated in previous studies [5]. Strain 812A1-131 was sensitive to sodium azide and could not grow in LB agar plates containing sodium azide (100 mg/L). The bacteria were cocultured at two temperatures, 37 °C or 28 °C, for 16 h. The mixed culture was diluted to 10−1~10−6 using PBS, and 10 μL of the above diluted solution was inoculated on LB agar plates containing tigecycline and sodium azide. The LB agar plates were incubated at 37 °C for 12~16 h [31]. Single colonies of transconjugants were randomly selected, and the tet(X4) gene was verified by PCR [5].

2.6. S1-PFGE and Southern Blotting

The tet(X4) strain was embedded and subsequently lysed using SeaKem Gold Agarose (Lonza Rockland, Inc., Maryland, MD, USA), with Salmonella H9812 as the marker, as described previously [32]. The treated DNA fragments were then separated at 14 °C for 18 h in 0.5× Tris-borate EDTA buffer. A pulse-field electrophoresis system (CHEF Mapper, Bio-Rad Laboratories, California, CA, USA) was used with a voltage of 6 V, an electric field angle of 120°, and a pulse time of 2.2 to 63.8 s. After the gel was removed at the end of the procedure, the gel blocks were stained and observed with a gel imager. The tet(X4)-specific probe was labeled according to the instructions of the DIG High Prime DNA marker and assay starter kit (Roche Diagnostics GmbH, Mannheim, Germany).

2.7. The Galleria mellonella Model and the Mouse Infection Model

To evaluate the virulence of 812A1-131, the Galleria mellonella infection model was used to conduct in vivo experiments. E. coli 649A1 containing the stx2 gene was used as the virulent control and stored in the laboratory. Sterilized PBS served as a blank control. The inactivated 812A1-131 strain (incubated at 65 °C for 30 min) and the avirulent strain DH5α were used as the negative controls to assess the virulence of the live strain 812A1-131 [43]. Larvae with symmetrical physiques were evenly divided into the PBS control group, the experimental group, the highly virulent control group, and the avirulent control group. Ten larvae were set in each group, and the larvae were injected with 10 μL PBS or 106 CFU/mL bacterial inoculum, as previously described [44]. The death of larvae in each group was recorded every 6 h and observed for 48 h. GraphPad software was used for the plotting and statistical analysis of the data.
The mouse infection model test was performed as described in a previous report [45]. Ten 5-week-old SPF ICR male mice (Hangzhou Qizhen Laboratory Animal Technology Co., Ltd., Hangzhou, China) were weighed (about 25 g per mouse) and randomly divided into two groups, with five animals in each group. The bacterial solution (109 CFU in 100 μL) or PBS containing 20% sucrose were injected into the stomachs of the mice. To obtain the highest number of E. coli, the mice were weighed and euthanized by cervical dislocation after 6 h, as previously described [46]. The ileum, cecum, and colon parts were collected. Moreover, the tissue was vigorously cleaned and ground with PBS. The homogenate was diluted 1000 times using PBS, and 100 μL was coated on a MacConkey plate (containing 2 μg/mL tigecycline) at 37 °C for 16 h. Then, colony counting was performed.

3. Results

3.1. Pathogen Detection and Bacterial Isolation

PEDV, TGEV, and RV were not detected in the dead pig. The cause of the pig’s death could not be determined. An E. coli strain was successfully isolated and identified by PCR.

3.2. Antimicrobial Susceptibility

Antimicrobial susceptibility testing showed that strain 812A1-131 was only sensitive to meropenem and trimethoprim/sulfamethoxazole and showed an intermediate reaction to colistin (Table 1). It was resistant to tigecycline, with an MIC of 16 μg/mL. The agar dilution method and E-test were used to further verify its resistance to tigecycline (Figure 1). Strain 812A1-131 is a multidrug-resistant (MDR) strain that is resistant to 10 different antibiotics from nine classes (AMP-A/C-CTX-AMK-GEM-CEF-CIP-TET-TIG-FFC).

3.3. Whole-Genome Sequencing and Sequence Analysis

The strain 812A1-131 has a genome consisting of one chromosome and three plasmids. The chromosome size was 4,665,801 bp, and the GC content was 50.9%. The MLST analysis showed that the strains belonged to E. coli ST10. There are three plasmids located in 812A1-131 that were named p812A1-tetX4-193K, p812A1-69K, and p812A1-65K. The plasmid p812A1-tetX4-193K has a size of 193,145 bp and a GC content of 46.21%, and it contains two types of replicons (IncHI1A and IncHI1B(R27)). p812A1-69K has no known replicon and is 69,262 bp with a 51.89% GC content. The size of p812A1-65K is 65,072 bp, and it contains the IncFIA(HI1)- and IncY-type replicons. In addition, the size of plasmid p812A1-tetX4-193K was verified using S1-PFGE, which was consistent with the whole-genome sequencing analysis (Figure 2).
Sixteen antimicrobial resistance genes (ARGs) were identified in 812A1-131, of which six were located on p812A1-tetX4-193K: tet(X4), blaTEM-1B, lnu(G), aadA22, qnrB, qnrS1, and floR. The plasmid p812A1-65K contains seven ARGs: fosA3, aph(4)-Ia, aac(3)-IV, sul2, tet(A), blaCTX-M-14, and floR. In addition, rmtB, blaTEM-141, blaCTX-M-55, and blaTEM-1B were located on the plasmid p812A1-69K (Table 1). A total of 16 virulence genes were identified, including astA, hha, hlyE, traT, yehA, and traJ. These virulence genes are related to heat-stable toxins, hemolysin, outer membrane protein virulence factor, and invasion of the blood–brain barrier, respectively. Strains with close homology (<1000 SNP) from different hosts of ST10 types were screened from the NCBI database. These strains were identified in humans, cows, pigs, chickens, and the environment. The phylogenetic tree showed that strain 812A1-131 is relatively independent compared to the others (Figure 3). Some strains carried multiple ARGs, but the tet(X4) gene was rare. Except in strain 812A1-131, the tet(X4) gene was also found in strain ECSW_09, which was identified from pigs (Figure 3). Moreover, these strains contained several virulence genes, alsA, csg, hlyE, nlpl, and traC (Figure 4).

3.4. Conjugative Transfer of 812A1-131

The recipient strain E. coli J53 was able to grow on an LB agar plate containing sodium azide and tigecycline after coculture with strain 812A1-131, confirming conjugative transfer. The tet(X4) gene was located in the transconjugants, which was verified by PCR. This result indicated that, by conjugation, strain 812A1-131 could transfer the plasmid harboring the tet(X4) gene to recipient bacteria. The conjugative transfer frequencies were different between different temperatures, being lower at 37 °C (1.87 ± 0.89 × 10−5) than at 28 °C (4.71 ± 5.64 × 10−3).

3.5. Genetic Environment Analysis of p812A1-tetX4-193K

The complete sequence of plasmid p812A1-tetX4-193K was obtained by sequencing. The highly similar plasmids (coverage > 97%, identity > 99%) compared with p812A1-tetX4-193K from different hosts were screened using BLAST from the GenBank database (Figure 5). They were all IncHI1-type plasmids that were identified from Enterobacter hormaechei (pGX4-8L (CP071877)), Citrobacter sp. (pSZ6R-tetX4 (MW940627)), Morganella morganii (pTQ28-tet(X4) (ON390816)), Enterobacter cloacae (pTECL_2-190k-tetX4 (MZ773210)), and Klebsiella pneumoniae (pTKPN3-186K-tetX4 (MZ773211)). These strains were isolated from swine feces, swine nasal swabs, and so on. pGX4-8L had the highest similarity to p812A1-tetX4-193K, and the other plasmids were found to lack a segment in the comparisons with p812A1-tetX4-193K (Figure 5). It is worth noting that these five plasmids came from different species in China. The IncHI1 plasmid has a higher risk of spreading the tet(X4) gene between bacteria.

3.6. Pathogenic Testing of Strain 812A1-131 in Galleria mellonella and Mouse

Strain 812A1-131 and the highly virulent E. coli strain 629A1 killed 80% and 100% of Galleria mellonella at 20 h, respectively. All Galleria mellonella died by 24 h after infection with strain 812A1-131. However, only 10% of Galleria mellonella died in the PBS or avirulent groups by 20 h. The fatality rate of strain 812A1-131 was significantly higher than that of the PBS and avirulent strains (Figure 6). The mice were subjected to gavage to observe the bacterial colonization ability. The change in weight was not significantly different between the different groups. E. coli 812A1-131 was isolated from the ileum, cecum and colon. The numbers of colonies were 2.58 × 103, 2.79 × 108, and 7.11 × 106 CFUs/g, respectively. This strain preferred to colonize the cecum and colon.

4. Discussion

Although tigecycline has not been used in breeding, reports of tigecycline-resistant bacteria carrying tet(X4) genes in animals have increased. This may be due to the widespread use of tetracycline in food animals. E. coli is an essential host of tet(X4) resistance genes. Notice that the host range of tet(X4) genes is increasingly extensive, and tet(X4) has even been found in pathogenic bacteria [47,48]. The risk of tigecycline therapy failure is increased when an MDR pathogen harboring tet(X4) infects humans. In this study, one E. coli strain belonging to ST10 carrying the tet(X4) resistance gene and the astA virulence gene was isolated from a dead piglet.
ST10 is a common E. coli sequence type that has been widely isolated from the environment, animals, and even humans [49]. E. coli ST10 is a potential food-borne pathogen that threatens human health and has been identified in the food chain [50,51]. There has been no direct evidence of the direct transmission of E. coli ST10 to humans through contact or the food chain [52]. However, previous studies have shown that the ST10 E. coli strain from the environment has a genome closely related to the strain from humans. The same ST10 was observed in human patients, pig feces, and pork samples [27,53]. This result suggested that ST10 has a broad host range and is a zoonotic pathogen. Moreover, ST10 strains are usually multidrug-resistant (MDR) bacteria [54]. The ST10 strain of E. coli has resistance to seven types of antibiotics, including colistin, and was isolated from Polish poultry [55]. MDR ST10 E. coli resistance to imipenem and colistin was identified in young clinical patients [56].
ARGs can be located on chromosomes or mobile genetic elements (MGEs) of bacteria. MGEs can help to rapidly spread ARGs that include integrons, transposons, insertion sequences, and plasmids [57]. Plasmids were the most common MGEs for transferring ARGs within the same family of bacteria. The tet(X4) gene was identified in an IncHI1 plasmid in this study. The IncHI1 plasmid is usually larger than 180 kb, and it always carries various ARGs and heavy-metal resistance genes [58]. It has a wide host range and has been widely isolated from various Enterobacteriaceae bacteria, especially Salmonella [59]. This plasmid plays an important role in tet(X4) transmission and has become the second most-prevalent plasmid for transferring the tet(X4) gene. The p812A1-tetX4-193K plasmids from the dead piglet were similar to those from the nonpathogen in different regions. This result indicated that the tet(X4)-positive IncHI1 plasmid is widespread and could transfer horizontally between bacteria in humans, animals, food, and the environment [60]. Furthermore, IncHI is a common plasmid type carrying the colistin resistance gene mcr [61]. It carries the risk of coharboring the tet(X) and mcr genes, which would be a serious threat to human health.
The emergence of pathogens with important AMR genes and virulence factors seriously threatens public health. Pathogenic K. pneumoniae carrying virulence genes hvKp and tet(X4) were isolated from pork samples and caused 100% death in mice 12 h after inoculation [62]. Foodborne pathogens can infect humans, threatening human health [63]. The coexistence of the virulence factor tdh and the AMR gene mcr-1 was also discovered in Vibrio parahemolyticus [64]. Pathogenic E.coli (UPEC) containing the blaNDM gene has caused multiple typical urinary tract infections in patients [65]. This result indicated that bacteria coharboring resistance genes and virulence factors are widespread, limiting antibiotic treatment. The astA gene has been identified in various DEC strains with outbreaks of diarrhea [66,67,68,69]. This study indicated that the astA gene might increase the strain’s virulence. The difficulty of treatment is increased when a pathogen acquires the tet(X4) gene. Therefore, monitoring the coexistence of resistance genes with virulence factors in bacteria is very important. Furthermore, limiting the use of tetracycline antibiotics in livestock and poultry may help reduce the spread of resistant bacteria. People should avoid contact with live animals and choose processed meats instead.
In conclusion, an E. coli strain coharboring tet(X4) and astA was isolated from a dead piglet in routine antimicrobial resistance surveillance. The 812A1-131 strain infection could lead to the death of Galleria mellonella, which indicates that astA might have virulence potential. The IncHI1 plasmid transferred tet(X4), which is increasingly common. The acquisition of tet(X4) by pathogenic bacteria would increase treatment difficulty in humans and animals, and the bacteria coharboring virulence and AMR genes need attention.

5. Accession Numbers

The whole-genome sequence of E. coli 812A1-131 has been submitted to GenBank with the accession numbers CP116046-CP116049.

Author Contributions

Conceptualization, B.T. and J.M.; Funding acquisition, B.T. and J.M.; Investigation, J.W., Y.H., C.G., J.L. and H.Y.; Methodology, J.W., C.G. and C.L.; Supervision G.Z., J.M. and B.T.; Visualization, G.Z., J.L. and B.T.; Writing—original draft, J.W. and Y.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Zhejiang Provincial Natural Science Foundation of China (LY23C180001), the “Leading Goose” R&D Program of Zhejiang Province (2023C03045), the Collaborative Extension Plan of Major Agricultural Technologies in Zhejiang Province (2021XTTGXM03), the Program of Zhejiang Agriculture and Rural Affairs (2023SNJF058, 2023SNJF062), and the One Health Interdisciplinary Research Project, Ningbo University (HZ202201).

Institutional Review Board Statement

All animal experiments were carried out according to animal welfare standards and approved by the Laboratory Animal Welfare Ethics Committee of Zhejiang Academy of Agricultural Sciences (approval number 2023ZAASLA01).

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated during and/or analyzed during the current study can be find in the main text.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. He, T.; Wang, R.; Liu, D.; Walsh, T.R.; Zhang, R.; Lv, Y.; Ke, Y.; Ji, Q.; Wei, R.; Liu, Z.; et al. Emergence of plasmid-mediated high-level tigecycline resistance genes in animals and humans. Nat. Microbiol. 2019, 4, 1450–1456. [Google Scholar] [CrossRef]
  2. Frey, L.; Tanunchai, B.; Glaser, B. Antibiotics residues in pig slurry and manure and its environmental contamination potential. A meta-analysis. Agron. Sustain. Dev. 2022, 42, 31. [Google Scholar] [CrossRef]
  3. Zhang, R.; Dong, N.; Shen, Z.; Zeng, Y.; Lu, J.; Liu, C.; Zhou, H.; Hu, Y.; Sun, Q.; Cheng, Q. Epidemiological and phylogenetic analysis reveals Flavobacteriaceae as potential ancestral source of tigecycline resistance gene tet(X). Nat. Commun. 2020, 11, 4648. [Google Scholar] [CrossRef] [PubMed]
  4. Cui, C.Y.; Li, X.J.; Chen, C.; Wu, X.T.; He, Q.; Jia, Q.L.; Zhang, X.J.; Lin, Z.Y.; Li, C.; Fang, L.X.; et al. Comprehensive analysis of plasmid-mediated tet(X4)-positive Escherichia coli isolates from clinical settings revealed a high correlation with animals and environments-derived strains. Sci. Total Environ. 2022, 806, 150687. [Google Scholar] [CrossRef] [PubMed]
  5. Guan, C.; Tang, B.; Yang, H.; Ma, J.; Huang, Y.; Liu, C. Emergence of plasmid-mediated tigecycline resistance gene, tet(X4), in Escherichia fergusonii from pigs. J. Glob. Antimicrob. Resist. 2022, 30, 249–251. [Google Scholar] [CrossRef] [PubMed]
  6. Zhai, W.; Tian, Y.; Shao, D.; Zhang, M.; Li, J.; Song, H.; Sun, C.; Wang, Y.; Liu, D.; Zhang, Y. Fecal carriage of Escherichia coli harboring the tet(X4)-IncX1 plasmid from a tertiary class-a hospital in Beijing, China. Antibiotics 2022, 11, 1068. [Google Scholar] [CrossRef]
  7. Li, R.; Liu, Z.; Li, Y.; Xiao, X.; Wang, Z. Characterization of blaNDM-positive Enterobacteriaceae reveals the clonal dissemination of Enterobacter hormaechei coharboring blaNDM and tet(X4) along the pork production chain. Int. J. Food Microbiol. 2022, 372, 109692. [Google Scholar] [CrossRef]
  8. Li, R.; Lu, X.; Munir, A.; Abdullah, S.; Liu, Y.; Xiao, X.; Wang, Z.; Mohsin, M. Widespread prevalence and molecular epidemiology of tet(X4) and mcr-1 harboring Escherichia coli isolated from chickens in Pakistan. Sci. Total Environ. 2022, 806, 150689. [Google Scholar] [CrossRef]
  9. Ma, J.; Zhou, W.; Wu, J.; Liu, X.; Lin, J.; Ji, X.; Lin, H.; Wang, J.; Jiang, H.; Zhou, Q.; et al. Large-scale studies on antimicrobial resistance and molecular characterization of Escherichia coli from food animals in developed areas of Eastern China. Microbiol. Spectr. 2022, 10, e0201522. [Google Scholar] [CrossRef] [PubMed]
  10. Chen, C.; Cui, C.Y.; Zhang, Y.; He, Q.; Wu, X.T.; Li, G.; Liao, X.P.; Kreiswirth, B.N.; Liu, Y.H.; Chen, L.; et al. Emergence of mobile tigecycline resistance mechanism in Escherichia coli strains from migratory birds in China. Emerg. Microbes Infect. 2019, 8, 1219–1222. [Google Scholar] [CrossRef] [Green Version]
  11. Sun, H.; Wan, Y.; Du, P.; Liu, D.; Li, R.; Zhang, P.; Wu, Y.; Fanning, S.; Wang, Y.; Bai, L. Investigation of tigecycline resistant Escherichia coli from raw meat reveals potential transmission among food-producing animals. Food Control 2021, 121, 107633. [Google Scholar] [CrossRef]
  12. Li, R.; Mohsin, M.; Lu, X.; Abdullah, S.; Munir, A.; Wang, Z. Emergence of plasmid-mediated resistance genes tet(X) and mcr-1 in Escherichia coli clinical isolates from Pakistan. mSphere 2021, 6, e0069521. [Google Scholar] [CrossRef] [PubMed]
  13. Kennedy, K.J.; Roberts, J.L.; Collignon, P. Escherichia coli bacteraemia in Canberra: Incidence and clinical features. Med. J. Aust. 2008, 188, 209–213. [Google Scholar] [CrossRef]
  14. Yayan, J.; Ghebremedhin, B.; Rasche, K. No Development of imipenem resistance in pneumonia caused by Escherichia coli. Medicine 2015, 94, e1020. [Google Scholar] [CrossRef]
  15. Bradford, P.A.; Petersen, P.J.; Fingerman, I.M.; White, D.G. Characterization of expanded-spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease. J. Antimicrob. Chemother. 1999, 44, 607–610. [Google Scholar] [CrossRef]
  16. Kemmett, K.; Williams, N.; Chaloner, G.; Humphrey, S.; Wigley, P.; Humphrey, T. The contribution of systemic Escherichia coli infection to the early mortalities of commercial broiler chickens. Avian Pathol. 2014, 43, 37–42. [Google Scholar] [CrossRef]
  17. Aminshahidi, M.; Arastehfar, A.; Pouladfar, G.; Arman, E.; Fani, F. Diarrheagenic Escherichia coli and Shigella with high rate of extended-spectrum Beta-lactamase production: Two predominant etiological agents of acute diarrhea in Shiraz, Iran. Microb. Drug Resist. 2017, 23, 1037–1044. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Angulo-Zamudio, U.A.; Gutiérrez-Jiménez, J.; Monroy-Higuera, L.; Flores-Villaseñor, H.; Leon-Sicairos, N.; Velazquez-Roman, J.; Vidal, J.E.; Tapia-Pastrana, G.; Canizalez-Roman, A. Non-diarrheagenic and diarrheagenic E. coli carrying supplementary virulence genes (SVG) are associated with diarrhea in children from Mexico. Microb. Pathog. 2021, 157, 104994. [Google Scholar] [CrossRef]
  19. Clements, A.; Young, J.C.; Constantinou, N.; Frankel, G. Infection strategies of enteric pathogenic Escherichia coli. Gut Microbes 2012, 3, 71–87. [Google Scholar] [CrossRef] [Green Version]
  20. Zajacova, Z.S.; Konstantinova, L.; Alexa, P. Detection of virulence factors of Escherichia coli focused on prevalence of EAST1 toxin in stool of diarrheic and non-diarrheic piglets and presence of adhesion involving virulence factors in astA positive strains. Vet. Microbiol. 2012, 154, 369–375. [Google Scholar] [CrossRef]
  21. Awad, W.S.; El-Sayed, A.A.; Mohammed, F.F.; Bakry, N.M.; Abdou, N.-E.M.I.; Kamel, M.S. Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves. Trop. Anim. Health Prod. 2020, 52, 3173–3185. [Google Scholar] [CrossRef]
  22. Clermont, O.; Gordon, D.; Denamur, E. Guide to the various phylogenetic classification schemes for Escherichia coli and the correspondence among schemes. Microbiology 2015, 161, 980–988. [Google Scholar] [CrossRef]
  23. Henriot, C.P.; Celle, H.; Klaba, V.; Biguenet, A.; Miège, C.; Daval, A.; Amiotte-Suchet, P.; Beugnot, J.-C.; Karbowiak, T.; Bertrand, X. Effect of a karst system (France) on extended spectrum beta-lactamase (ESBL)-producing Escherichia coli. Water Res. 2023, 230, 119582. [Google Scholar] [CrossRef] [PubMed]
  24. Seenama, C.; Thamlikitkul, V.; Ratthawongjirakul, P. Multilocus sequence typing and blaESBL characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolated from healthy humans and swine in Northern Thailand. Infect. Drug Resist. 2019, 12, 2201–2214. [Google Scholar] [CrossRef] [Green Version]
  25. Dos Santos Alves, T.; Rosa, V.S.; da Silva Leite, D.; Guerra, S.T.; Joaquim, S.F.; Guimarães, F.F.; de Figueiredo Pantoja, J.C.; Lucheis, S.B.; Rall, V.L.M.; Hernandes, R.T. Genome-based characterization of multidrug-resistant Escherichia coli isolated from clinical bovine mastitis. Microbiol. Res. 2023, 80, 89. [Google Scholar] [CrossRef]
  26. Jonare, L.; Östlund, E.; Söderlund, R.; Hansson, I.; Aspán, A.; Jansson, D.S. Core genome multilocus sequence typing (cgMLST) confirms systemic spread of avian pathogenic Escherichia coli (APEC) in broilers with cellulitis. Veter. Microbiol. 2023, 282, 109755. [Google Scholar] [CrossRef]
  27. Lu, X.; Zhang, P.; Du, P.; Zhang, X.; Wang, J.; Yang, Y.; Sun, H.; Wang, Z.; Cui, S.; Li, R. Prevalence and genomic characteristics of mcr-positive Escherichia coli strains isolated from humans, pigs, and foods in China. Microbiol. Spectr. 2023, 12, e04569-22. [Google Scholar] [CrossRef] [PubMed]
  28. García-Meniño, I.; García, V.; Mora, A.; Díaz-Jiménez, D.; Flament-Simon, S.C.; Alonso, M.P.; Blanco, J.E.; Blanco, M.; Blanco, J. Swine enteric colibacillosis in Spain: Pathogenic potential of mcr-1 ST10 and ST131 E. coli isolates. Front. Microbiol. 2018, 9, 2659. [Google Scholar] [CrossRef]
  29. Wen, F.; Yang, J.; Li, A.; Gong, Z.; Yang, L.; Cheng, Q.; Wang, C.; Zhao, M.; Yuan, S.; Chen, Y.; et al. Genetic characterization and phylogenetic analysis of porcine epidemic diarrhea virus in Guangdong, China, between 2018 and 2019. PLoS ONE 2021, 16, e0253622. [Google Scholar] [CrossRef] [PubMed]
  30. Lindsey, R.L.; Garcia-Toledo, L.; Fasulo, D.; Gladney, L.M.; Strockbine, N. Multiplex polymerase chain reaction for identification of Escherichia coli, Escherichia albertii and Escherichia fergusonii. J. Microbiol. Methods 2017, 140, 1–4. [Google Scholar] [CrossRef] [Green Version]
  31. Lin, J.; Tang, B.; Zheng, X.; Chang, J.; Ma, J.; He, Y.; Yang, H.; Wu, Y. Emergence of Incl2 plasmid-mediated colistin resistance in avian Escherichia fergusonii. FEMS Microbiol. Lett. 2022, 369, fnac016. [Google Scholar] [CrossRef]
  32. Tang, B.; Wang, J.; Zheng, X.; Chang, J.; Ma, J.; Wang, J.; Ji, X.; Yang, H.; Ding, B. Antimicrobial resistance surveillance of Escherichia coli from chickens in the Qinghai Plateau of China. Front. Microbiol. 2022, 13, 885132. [Google Scholar] [CrossRef] [PubMed]
  33. Peng, K.; Yin, Y.; Li, Y.; Qin, S.; Liu, Y.; Yang, X.; Wang, Z.; Li, R. QitanTech Nanopore long-read sequencing enables rapid resolution of complete genomes of multi-drug resistant pathogens. Front. Microbiol. 2022, 13, 778659. [Google Scholar] [CrossRef] [PubMed]
  34. Yu, Y.; Tang, B.; Dai, R.; Zhang, B.; Chen, L.; Yang, H.; Zhao, G.; Ding, X. Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd. Appl. Microbiol. Biotechnol. 2018, 102, 2621–2633. [Google Scholar] [CrossRef] [PubMed]
  35. Zhou, W.; Lin, R.; Zhou, Z.; Ma, J.; Lin, H.; Zheng, X.; Wang, J.; Wu, J.; Dong, Y.; Jiang, H.; et al. Corrigendum: Antimicrobial resistance and genomic characterization of Escherichia coli from pigs and chickens in Zhejiang, China. Front. Microbiol. 2023, 14, 1102931. [Google Scholar] [CrossRef] [PubMed]
  36. Tang, B.; Siddique, A.; Jia, C.; Ed-Dra, A.; Wu, J.; Lin, H.; Yue, M. Genome-based risk assessment for foodborne Salmonella enterica from food animals in China: A One Health perspective. Int. J. Food Microbiol. 2023, 390, 110120. [Google Scholar] [CrossRef] [PubMed]
  37. Brettin, T.; Davis, J.J.; Disz, T.; Edwards, R.A.; Gerdes, S.; Olsen, G.J.; Olson, R.; Overbeek, R.; Parrello, B.; Pusch, G.D.; et al. RASTtk: A modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci. Rep. 2015, 5, 8365. [Google Scholar] [CrossRef] [Green Version]
  38. Zankari, E.; Hasman, H.; Cosentino, S.; Vestergaard, M.; Rasmussen, S.; Lund, O.; Aarestrup, F.M.; Larsen, M.V. Identification of acquired antimicrobial resistance genes. J. Antimicrob. Chemother. 2012, 67, 2640–2644. [Google Scholar] [CrossRef]
  39. Carattoli, A.; Zankari, E.; Garcìa-Fernandez, A.; Larsen, M.; Lund, O.; Voldby Villa, L.; Møller Aarestrup, F.; Hasman, H. In silico detection and typing of plasmids. Antimicrob using PlasmidFinder and plasmid multilocus sequence typing. Agents Chemother. 2014, 58, 3895–3903. [Google Scholar] [CrossRef] [Green Version]
  40. Joensen, K.G.; Tetzschner, A.M.; Iguchi, A.; Aarestrup, F.M.; Scheutz, F. Rapid and Easy In Silico Serotyping of Escherichia coli isolates by use of whole-genome sequencing data. J. Clin. Microbiol. 2015, 53, 2410–2426. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  41. Feng, Y.; Zou, S.; Chen, H.; Yu, Y.; Ruan, Z. BacWGSTdb 2.0: A one-stop repository for bacterial whole-genome sequence typing and source tracking. Nucleic Acids Res. 2021, 49, D644–D650. [Google Scholar] [CrossRef] [PubMed]
  42. Alikhan, N.-F.; Petty, N.K.; Ben Zakour, N.L.; Beatson, S.A. BLAST Ring Image Generator (BRIG): Simple prokaryote genome comparisons. BMC Genom. 2011, 12, 402. [Google Scholar] [CrossRef] [Green Version]
  43. Jønsson, R.; Struve, C.; Jenssen, H.; Krogfelt, K.A. The wax moth Galleria mellonella as a novel model system to study Enteroaggregative Escherichia coli pathogenesis. Virulence 2017, 8, 1894–1899. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  44. Tsai, C.J.-Y.; Loh, J.M.S.; Proft, T. Galleria mellonella infection models for the study of bacterial diseases and for antimicrobial drug testing. Virulence 2016, 7, 214–229. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Mohawk, K.L.; Melton-Celsa, A.R.; Zangari, T.; Carroll, E.E.; O’Brien, A.D. Pathogenesis of Escherichia coli O157:H7 strain 86-24 following oral infection of BALB/c mice with an intact commensal flora. Microb. Pathog. 2010, 48, 131–142. [Google Scholar] [CrossRef] [Green Version]
  46. Yang, B.; Feng, L.; Wang, F.; Wang, L. Enterohemorrhagic Escherichia coli senses low biotin status in the large intestine for colonization and infection. Nat. Commun. 2015, 6, 6592. [Google Scholar] [CrossRef] [Green Version]
  47. Wang, Y.; Liu, F.; Xu, X.; Huang, H.; Lyu, N.; Ma, S.; Chen, L.; Mao, M.; Hu, Y.; Song, X. Detection of plasmid-mediated tigecycline resistance gene tet(X4) in a Salmonella enterica Serovar Llandoff isolate. Infect. Microbes Dis. 2021, 3, 198–204. [Google Scholar] [CrossRef]
  48. Zhai, W.; Tian, Y.; Lu, M.; Zhang, M.; Song, H.; Fu, Y.; Ma, T.; Sun, C.; Bai, L.; Wang, Y.; et al. Presence of mobile tigecycline resistance gene tet(X4) in Clinical Klebsiella pneumoniae. Microbiol. Spectr. 2022, 10, e0108121. [Google Scholar] [CrossRef]
  49. Nguyen, M.N.; Hoang, H.T.T.; Xavier, B.B.; Lammens, C.; Le, H.T.; Hoang, N.T.B.; Nguyen, S.T.; Pham, N.T.; Goossens, H.; Dang, A.D. Prospective One Health genetic surveillance in Vietnam identifies distinct blaCTX-M-harbouring Escherichia coli in food-chain and human-derived samples. Clin. Microbiol. Infect. 2021, 27, 1515.e1–1515.e8. [Google Scholar] [CrossRef] [PubMed]
  50. García, V.; García-Meniño, I.; Mora, A.; Flament-Simon, S.C.; Díaz-Jiménez, D.; Blanco, J.E.; Alonso, M.P.; Blanco, J. Co-occurrence of mcr-1, mcr-4 and mcr-5 genes in multidrug-resistant ST10 Enterotoxigenic and Shiga toxin-producing Escherichia coli in Spain (2006–2017). Int. J. Antimicrob. Agents 2018, 52, 104–108. [Google Scholar] [CrossRef]
  51. Day, M.J.; Hopkins, K.L.; Wareham, D.W.; Toleman, M.A.; Elviss, N.; Randall, L.; Teale, C.; Cleary, P.; Wiuff, C.; Doumith, M. Extended-spectrum β-lactamase-producing Escherichia coli in human-derived and foodchain-derived samples from England, Wales, and Scotland: An epidemiological surveillance and typing study. Lancet Infect. Dis. 2019, 19, 1325–1335. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  52. Madec, J.-Y.; Haenni, M.; Nordmann, P.; Poirel, L. Extended-spectrum β-lactamase/AmpC-and carbapenemase-producing Enterobacteriaceae in animals: A threat for humans? Clin. Microbiol. Infect. 2017, 23, 826–833. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Ali, A.; Fontana, H.; Sano, E.; Li, R.; Humayon, M.; Rahman, S.; Lincopan, N.; Mohsin, M. Genomic features of a high-risk mcr-1.1-positive Escherichia coli ST10 isolated from cattle farm environment. Environ. Sci. Pollut. Res. 2021, 28, 54147–54152. [Google Scholar] [CrossRef]
  54. Dos Anjos, C.; Sabino, C.P.; Bueris, V.; Fernandes, M.R.; Pogliani, F.C.; Lincopan, N.; Sellera, F.P. Antimicrobial blue light inactivation of international clones of multidrug-resistant Escherichia coli ST10, ST131 and ST648. Photodiagnosis Photodyn. Ther. 2019, 27, 51–53. [Google Scholar] [CrossRef]
  55. Ćwiek, K.; Woźniak-Biel, A.; Karwańska, M.; Siedlecka, M.; Lammens, C.; Rebelo, A.R.; Hendriksen, R.S.; Kuczkowski, M.; Chmielewska-Władyka, M.; Wieliczko, A. Phenotypic and genotypic characterization of mcr-1-positive multidrug-resistant Escherichia coli ST93, ST117, ST156, ST10, and ST744 isolated from poultry in Poland. Braz. J. Microbiol. 2021, 52, 1597–1609. [Google Scholar] [CrossRef]
  56. Zhang, X.; Fang, C.; Zhang, J.; Hua, W.; He, R.; Zhou, M. Carbapenemase- and colistin resistant Escherichia coli strains from children in China: High genetic diversity and first report of bla(NDM-5), bla(CTX-M-65), bla(OXA-10), bla(TEM-1), and mcr-1.1 genes co-occurrence in E. coli ST156. Infect. Drug Resist. 2022, 15, 5315–5320. [Google Scholar] [CrossRef]
  57. Lekunberri, I.; Balcázar, J.L.; Borrego, C.M. Metagenomic exploration reveals a marked change in the river resistome and mobilome after treated wastewater discharges. Environ. Pollut. 2018, 234, 538–542. [Google Scholar] [CrossRef]
  58. Dolejska, M.; Villa, L.; Poirel, L.; Nordmann, P.; Carattoli, A. Complete sequencing of an IncHI1 plasmid encoding the carbapenemase NDM-1, the ArmA 16S RNA methylase and a resistance–nodulation–cell division/multidrug efflux pump. J. Antimicrob. Chemother. 2013, 68, 34–39. [Google Scholar] [CrossRef] [Green Version]
  59. Kubasova, T.; Cejkova, D.; Matiasovicova, J.; Sekelova, Z.; Polansky, O.; Medvecky, M.; Rychlik, I.; Juricova, H. Antibiotic resistance, core-genome and protein expression in IncHI1 plasmids in Salmonella Typhimurium. Genome Biol. Evol. 2016, 8, 1661–1671. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Lawley, T.D.; Gilmour, M.W.; Gunton, J.E.; Tracz, D.M.; Taylor, D.E. Functional and mutational analysis of conjugative transfer region 2 (Tra2) from the IncHI1 plasmid R27. J. Bacteriol. 2003, 185, 581–591. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Sennati, S.; Di Pilato, V.; Riccobono, E.; Di Maggio, T.; Villagran, A.L.; Pallecchi, L.; Bartoloni, A.; Rossolini, G.M.; Giani, T. Citrobacter braakii carrying plasmid-borne mcr-1 colistin resistance gene from ready-to-eat food from a market in the Chaco region of Bolivia. J. Antimicrob. Chemother. 2017, 72, 2127–2129. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Li, Y.; Wang, Z.; Dong, H.; Wang, M.; Qin, S.; Chen, S.; Li, R. Emergence of tet(X4)-positive hypervirulent Klebsiella pneumoniae of food origin in China. LWT 2023, 173, 114280. [Google Scholar] [CrossRef]
  63. Heredia, N.; García, S. Animals as sources of food-borne pathogens: A review. Anim. Nutr. 2018, 4, 250–255. [Google Scholar] [CrossRef]
  64. Lei, T.; Zhang, J.; Jiang, F.; He, M.; Zeng, H.; Chen, M.; Wu, S.; Wang, J.; Ding, Y.; Wu, Q. First detection of the plasmid-mediated colistin resistance gene mcr-1 in virulent Vibrio parahaemolyticus. Int. J. Food Microbiol. 2019, 308, 108290. [Google Scholar] [CrossRef] [PubMed]
  65. Sumbana, J.J.; Santona, A.; Fiamma, M.; Taviani, E.; Deligios, M.; Zimba, T.; Lucas, G.; Sacarlal, J.; Rubino, S.; Paglietti, B. Extraintestinal pathogenic Escherichia coli ST405 isolate coharboring blaNDM-5 and blaCTXM-15: A new threat in Mozambique. Microb. Drug Resist. 2021, 27, 1633–1640. [Google Scholar] [CrossRef]
  66. Turchi, B.; Dec, M.; Bertelloni, F.; Winiarczyk, S.; Gnat, S.; Bresciani, F.; Viviani, F.; Cerri, D.; Fratini, F. Antibiotic susceptibility and virulence factors in Escherichia coli from sympatric wildlife of the Apuan Alps regional park (Tuscany, Italy). Microb. Drug Resist. 2019, 25, 772–780. [Google Scholar] [CrossRef] [Green Version]
  67. Ma, J.; Tang, B.; Lin, J.; Ed-Dra, A.; Lin, H.; Wu, J.; Dong, Y.; Yang, H.; Yue, M. Genome assessment of carbapenem-and colistin-resistant Escherichia coli from patients in a sentinel hospital in China. Cells 2022, 11, 3480. [Google Scholar] [CrossRef]
  68. Montealegre, M.C.; Rodríguez, A.T.; Roy, S.; Hossain, M.I.; Islam, M.A.; Lanza, V.F.; Julian, T.R. High genomic diversity and heterogenous origins of pathogenic and antibiotic-resistant Escherichia coli in household settings represent a challenge to reducing transmission in low-income settings. Msphere 2020, 5, e00704-19. [Google Scholar] [CrossRef] [Green Version]
  69. Yatsuyanagi, J.; Saito, S.; Miyajima, Y.; Amano, K.-I.; Enomoto, K. Characterization of Atypical Enteropathogenic Escherichia coli strains harboring the astA gene that were associated with a waterborne outbreak of diarrhea in Japan. J. Clin. Microbiol. 2003, 41, 2033–2039. [Google Scholar] [CrossRef] [Green Version]
Pathogens 12 00903 g001 550
Figure 1. The antimicrobial resistance of strain 812A1-131 to tigecycline. (A) The growth of strain 812A1-131 on LB plates with different tigecycline concentrations. (B) E-test analysis of strain 812A1-131. E. coli ATCC 25922 was used as the control.
Figure 1. The antimicrobial resistance of strain 812A1-131 to tigecycline. (A) The growth of strain 812A1-131 on LB plates with different tigecycline concentrations. (B) E-test analysis of strain 812A1-131. E. coli ATCC 25922 was used as the control.
Pathogens 12 00903 g001
Pathogens 12 00903 g002 550
Figure 2. S1-PFGE (A) and Southern blot (B) of strain 812A1-131. The plasmid harboring the tet(X4) gene is indicated with arrows.
Figure 2. S1-PFGE (A) and Southern blot (B) of strain 812A1-131. The plasmid harboring the tet(X4) gene is indicated with arrows.
Pathogens 12 00903 g002
Pathogens 12 00903 g003 550
Figure 3. Phylogenetic tree of ST10 strains similar to 812A1-131 based on SNPs of core genomes.
Figure 3. Phylogenetic tree of ST10 strains similar to 812A1-131 based on SNPs of core genomes.
Pathogens 12 00903 g003
Pathogens 12 00903 g004 550
Figure 4. Virulence gene profiles of ST isolates closely related to 812A1-131.
Figure 4. Virulence gene profiles of ST isolates closely related to 812A1-131.
Pathogens 12 00903 g004
Pathogens 12 00903 g005 550
Figure 5. Plasmid characteristics and genetic environment of plasmid p812A1-tetX4-193K. Ring comparison of p812A1-tetX4-193K with similar plasmids was conducted using BRIG.
Figure 5. Plasmid characteristics and genetic environment of plasmid p812A1-tetX4-193K. Ring comparison of p812A1-tetX4-193K with similar plasmids was conducted using BRIG.
Pathogens 12 00903 g005
Pathogens 12 00903 g006 550
Figure 6. Survival curve of Galler mellonella. The black line indicates the Galler mellonella survival rate of the PBS control group. The blue and red lines indicate the experimental group (812A1-131) and the highly virulent control group (637A2), respectively. The green (DH5α) and purple (inactivated 812A1-131) lines indicate the two avirulent control groups.
Figure 6. Survival curve of Galler mellonella. The black line indicates the Galler mellonella survival rate of the PBS control group. The blue and red lines indicate the experimental group (812A1-131) and the highly virulent control group (637A2), respectively. The green (DH5α) and purple (inactivated 812A1-131) lines indicate the two avirulent control groups.
Pathogens 12 00903 g006
Table
Table 1. MIC and AMR genes of strain 812A1-131.
Table 1. MIC and AMR genes of strain 812A1-131.
IDsAntibiotics812A1-131
MIC (μg/mL)ARGs
1Ampicillin>128 RblaTEM-1B, blaTEM-141, blaTEM-206, blaCTX-M-55, blaOXA-10, blaCTX-M-14
2Amoxicillin/clavulanic acid>128/64 R
3Cefotaxime>8 RblaCTX-M-55, blaCTX-M-14
4Meropenem1 S/
5Amikacin>64 RrmtB
6Gentamicin>32 Raac(3)-IV, rmtB
7Colistin1 S/
8Ceftiofur>32 R
9Ciprofloxacin>8 RqnrS1, parC, gyrA
10Trimethoprim/sulfamethoxazole>0.5/9.5 Rsul2
11Tetracycline>64 Rtet(X4), tet(A)
12Tigecycline>16 Rtet(X4)
13Florfenicol128 RfloR
R: resistance; S: susceptibility; /: no AMR gene. The results are based on CLSI guidelines.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, J.; Huang, Y.; Guan, C.; Li, J.; Yang, H.; Zhao, G.; Liu, C.; Ma, J.; Tang, B. Characterization of an Escherichia coli Isolate Coharboring the Virulence Gene astA and Tigecycline Resistance Gene tet(X4) from a Dead Piglet. Pathogens 2023, 12, 903. https://doi.org/10.3390/pathogens12070903

AMA Style

Wang J, Huang Y, Guan C, Li J, Yang H, Zhao G, Liu C, Ma J, Tang B. Characterization of an Escherichia coli Isolate Coharboring the Virulence Gene astA and Tigecycline Resistance Gene tet(X4) from a Dead Piglet. Pathogens. 2023; 12(7):903. https://doi.org/10.3390/pathogens12070903

Chicago/Turabian Style

Wang, Jianmei, Yuting Huang, Chunjiu Guan, Jie Li, Hua Yang, Guoping Zhao, Canying Liu, Jiangang Ma, and Biao Tang. 2023. "Characterization of an Escherichia coli Isolate Coharboring the Virulence Gene astA and Tigecycline Resistance Gene tet(X4) from a Dead Piglet" Pathogens 12, no. 7: 903. https://doi.org/10.3390/pathogens12070903

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop