# THE MOLECULAR MECHANISMS OF ANTIBIOTIC RESISTANCE IN AQUATIC PATHOGENS

EDITED BY : Xiangmin Lin, Patrícia Poeta, Bo Peng and Xiao-Peng Xiong PUBLISHED IN : Frontiers in Cellular and Infection Microbiology

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ISSN 1664-8714 ISBN 978-2-88966-169-5 DOI 10.3389/978-2-88966-169-5

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# THE MOLECULAR MECHANISMS OF ANTIBIOTIC RESISTANCE IN AQUATIC PATHOGENS

Topic Editors:

Xiangmin Lin, Fujian Agriculture and Forestry University, China Patrícia Poeta, University of Trás-os-Montes and Alto Douro, Portugal Bo Peng, Sun Yat-sen University, China Xiao-Peng Xiong, Sanford Burnham Institute for Medical Research, United States

Citation: Lin, X., Poeta, P., Peng, B., Xiong, X.-P., eds. (2020). The Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-169-5

# Table of Contents

*05 Editorial: The Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens*

Xiangmin Lin, Patrícia Poeta and Bo Peng

*07 Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of* Aeromonas veronii

Dan Wang, Hong Li, Xiang Ma, Yanqiong Tang, Hongqian Tang, Xinwen Hu and Zhu Liu

*19 Genetic Features of Antarctic* Acinetobacter radioresistens *Strain A154 Harboring Multiple Antibiotic-Resistance Genes*

Andrés Opazo-Capurro, Paul G. Higgins, Julia Wille, Harald Seifert, Camila Cigarroa, Paulina González-Muñoz, Mario Quezada-Aguiluz, Mariana Domínguez-Yévenes, Helia Bello-Toledo, Luis Vergara and Gerardo González-Rocha

Hossam Abdelhamed, Reshma Ramachandran, Ozan Ozdemir, Geoffrey Waldbieser and Mark L. Lawrence

*47 Antibiotic Resistance Profile, Outer Membrane Proteins, Virulence Factors and Genome Sequence Analysis Reveal Clinical Isolates of* Enterobacter *are Potential Pathogens Compared to Environmental Isolates*

Mitali Mishra, Sasmita Panda, Susmita Barik, Arup Sarkar, Durg Vijai Singh and Harapriya Mohapatra

*60 5-Methylindole Potentiates Aminoglycoside Against Gram-Positive Bacteria Including* Staphylococcus aureus *Persisters Under Hypoionic Conditions*

Fengqi Sun, Mengmeng Bian, Zhongyan Li, Boyan Lv, Yuanyuan Gao, Yan Wang and Xinmiao Fu

Shihui Zhou, Xueting Tu, Huanying Pang, Rowena Hoare, Sean J. Monaghan, Jiajun Luo and Jichan Jian

*95 SmpB and tmRNA Orchestrate Purine Pathway for the Trimethoprim Resistance in* Aeromonas veronii

Dan Wang, Hong Li, Wasi Ullah Khan, Xiang Ma, Hongqian Tang, Yanqiong Tang, Dongyi Huang and Zhu Liu

*102 Identification of Antimicrobial Resistance Determinants in* Aeromonas veronii *Strain MS-17-88 Recovered From Channel Catfish (*Ictalurus punctatus*)*

Hasan C. Tekedar, Mark A. Arick II, Chuan-Yu Hsu, Adam Thrash, Jochen Blom, Mark L. Lawrence and Hossam Abdelhamed

# Editorial: The Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens

#### Xiangmin Lin1 \*, Patrícia Poeta2 and Bo Peng3

1 School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China, 2 Microbiology and Antibiotic Resistance Team (MicroART), Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Vila Real, Portugal, 3 School of Life Sciences, Sun Yat-sen University, Guangzhou, China

Keywords: aquatic pathogens, antibiotic resistance, mechanisms, multidrug resistance, prevention strategies

#### **Editorial on the Research Topic**

#### **The Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens**

Bacterial antibiotic resistance has become a formidable problem in the treatment of infectious diseases worldwide. The molecular mechanisms of antibiotic resistance remain elusive, especially for aquatic pathogens. Antibiotic resistance and virulence determinants in environmental/aquatic bacterial pathogens are the subject of five research articles published in this Research Topic. Mishra et al. have investigated the influence of outer membrane proteins (OMPs) on the antibiotic resistance profile and virulence factors of Enterobacter isolated from aquatic environments and clinical settings. The environmental Enterobacter isolates lack OmpC, are unable to invade host cells, and induce low amounts of reactive oxygen species (ROS) production by neutrophils. In contrast, a clinical Enterobacter isolate described possesses OmpF, has better invasive and adhesive capabilities, and stimulates a higher amount of ROS production. Furthermore, some clinical Enterobacter isolates which have the ompA gene also have the strong capacity to form biofilms. Taking a different angle, Abdelhamed et al. and Opazo-Capurro et al. have sequenced the whole genomes of the Edwardsiella piscicida MS 18-99 strain possessing a 117.4 kb conjugative plasmid pEPMS-1899, and Acinetobacter radioresistens A154 harboring several plasmids, respectively. The genome analysis revealed that several antimicrobial resistance genes/elements confer resistance to various antibiotics, such as tetA&R, strA&B, sul2, and floR in MS 18-99, arsA and arsD on pEPMS-1899, and tetB, strA, strB, aph(3′ )-Vla, aac(3)-IIa, blaSCO-1, blaTEM-1B, and blaPER-2 in A. radioresistens. A study by Montso et al. describes the presence of genes such as shiga toxins (stx) and bundle-forming pili (bfpA) that are related to virulence determinants and antibiotic resistance in atypical enteropathogenic Escherichia coli (aEPEC) O177, the first reported occurrence of the aEPEC O177 strain in South African cattle. Tekedar et al. have characterized genome sequences of Aeromonas veronii MS 17-88 strains isolated from channel catfish to uncover the mechanism of multidrug resistance. This strain has several antimicrobial resistance genes/elements such astet(34), tet(35), tet(E), tetR, mcr-7.1, and floR. These five studies have all contributed further evidence of the abundance and prevalence of antibiotic resistance genes in different bacterial genomes.

Proteins are the fundamental and vital material of life. Ribosome rescue is a mechanism to maintain protein synthesis when mistakes or stalling occur. Quality control of protein synthesis is part of this process, which requires transfer-messenger RNA (tmRNA) and small protein B (SmpB). In this Research Topic, there are three articles from Prof. Liu's group describing the involvement of tmRNA and SmpB in the antibiotic resistance and persistence development of A. veronii. The transcriptomic data presented reveal the downregulation of protein secretion systems, such as the type VI secretion system (T6SS) and type IV pilus assembly, in the SmpB deficient strain.

#### Edited and reviewed by:

John S. Gunn, The Research Institute at Nationwide Children's Hospital, United States

> \*Correspondence: Xiangmin Lin<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology

Received: 23 July 2020 Accepted: 20 August 2020 Published: 24 September 2020

#### Citation:

Lin X, Poeta P and Peng B (2020) Editorial: The Molecular Mechanisms of Antibiotic Resistance in Aquatic Pathogens. Front. Cell. Infect. Microbiol. 10:586460. doi: 10.3389/fcimb.2020.586460

This downregulation attenuated the production of virulence factors and the colonization ability of A. veronii. However, the alternative rescue factor A (AvrA) acts as a substitute for virulence compensation. The decreased level of avrA expression increases the expression level of iron-sulfur protein activator iscR, resulting in an increase in bacterial antioxidant ability through enhancement of the synthesis of the ironsulfur protein and assembly of iron-sulfur clusters (Santos et al., 2014). SmpB deficiency showed attenuated virulence and enhanced tolerance to oxidative stress. In another study, the deletion of the smpB gene in A. veronii enhanced the expression of enzymes related to adenosine metabolism, which leads to increases in the products such as adenosine AMP, cAMP and deoxyadenosine. By comparison, the deletion of ssrA (tmRNA) upregulated the expression of guanosine metabolism and hypoxanthine synthesis, eventually increasing the levels of the metabolic product xanthine, which in turn downregulated the genes related to the efflux pump such as acrA and acrB. With the ssrA and smpB genes deleted, the minimal inhibitory concentration of trimethoprim against A. veronii was lower. Therefore, this study demonstrates the effect of SmpB and tmRNA on different branches of purine metabolism and tolerance to trimethoprim (Wang et al.). A study by Yu et al. showed that the deletion of ssrA (tmRNA) increased the capacity of A. veronii to form persister cells. Moreover, 1ssrA significantly raised the intercellular levels of Nacetylglucosamine production, promoting NaCl osmotic stress tolerance. To confirm this, the authors exogenously added Nacetylglucosamine to the medium and observed resistance to osmotic pressure and higher persistence to cefotaxime in A. veronii. Overall, these three research articles suggest that the trans-translation system might be an effective target in the treatment of A. veronii and other multidrug resistant aquatic bacterial pathogens.

#### REFERENCES

Santos, J. A., Alonso-García, N., Macedo-Ribeiro, S., and Pereira, P. J.B. (2014). The unique regulation of iron-sulfur cluster biogenesis in a Gram-positive bacterium. Proc. Natl. Acad. Sci. U.S.A. 111, 2251–2260. doi: 10.1073/pnas.1322728111

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Enhancing the efficiency of traditional antibiotics is a vital strategy for combatting the antibiotic resistance in bacterial pathogens. A study by Sun et al. has revealed that indole and its derivative 2,5-methylindole potentiate the killing efficacy of tobramycin against persister cells of Staphylococcus aureus. Furthermore, combinations of indole and its derivatives with aminoglycoside antibiotics were effective in inhibiting the growth of many Gram-positive bacterial pathogens such as Streptococcus pyogenes and Enterococcus faecalis. Finally, Zhou et al. have studied the role of the type III secretion system (T3SS) on the pathogenicity of Vibrio alginolyticus. Swarming motility is inhibited in the T3SS mutant strain tyeA and its virulence toward zebrafish is also reduced. Further, the tyeA mutant strain is extremely susceptible to cefoperazone, minocycline, amikacin, and gentamicin. The effects of a live attenuated vaccine made from the mutant strain on the survival of zebrafish was tested. The data presented show that the survival of zebrafish infected with mutant was enhanced compared to the control infected with the wild-type strain. Both of these approaches may pave the way for the development of an effective treatment strategy to combat antibiotic resistance in bacterial pathogens.

#### AUTHOR CONTRIBUTIONS

XL and PP wrote the manuscript. BP revised and approved the final version of the manuscript. All authors conceived the outline of the manuscript.

#### ACKNOWLEDGMENTS

This work was sponsored by grants from NSFC projects (Nos. 31670129). We thank Dr. Xiaopeng Xiong for suggesting on the manuscript.

Copyright © 2020 Lin, Poeta and Peng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii

Dan Wang, Hong Li, Xiang Ma, Yanqiong Tang, Hongqian Tang, Xinwen Hu\* and Zhu Liu\*

The superbacteria *Aeromonas veronii* displays not only a strong pathogenicity but also

*Key Laboratory of Tropical Biological Resources of Ministry of Education, School of Life and Pharmaceutical Sciences, Hainan University, Haikou, China*

the resistance to nine kinds of antibiotics, resulting in the economic losses and health hazards. Small Protein B (SmpB) plays an important role in protein quality control, virulence, and stress reactions. Transcriptomic data revealed that expressions of the type IV pilus assembly and type VI secretion system (T6SS) proteins were downregulated in SmpB deficiency, indicating that the virulence of *A. veronii* might be attenuated. Although SmpB deletion decreased colonization in the mouse spleen and liver, LD50 of the *smpB* mutant was not altered as expected, compared with the wild type. Further, the transcriptomic and quantitative RT-PCR analyses showed that the combination of the downregulated AvrA and the upregulated iron-sulfur protein activator IscR, mediated the oxidative tolerance in *smpB* deletion. Next a reporter plasmid was constructed in which the promoter of *iscR* was applied to control the expression of the enhanced green fluorescent protein (eGFP) gene. When the reporter plasmid was co-expressed with the AvrA expression into *E. coli*, the relative fluorescence intensity was decreased significantly, suggesting that AvrA bound to *iscR* mRNA by base pairing, which in turn relieved the inhibition of *iscR* and intensified the downstream iron-sulfur proteins. Collectively, the *smpB* mutant exhibited an attenuated virulence in mice and enhanced tolerances to oxidative stress. This study demonstrates the complexity of gene regulation networks mediated by sRNA in systems biology, and also reflects the strong adaptability of superbacteria *A. veronii* in the process of evolution.

Keywords: oxidative, stress, iron deficiency, iron/sulfur clusters, virulence

# INTRODUCTION

With the application of antimicrobial drugs, a wide range of mechanisms have evolved for bacteria to combat the huge selection pressures of antibacterial agents. Superbacteria have accumulated in a long evolutionary history, while the abuse of antibiotics has accelerated its formation. The superbacterium Aeromonas veronii (A. veronii) has been isolated, which has displayed not only a strong pathogenicity but also the resistance to nine kinds of antibiotics (Liu et al., 2016, 2018). As A. veronii infects fish, it causes fish canker and perforated disease, eventually resulting in economic losses. Furthermore, A. veronii increases human diseases such as neonatal sepsis, diarrhea and

#### Edited by:

*Xiangmin Lin, Fujian Agriculture and Forestry University, China*

#### Reviewed by:

*Shengkang Li, Shantou University, China Zhihong Xie, Yantai Institute of Coastal Zone Research (CAS), China*

#### \*Correspondence:

*Xinwen Hu<EMAIL_ADDRESS>Zhu Liu<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology*

> Received: *14 March 2019* Accepted: *17 April 2019* Published: *08 May 2019*

#### Citation:

*Wang D, Li H, Ma X, Tang Y, Tang H, Hu X and Liu Z (2019) Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii. Front. Cell. Infect. Microbiol. 9:142. doi: 10.3389/fcimb.2019.00142* Wang et al. sRNA Regulation Increases Stress Tolerances

dysentery (Aguilera-Arreola et al., 2007). In view of the wide array of infections to the host and a strong tolerance to antibiotics, A. veronii is worth researching further, as a model strain.

Proteins are the material basis of life. If there is a mistake in the protein synthesis process, ribosome rescue should occur in order to maintain life. Trans-translation which is composed of small protein B(SmpB) and transfer-message RNA (tmRNA) is the predominant form of ribosome rescue (Himeno et al., 2014). The malfunction of trans-translation exhibits more sensitivity to virulence and tolerance in most pathogens such as Mycobacterium tuberculosis, Legionella pneumophila, and Neisseria gonorrhoeae (Keiler, 2008, 2015). However, the alternative pathways have evolved to compensate for the deficiency of trans-translation. For example, the alternative rescue factor A (ArfA) acts to release the stocked mRNA in Proteobacteria and mycobacteria, and the alternative rescue factor B (ArfB) appears in 34% of bacteria (Kurita et al., 2014; Huter et al., 2017; Fiedler et al., 2018). Therefore, superbacteria might have alternate ways for SmpB defects, while no reports indicate that other functions of SmpB are maintained by small RNA (sRNA).

In this study, the transcriptome analysis was compared between wild type and smpB knockout strains, showing that the expressions of the pilus and virulence protein were downregulated after smpB deletion. However, the capacities of oxidative stress and iron were increased by sRNA downregulation in smpB knockout. Non-coding sRNA modulates bacterial metabolism and stress by base pairing with target genes and reacts faster than transcription factor regulation (Durand et al., 2015; Oliva et al., 2015). Here a sRNA designated as AvrA was revealed to negatively regulate the expression of IscR (ironsulfur cluster assembly transcriptional regulator) IscR governs iron hemostasis as a global regulator during growth and stress responses (Aguilera-Arreola et al., 2007), which exhibits a resistance to oxidants (Mandin et al., 2016), and represses ironsulfur metabolism under iron starvation (Haines et al., 2015; Carrier et al., 2017). The overproduction of IscR favored the tolerance under the nutrient deficiency and oxidative, ironlimited and membrane stresses when smpB was knocked out and followed by AvrA downregulation.

Survival under detrimental stresses is preliminary when pathogens are infected to the host and escape from macrophage recognition in the immune system (Mandin et al., 2016). And then the bacteria are transported to tissues using blood and lymph as channels, eventually causing tissue damage. Iron metabolism is of vital importance to bacteria. The proteins composed of iron/sulfur clusters participate in multiple cellular processes, such as gene expression, DNA repair, RNA modification, central metabolism and respiration (Roche et al., 2013). Iron sulfur proteins also relate to oxidoreductase which is involved in the basic metabolism of bacteria (Xu and Moller, 2008; Ilbert and Bonnefoy, 2013). So, overexpression of IscR is vital for bacteria to survive in adverse conditions.

In summary, SmpB plays an important role in protein quality control, virulence, gene transcription, and antibiotic and stress reaction (Liang and Deutscher, 2010; Li et al., 2013; Mu et al., TABLE 1 | Strains and vector used in this paper.

2013; Mraheil et al., 2017). Unlike Salmonella Typhimurium, the virulence of the A. veronii 1smpB strain did not become avirulent but maintained a strong pathogenicity. Loss of function caused by smpB deletion can be compensated through different pathways. For instance, some bacteria employ ArfA and ArfB to rescue stalled ribosomes when smpB is deficient (Keiler, 2015; Liu et al., 2016; Huter et al., 2017). Additionally, sRNA AvrA releases the trapped IscR promotor to activate the expression of iron sulfur proteins, remedying the iron deficiency after smpB destruction. In the evolutionary history of bacteria, it offers additional evidence for the formation of superbacteria.

#### MATERIALS AND METHODS

#### Plasmids

The plasmids and primers are listed in **Tables 1**, **2**, respectively.

For the construction of plasmid piscR-eGFP, the 200 bp-DNA fragments containing the promoter of iscR was amplified from the genomic DNA of A. veronii with primers proiscR\_F/proiscR\_R. In the meantime, the 750 bp-encoding region of eGFP was amplified from a plasmid pDH114 with primer eGFP\_F/eGFP\_R. The fusion was produced using the above two fragments as the templates by overlapping the PCR with primers F\_PiscR/eGFP\_R and by insertion into the backbone of the plasmid pUC19 to construct the 8(piscReGFP) vector. The plasmid pavrA was constructed in a similar manner using the primers proavrA\_F and proavrA\_R. To TABLE 2 | Primers used in vector construction and real-time qPCR.

construct the gene knockout strain of 1avrA, the flanking sequences of 1avrA were amplified and inserted into the plasmid pRE112, resulting in the gene knockout vector pRE112 avrAdel. The complementary plasmid pBBR-avrA was applied to express the extra AvrA using plasmid pBBR1MCS-2 as the backbone.

#### Strains and Culture Conditions

The strains used are listed in **Table 1**. The derivative A. veronii strains were picked from frozen stocks and cultured at 30◦C in Luria-Bertani (LB) broth or M9 medium with aeration. The Antibiotics included 50µg/mL ampicillin, 25µg/mL chloramphenicol, 50µg/mL kanamycin. The E. Coli Felden strain was chosen for the expression of eGFP and sRNA (Liu et al., 2015). E. Coli WM3064 was selected for gene knockout by homologous recombination, which is capable of growth at 37◦C in LB broth with 0.3 mM diaminopimelic acid (DAP).

The plasmid pRE112-avrAKO was transformed into the competent E. coli WM3064 and delivered into A. veronii by conjugation (Liu et al., 2016). The A. veronii 1avrA was screened on LB agar supplemented with 6% sucrose and ampicillin and was further checked by sequencing.

The plasmid pBBR-avrA was transformed into the competent E. coli WM3064, and mobilized into the 1avrA strain by conjugation, resulting in complementary A. veronii 1avrA::avrA.

#### RNA Sequencing and Bioinformatics Analysis

Strains were grown in a 10 mL M9 medium containing 50µg/mL ampicillin at 30◦C, 150 rpm for 20 h. The cells were collected and lysed. RNA samples were extracted by phenol-chloroform. The concentration and quality of RNA was detected by the Agilent 2100 Bio analyzer. The samples were treated with DNase I to eliminate double-stranded and single-stranded DNA, followed by the depletion of rRNA with the Ribo-Zero Magnetic Kit. First-strand cDNA was generated using a random primer reverse transcription, followed by a secondstrand cDNA synthesis. The end of the synthesized cDNA was subjected to repair and adenylate. The adapters were ligated to the ends of these 3′ -adenylated cDNA fragments. The cDNA fragments were enriched by PCR amplification with a PCR Primer Cocktail, and the purified products were sequenced on the Hiseq Xten (Illumina, San Diego, CA, USA) using the gel-free protocol. Libraries were sequenced as 100 bp pairedend reads to a minimum target depth of 2G clean data per sample. The software HISAT (V2.0.1-beta) was used for

the reference genome alignment, and CPC (coding potential calculator) was used to characterize the translation possibility of new transcripts. To identify the differential expression between wild type and 1smpB, Bowtie 2(v2.2.5) was used to analyze the mRNA expression. Fragments per kilobase of transcript per million fragments sequenced (FPKM) were used to normalize the gene expression levels. For each gene, the pvalue was evaluated, and Benjamini-Hochberg false discovery rate (FDR) was applied for the correction. The differential expressions of the transcripts were estimated by the virtue of ≥1 absolute log2 -fold change and < 0.001 FDR adjusted p-value. These results were sorted by GO categories using Perl scripts. The GO and KEGG pathway enrichments of differential uni-genes were analyzed. The hypergeometric test was used to compare differential expression genes in the pathway with the entire genome background. Pathways with a p ≤ 0.05 were considered as significant. GEO accession number **GSE120603**.

# LD50 Test in Mice

The derivative of A. veronii was streaked onto M9 medium and grown for 18–20 h at 30◦C. The cells were re-suspended in sterile PBS. ICR mice (male, 6–8 weeks old) were infected by intraperitoneal injection with 103–107 CFU/g of each strain (n = 8) and monitored daily for weight loss and signs of illness. The animals were euthanized if they accorded with alternate endpoint criteria including the loss of mobility. The 50% lethal dose was calculated by linear regression (Torii et al., 2015).

type and 1*smpB*. (B) Transcriptomic comparisons of *iscR* operon genes between wild type and 1*smpB*. (C) Realtime qPCR detection of *iscR* operon and AvrA. (D) The growth curves of the derivative *A. veronii*. (E) The survivals of 1*smpB* and wild type in H2O2 concentrations ranging from 0 to 10 mM. \*\**p* < 0.01, \**p* < 0.05, following by one-way ANOVA, independent *t*-tests.

#### Organ Coefficient

The coefficient of organs was calculated from the ratio of organ weight and body weight of the same mouse. The change of coefficient was used to characterize the degree of injury by contrasting the pathological with the normal tissue (Zhang et al., 2014).

All animal experiments were approved by the Committee of the Ethics on Animal Care and Experiments at Hainan

University, and were carried out in accordance with the university guidelines.

#### Measurement of Growth Curve

The condition of iron deficiency was equipped with LB medium containing 200 mM dipyridyl. M9 medium was considered as the nutrient limited condition. The strains were cultured at logarithmic phase, and then inoculated into 50 ml media at an initial OD600 of 0.05. The ODs were monitored with a spectrophotometer (INESA 970CRT, Shanghai, China) at regular intervals.

#### Survival Under Oxidative Stress

The derivative of A. veronii was grown until to the OD600 value of 0.6 in the LB broth appended with 200 mM dipyridyl and subsequently challenged with 0–10 mM H2O2. The cultures were continuously incubated at 30◦C with shaking for 10 min, and the survival colonies were counted on the LB agar plate with 50 µg/mL ampicillin.

#### Real-Time PCR Experiment

The wild type and mutant strains of A. veronii were grown in LB and M9 supplemented with 50µg/mL ampicillin at 30◦C till to stationary stage. The total amount of RNA was extracted for relative expression analysis of the genes, including smpB, the predicted targets of AvrA (t15190, t21425, t18380, t22295, t14250), Fe/S cluster assembly regulation factor (iscR) and its regulated targets iscA, iscU, iscS.

#### Florescent Measurement

The cells conferring eGFP expression were suspended in PBS buffer, and loaded to a 96 well microtiter plate (Greiner BIOONE, Nurnberg, Germany). The fluorescent intensity was measured at 488 nm excitation and 540 nm emission wavelengths with a fluorescence microplate reader (Infinite R 200 PRO, Tecan, Shanghai, China). The relative fluorescence was calculated as the total fluorescence divided by OD600 value.

#### Statistical Analysis

Statistical data were analyzed using the statistical Package for the Social Science (SPSS) version 20.0 (SPSS, Chicago, IL, United States) and GraphPad Prism version 6.0 (GraphPad, San Diego, CA, United States). The results were presented as mean values of three independent experiments with standard deviation (SD) using one-way analysis of variance (ANOVA). P < 0.05 or 0.01 were represented as significant or extremely significant.

#### RESULTS

#### Transcriptomic Analysis

To determine the differences in pathways and gene expressions between wild type and smpB knockout (1smpB) in the M9 medium, the transcriptome assembly of A. veronii was generated from 91.17 million paired-end RNA-seq reads using Illumina Xten technology and a total of 22,055 transcripts were mapped by genome NZ\_CP012504.1 (**Figure 1A**). Sixteen pathways were significantly affected by SmpB knockout, including sulfur metabolism, biosynthesis of siderophore group non-ribosomal peptides, bacterial chemotaxis, arginine and peptidoglycan biosynthesis, and microbial metabolism in a diverse environment (**Figure 1B**). Among them, 21 and 20 genes were downregulated, respectively in the type IV pilus assembly and the type VI secretion system at the stationary phase (**Figure 1C**). The type IV pili are ubiquitously expressed on the surface of many Gram-negative bacteria and are important virulence factors that facilitate host-pathogen interactions and persistence of infection. T6SS is the molecular machine, which transports the virulence effectors from the interior cytoplasm or cytosol into an adjacent target cell across the cellular envelope. The downregulated pilus and secretion system proteins revealed that toxicity might be attenuated in 1smpB.

#### SmpB Deletion Decreases the Colonization in Host Tissue

To compare the virulence between wild type and 1smpB, Institute of Cancer Research (ICR) mice were divided into 10 groups with 104–108 CFU/g bacterial infection. The survival rate of ICR mice revealed that the virulence of the wild type was no different compared with the smpB deletion (**Figures 2A,B**).

After the mice were intraperitoneally injected with 5 × 105 CFU/g bacterial strains, the liver, spleen and kidneys were collected, and tissue suspension was cultured on LB plates supplemented with ampicillin. The wild type was colonized significantly more than 1smpB in the kidney, liver, and spleen (**Figure 2C**), and the results agreed with the downregulated expressions of the type IV pilus assembly proteins (**Figure 1C**). Organ coefficient is a common toxicology indicator of ratio of organ and body weight reflecting animal damage (Zhang et al., 2014). In our study, the coefficients of spleen and liver infected by the wild type were 3.68 ± 0.45 and 58.65 ± 7.94 mg/g, which were significantly higher than the control group (2.93 ± 0.23 and 47.38 ± 5.27 mg/g) and 1smpB group (3.08 ± 0.38 and 50.65 ± 5.62 mg/g) (**Figures 2D,E**), while little difference was observed in the kidney (**Figure 2E**). Next the organ coefficient was evaluated for the visceral injury between the treatment and the control group. The coefficients of the spleen and liver infected by the wild type were significantly higher than the control group and 1smpB group (**Figures 2D–F**), while little difference was

observed in the kidney (**Figure 2E**). This also suggested that the virulence of wild type A. veronii was not affected by smpB deletion. Consistently, the acute LD50 for 1smpB strain in mice was calculated as 5 × 105 CFU/g, which was close to that of 106 CFU/g for the wild type. Because of the contradiction between the decreased pathogenicity and unvaried lethality, we speculated that the bacterial resistance had enhanced in the host.

# The Combination of the Downregulated AvrA and Upregulated IscR Mediates the Oxidative Tolerance in smpB Deletion

Expressions of transcripts were compared between the wild type and smpB mutant in the transcriptomic data. A total of 466 non-coding transcripts were defined as sRNA, including 50–500 nt. Among them, 11 sRNAs were analyzed to express specifically between the wild type and 1smpB at the stationary stage in the M9 medium (**Figure 3A**). There were 62 differentially expressed target genes of the 11 sRNAs involved in stress response pathways, pathogenesis, and also the regulations of metabolism, transport, quorum sensing (**Supplementary Table 1**). The pathways were associated with the synthesis of cell membrane, protein translocation, ATPase, Fe/S protein assembly and energy metabolism (**Table 3**). The candidate\_80 sRNA, designated as AvrA (Aeromonas veronii non-coding RNA A) was potentially associated with the target gene iscR in Fe/S assembly. After smpB was knocked out, AvrA expression declined (**Figure 3A**) resulting in the increased expression of the predicted targets isc operon genes which encoded iron-sulfur proteins (**Figure 3B**). The similar tendency of AvrA and its target genes was also turned out by Real-time PCR (**Figure 3C**).

To evaluate whether smpB mutation lived better in stress conditions than the wild type, the iron deficiency condition was performed to mimic the host environment

and the tolerance to hydrogen peroxide oxidation (H2O2) was tested. The smpB mutation exhibited a slow growth compared with the wild type and the complementary strain (**Figure 3D**). The 1smpB survived better than the wild type when the H2O2 concentration ranged from 6 to 10 mM, revealing that 1smpB was endowed

FIGURE 6 | Regulation of SmpB in virulence regulation. The virulence of *A. veronii* was affected by the expression of virulent proteins the ability of host colonization and the resistance to adversity. Type VI secretion system and pilus assembly were affected on the transcription and translation level by SmpB regulation directly. The sRNA AvrA functioned the stress resistance directly, which was mediated by SmpB indirectly. The arrows in blue showed the positive regulation, and the line in red showed repression. The symbol "+", "–" indicated upregulation and downregulation, respectively.

with a stronger capability of growth under adverse conditions (**Figure 3E**).

#### AvrA Deletion Increases the Tolerances to Iron Deficiency and Oxidative Stress by Upregulating Iron-Sulfur Gene Expression

AvrA was a non-coding RNA of 253nt, and its secondary structure was predicted by the software RNAfold (http://rna.tbi. univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) (**Figure 4A**). The growth results demonstrated that the AvrA deletion presented a better ability to grow under dipyridyl (DIP)-chelated iron deficiency state (**Figure 4B**). Also, the oxidation resistance of 1avrA was evaluated under 0–10 mM H2O2 in comparison with the wild type and the complemented strains, showing that the antioxidation capacity was enhanced after AvrA knockout (**Figure 4C**). Realtime qPCR results showed that the transcriptional expressions of the iron-sulfur genes iscR, iscU, iscS, and iscA were significantly increased (**Figure 4D**).

# AvrA Binds and Downregulates iscR mRNA by Base Pairing

The potential interaction site was predicted between AvrA and the iscR promotor using the RNAplex program. Interestingly, the base-pairing region encompassed the ribosome binding site and the start codon AUG of iscR, and the binding site spanned from C37 to A63 of 5′ -end AvrA (**Figure 5A**). Two mutations, P1 (from A2 to C5, ACUC) and P2 (from C−18 to G−13, GCGUUC), were introduced in the iscR promoter (piscR) and fused with the enhanced green fluorescent protein (eGFP) to produce 8(piscR mutP1-eGFP) and 8(piscRmutP2 eGFP), respectively (**Figures 5A,B**). When AvrA was coexpressed with 8(piscR-eGFP), the fluorescence was repressed, suggesting that AvrA could interact with the promotor of iscR by base pairing (**Figure 5C**). Furthermore, the fluorescence recovered when AvrA was co-transformed with 8(piscRmutP1 eGFP), revealing that P1 was the exact site for interaction (**Figure 5C**). Next, a mutation named pavrAmutP1 was introduced in the AvrA expression plasmid pavrA to match 8 (piscR mutP1-eGFP), resulting in fluorescent repression. Conversely the mutated pavrAmutP1 did not reduce the fluorescence when co-expressed with the wild type 8(iscR-eGFP) (**Figure 5D**). The results concluded that AvrA repressed the expression of iscR by base pairing with the iscR translation initiation region P1.

### DISCUSSION

SmpB is not only the key component of the trans-translation system, but also plays an important role as a transcription regulator in the global regulation of bacteria (Liu et al., 2016). In our study, the smpB deletion affected the transcriptional expression of the genes related to the bacterial growth, metabolism and protein secretion in multiple metabolic pathways in A. veronii. Previously Type II, III, and VI secretion systems were reported in multiple strains of Aeromonas bacteria, which function by exporting bacterial virulence proteins and by infecting host cells (Suarez et al., 2009; Journet and Cascales, 2016). The gene expression of the Type III secretion system had extremely low abundance, while those of Type VI performed higher in the transcriptome**.** Most of all, virulence-related genes which mediated the pili assembly (type IV pilus) and protein secretion (type VI secretion system, TSSS) were totally downregulated (**Figure 1**). Previously, the type VI secretion system was identified to function as the virulence factor in Vibrio hollisae and Aeromonas hydrophila (Russell et al., 2014). The colonization of wild type A. veronii was significantly higher than that of 1smpB. However, the survival rates of the infected mice showed no significant differences between the wild type and 1smpB strain. Previously the smpB deletion mutant of Listeria monocytogenes and Salmonella Typhimurium were avirulent and deficient in the intramacrophage survival (Kivisaar, 2003; Mraheil et al., 2017) The results were inconsistent with past reports, indicating that there are other pathways maintaining the virulence in A. veronii smpB mutants. Subsequently, sRNA AvrA was revealed as an alternative for virulence compensation. The downregulation of AvrA affected the expression of IscR in 1smpB A. veronii. In case of iron deficiency, IscR enhanced iron-sulfur cluster assembly and iron sulfur protein synthesis (Santos et al., 2014), and amended the antioxidant ability of A. veronii.

When bacteria infect the host, they are subjected to stressful conditions such as strong oxidization, deficient nutrition and metal ion. The harsh conditions cause starvation, accumulation of secondary metabolites, decreased fidelity of DNA replication and the reduction in DNA repair activity which are similarly encountered during the stationary stage (De Biase et al., 1999; Kivisaar, 2003). Most sRNAs are expressed as important regulators in the process of gene regulation under adverse stress (Michaux et al., 2014; Amin et al., 2016; Holmqvist and Wagner, 2017). The sRNA AvrA was uncovered to function by base pairing with its target gene iscR. IscR regulated a set of genes involved in iron-sulfur cluster assembly in adversity like iron deficiency and oxidative stress (**Figure 6**). However, the transcription factors associated with oxidative stress, including Fur (ferric uptake regulation protein), OxyR (hydrogen peroxide-inducible genes activator), SoxR (hydrogen peroxide-inducible genes activator) were insignificantly expressed by comparing 1smpB strains the with wild type strains (**Supplementary Table 2**). The mutation of smpB resulted in the downregulation of sRNA AvrA, which leads to the upregulation of iscR expression and the enhancement of the adverse resistance. Combined with the attenuated virulence and the increased survival in adversity in 1smpB, the strains performed a similar lethality in mice compared with the wild type. Virulence is a trait that has been selected in evolutionary history (Rafaluk et al., 2015; Gerstein and Nielsen, 2017). Therefore, the survival viability in the macrophage and the tolerance to adverse environments caused by smpB mutation compensates for the loss of function in virulence, causing A. veronii to evolve as a superbug.

#### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

#### REFERENCES

#### AUTHOR CONTRIBUTIONS

ZL, XM, and DW contributed the conception and design of the study. DW, HL, YT, HT, and ZL performed the statistical analysis. DW, HL, XH, and ZL drafted the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.

#### FUNDING

This work was supported by grants from the Hainan Natural Science Foundation Nos. 317015 (to YT) and 319QN161 (to HL), and the Science and Technology Program of Hainan Province ZDYF2017020 (to ZL).This work was also partially supported by grants from the National Natural Science Foundation of China Nos. 31560021, 31772887 (to ZL), and 31860676 (to YT).

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb. 2019.00142/full#supplementary-material

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Wang, Li, Ma, Tang, Tang, Hu and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

,

# Genetic Features of Antarctic Acinetobacter radioresistens Strain A154 Harboring Multiple Antibiotic-Resistance Genes

Andrés Opazo-Capurro1,2, Paul G. Higgins 3,4, Julia Wille3,4, Harald Seifert 3,4 Camila Cigarroa1 , Paulina González-Muñoz 1,2,5, Mario Quezada-Aguiluz 1,2 , Mariana Domínguez-Yévenes 1,2, Helia Bello-Toledo1,2, Luis Vergara5 and Gerardo González-Rocha1,2 \*

#### Edited by:

Xiangmin Lin, Fujian Agriculture and Forestry University, China

#### Reviewed by:

Hossam Abdelhamed, Mississippi State University, United States José Alejandro Di Conza, University of Buenos Aires, Argentina

#### \*Correspondence:

Gerardo González-Rocha<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology

Received: 14 June 2019 Accepted: 02 September 2019 Published: 13 September 2019

#### Citation:

Opazo-Capurro A, Higgins PG, Wille J, Seifert H, Cigarroa C, González-Muñoz P, Quezada-Aguiluz M, Domínguez-Yévenes M, Bello-Toledo H, Vergara L and González-Rocha G (2019) Genetic Features of Antarctic Acinetobacter radioresistens Strain A154 Harboring Multiple Antibiotic-Resistance Genes. Front. Cell. Infect. Microbiol. 9:328. doi: 10.3389/fcimb.2019.00328 1 Laboratorio de Investigación en Agentes Antibacterianos (LIAA), Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile, 2 Millennium Nucleus for Collaborative Research on Bacterial Resistance (MICROB-R), Santiago, Chile, 3 Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany, 4 German Center for Infection Research (DZIF), Partner Site Bonn-Cologne, Cologne, Germany, 5 Departamento de Ciencias Biológicas y Químicas, Facultad de Medicina y Ciencia, Universidad San Sebastián, Concepción, Chile

While antibiotic-resistant bacteria have been detected in extreme environments, including Antarctica, to date there are no reports of Acinetobacter species isolated from this region. Here, we characterized by whole-genome sequencing (WGS) the genetic content of a single antibiotic-resistant Acinetobacter spp. isolate (A154) collected in Antarctica. The isolate was recovered in 2013 from soil samples at Fildes Peninsula, Antarctica, and was identified by detection of the intrinsic OXA-23 gene, and confirmed by Tetra Correlation Search (TCS) and WGS. The antibiotic susceptibility profile was determined by disc diffusion, E-test, and broth microdilution methods. From WGS data, the acquired resistome and insertion sequence (IS) content were identified by in silico analyses. Plasmids were studied by the alkaline lysis method followed by pulsed-field gel electrophoresis and conventional PCR. The A154 isolate was identified as A. radioresistens by WGS analysis and displayed >99.9 of similarity by TCS in relation with the databases. Moreover, it was resistant to ampicillin, ceftriaxone, ceftazidime, cefepime, cefotaxime, streptomycin, and kanamycin. Likewise, in addition to the intrinsic blaOXA−23−like gene, A154 harbored the plasmid-encoded antibiotic-resistance genes blaPER−2, tet(B), aph(3′ )-Vla, strA, and strB, as well as a large diversity of ISs. This is the first report of antibiotic-resistant A. radioresistens in Antarctica. Our findings show the presence of several resistance genes which could be either intrinsic or acquired in the region.

Keywords: antibiotic-resistance genes, Acinetobacter, Antarctica, whole-genome sequencing, resistance plasmid, ecotoxicology

# INTRODUCTION

Antarctica possesses one of the most extreme environmental conditions on earth, where low temperatures, high levels of UV radiation, and low water availability, limit the growth of living organisms, including bacteria (Convey et al., 2008). In these ecosystems, diverse survival strategies to ward off competing microorganisms are present, including the production of antibiotics by environmental microbiota (Aronson et al., 2011). Lately, it has been determined that antibiotic production and resistance have ancient origins in nature, ranging from 2 billion years to 40 million years (Dcosta et al., 2011). In this sense, several antibiotic resistance genes (ARGs) have been identified in uncontaminated soil in Antarctica, confirming that this continent possesses a natural microbial community associated with a local resistome (Van Goethem et al., 2018). Although this natural phenomenon is old, the high use of antibiotics in human practice, as well as the rise of anthropic activity around the globe, have favored to increase the selection and dissemination of antibiotic resistant bacteria and antibiotic resistance determinants (Rabbia et al., 2016).

Although Antarctica has been historically considered as a pristine unexplored continent, the anthropogenic activity has been increasing recently, which could potentially have an impact on the resistome of the natural bacterial community (Rabbia et al., 2016). It has been demonstrated that the presence of military bases, scientific programs, and tourism, promotes the dissemination of ARGs possibly due to the introduction of human-related microorganisms (Hernandez and González-Acuña, 2016). For instance, we recently found that Escherichia coli isolates collected from seawater and waste-water treatment plants located in the Antarctic Treaty area showed resistance to β-lactams, aminoglycosides, tetracycline, and trimethoprimsulphonamide, which is probably associated with discharged of inefficiently treated water emanating from waste-water plants (Rabbia et al., 2016).

Acinetobacter radioresistens is able to survive extreme levels of oxidative stress, desiccation, and irradiation (Touchon et al., 2014; Sacher et al., 2018) and has primarily been isolated from environmental sources (i.e., cotton, water, and soil), and has also been identified as part of the human skin microbiota of healthy people (Seifert et al., 1997), as well as from fecal samples from chickens (Ngaiganam et al., 2019). Few studies on the presence of ARGs in A. radioresistens are published, and the species is considered largely susceptible to antibiotics.

A. radioresistens isolate A154 was collected from a soil sample in Antarctica in 2013. Interestingly, it displayed an unusual resistance pattern to diverse antibiotics, including β-lactams and aminoglycosides. Hence, to further understand the genetic features of the Antarctic A154 isolate containing multiple ARGs, we report its genome sequence.

#### MATERIALS AND METHODS

#### Bacterial Source

Acinetobacter spp. isolate A154 was recovered from ornitogenic soil (superficial layer) in Ardley Island (−62◦ 12′ 60.00′′S −58◦ 55′ 59.99′′W) in Fildes Peninsula, Antarctica, during a scientific expedition in 2013. Specifically, the isolate was recovered in a soil sample from a lagoon shore. Fildes Peninsula is located on King George Island and holds the largest number of scientific research bases in the Antarctic Treaty Area (Rabbia et al., 2016). The isolate was grown on trypticase soy agar incubated overnight at 30◦C.

### PCR Experiments

Total DNA was extracted by the boiling water method and utilized as template for preliminary species identification by the detection of the intrinsic chromosomally-encoded blaOXA−23−like gene.

#### Susceptibility Testing

Susceptibility testing were performed by disc diffusion following the CLSI recommendations, for ampicillin (AMP, 10 µg), cefotaxime (CTX, 30 µg), ceftriaxone (CRO, 30 µg), ceftazidime (CAZ, 30 µg), cefepime (FEP, 30 µg), imipenem (IPM, 10 µg), meropenem (MEM, 10 µg), gentamicin (GEN, 10 µg), amikacin (AMK, 30 µg), kanamycin (KAN, 30 µg), streptomycin (STR, 10 µg), tetracycline (TET, 30 µg), and ciprofloxacin (CIP, 5 µg) (Clinical Laboratory Standards Institute, 2017). Minimum inhibitory concentrations (MICs) were determined by E-test and broth microdilution for AMP, CTX, IPM, MEM, GEN, AMK, and KAN (Clinical Laboratory Standards Institute, 2017).

#### Whole-Genome Sequencing

Total DNA for whole-genome sequencing (WGS) was extracted using the Wizard R Genomic DNA Purification kit (Promega, USA) following the manufacturer's protocol. DNA concentration and integrity were verified using a Take3 plate (BioTek Instruments). WGS was performed by the Illumina MiSeq platform (2 × 250 bp paired end reads) with libraries prepared by the NexteraXT kit (Illumina).

#### In silico Genome Analyses

De novo assembly was performed by using the SPades software version 3.9 available at the CGE server (https://cge.cbs. dtu.dk/services/SPAdes/), utilizing default values (Nurk et al., 2013). Species identification was carried out by the SpeciesFinder service, version 1.3 (https://cge.cbs.dtu.dk/ services/SpeciesFinder/), which is based on the 16S rRNA sequence. Genome annotation was accomplished using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) web-service (http://www.ncbi.nlm.nih.gov/genome/annotation\_prok).

Moreover, the draft genome was analyzed by the Comprehensive Genome Analysis service (https://www.patricbrc.org/app/ ComprehensiveGenomeAnalysis), which incorporates the RAST tool kit (RASTtk). The genetic contexts of the contigs in which plasmid-encoded ARGs were detected, were plotted using the Artemis software, version 18.0.2 (Carver et al., 2012).

In order to establish the taxonomic relatedness of A154, we used the Tetra Correlation Search (TCS), which allows to compare the draft genome of this isolate against the genomes reference database GenomesDB (http://jspecies.ribohost.com/ jspeciesws/#genomesdb), which contains >30.000 whole and draft genomes with pre-calculated overall genome relatedness indices (Richter et al., 2015). In addition, we determined the acquired resistome, insertion sequences (ISs) content and toxinantitoxin (TA) systems through ResFinder (https://cge.cbs.dtu. dk/services/ResFinder/), RASTtk and Pathosystems Resource Integration Center (PATRIC) platforms. Plasmid replicons were searched using the PlasmidFinder tool (Carattoli et al., 2014) and the PATRIC platform. The genome of the isolate A154 was deposited at DDBJ/EMBL/GenBank under the accession number PXJD00000000.1.

#### Molecular Typing

Additionally, from WGS data we extracted the seven alleles according to the Pasteur MLST scheme which were submitted to the PubMLST database (https://pubmlst.org/) in order to assign a specific sequence type (ST).

#### Plasmids Studies

Plasmids were isolated by the alkaline lysis method according to a standard protocol (Kado and Liu, 1981). Afterwards, DNA concentration of the plasmids extracts were measured by spectrophotometry (BioTek Instruments) and chromosomal contamination was evaluated by detecting the 16S rDNA gene, which is chromosomally encoded. After, ARGs previously detected in total DNA were screened using the pure DNA plasmids extracts. Moreover, we performed mating and plasmid curing experiments (Trevors, 1986; Leungtongkam et al., 2018).

### RESULTS AND DISCUSSION

The A154 isolate was identified as A. radioresistens according to its 16S rRNA sequence, and displayed >99.9 of similarity by TCS with the genome of A. radioresistens strain SK82 (NCBI: txid596318), thus confirming that A154 belongs to this species. Additionally, it was resistant to AMP, CRO, CTX, CAZ, FEP, KAN, and STR, intermediate to GEN, and susceptible to IMP, MEM, CIP, TET, and AMK. MICs values of AMP, CAZ, CTX, FEP, GEN, and KAN were >128, >128, 128, 32, 8, and 128µg/mL, respectively (**Table 1**).

From in silico analyses, we determined that the WGS data generated resulted in 205 contigs and a N50 contig size of 131,303 bp. Moreover, A154 genome comprised 3,448,645 bp with an average GC content of 41.5%, 3,444 coding sequences (CDS), 64 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes.

From RASTtk results, we determined that the resistome of A154 was comprised of aminoglycoside modifying enzymes (AMEs) encoded by aph(3′ )-VIa, aac(3)-IIa, strA (aph(3′′)- Ia), and strB (aph(6)-Id), as well as the β-lactamase genes blaOXA−23−like (intrinsic to this species), blaTEM−1B, blaSCO−1 and blaPER−2, and the tetracycline-resistance gene tet(B) (**Table 1**). ARGs positions on the genome of A154 are shown in **Figure 1**. Interestingly, the blaOXA−23−like allele identified in A154 did not match with any variant from the database, sharing a 98.18% identity with blaOXA−565 (accession number KY883665). SCO-1 encodes for a RTG-type carbenicillinase identified in Escherichia coli and Acinetobacter spp. (Papagiannitsis et al., 2007; Poirel et al., 2007). To the author's knowledge, the single report of this enzyme in Acinetobacter genus occurred in isolates collected in Argentina (Poirel et al., 2007); thus its presence could be associated to isolates from South America and nearby regions. According to Van Goethem et al. (2018), resistance determinants present in environmental soil, especially from extreme and remote ecosystems, are still underrepresented, thus our findings represent the very first description of A. radioresistens resistant to multiple antibiotics in this area.

Importantly, A. radioresistens has been rarely associated with human infections and the species is normally highly susceptible to antibiotics. In this sense, Visca et al. described a communityacquired bacteraemia caused by this species, but the isolate was susceptible to cephalosporins and aminoglycosides (Visca et al., 2001). Similarly, Poirel et al. published a study that included five A. radioresistens isolates that were fully susceptible to all antibiotics tested, including penicillins and carbapenems, despite the detection of blaOXA−23−like gene (Poirel et al., 2008). On the other hand, Higgins et al. identified two carbapenemresistant A. radioresistens isolates that were recovered from the same patient 17 days apart, which were also resistant to fluoroquinolones (Higgins et al., 2013). Additionally, two A. radioresistens isolates collected from companion animal infections, which harbored blaIMP−1, sul1, aadA1, and aac(6)- 31, were resistant to carbapenems and fluoroquinolones (Kimura et al., 2017), but despite the presence of AMEs, the isolates were susceptible to GEN and AMK.

Our findings show that the isolate A154 was resistant to β-lactams, except carbapenems, which could be explained by the presence of the blaTEM−1B, blaPER−2, and blaSCO−1 genes. Interestingly, PER-2 corresponds to an extended-spectrum βlactamase (Pasteran et al., 2006; Gutkind et al., 2012), while TEM-1B and SCO-1 are class A narrow-spectrum β-lactamases (Poirel et al., 2007), which could be mediating the resistance displayed against β-lactams, such as cephalosporins (Gutkind et al., 2012). These findings are particularly interesting since although β-lactamase genes are common components of the soil resistome, they are not highly abundant. Accordingly, a study of soil samples from the Mackay Glacier in Antarctica showed a very low β-lactamase abundance (Van Goethem et al., 2018). The same study also demonstrated that β-lactamase genes are more common in soil samples from regions that are more likely to have been exposed to human activity (Van Goethem et al., 2018), representing a possible source of selective pressure.

Importantly, A154 was resistant to KAN and STR, and intermediate to GEN. Although the isolate was classified as AMK susceptible, the MIC value (16µg/mL) was only one dilution below the CLSI breakpoint for intermediate (32µg/mL). This particular phenotype is mainly explained by the activity the APH(3′ )-VIa enzyme, which was originally described in A. baumannii and confers resistance to AMK and KAN (Ramirez and Tolmasky, 2010). Additionally, other AME genes detected were strA and strB which confer resistance to STR (Ramirez and Tolmasky, 2010), in concordance with the observed phenotype. We also identified the aac(3)-IIa gene, which is related to resistance to GEN and tobramycin (TOB) (Ramirez and Tolmasky, 2010). Similarly, the identified resistome included the tet(B) gene, however, the isolate was susceptible to tetracycline. These inconsistencies suggest that A. radioresistens might need a complementary mechanism TABLE 1 | Antibiotic susceptibilities and genetic features of isolate A154 from Antarctica.

MIC, minimum inhibitory concentration; AMP, ampicillin; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; GEN, gentamicin; KAN, kanamycin; ARGs, antibiotic-resistance genes; ST, sequence-type; pl, plasmid-encoded.

FIGURE 2 | Linear map of A154 contigs 35 (A) and 10 (B). DNA strands sense and anti-sense are represented. Hyp., hypothetical protein. The linear representation was generated by using Artemis software.

of aminoglycosides/tetracycline resistance or to regulate the expression of ARGs to develop the resistance phenotype. This assumption is reaffirmed by the presence of the intrinsic blaOXA−23−like gene in carbapenem-susceptible A. radioresistens, which when present in A. baumannii, confers resistance to carbapenems (Evans and Amyes, 2014), when over-expressed (Higgins et al., 2013). Therefore, expression experiments of the ARGs are required in order to understand the potential influence of these determinants on the resistance level to antimicrobials in the species described.

On the other hand, A154 was assigned to the ST1207 in PubMLST (https://pubmlst.org/), which corresponds to a novel ST. This is a novel lineage which corresponds to a singleton, since is not associated to any described ST of the database. Accordingly, it is important to analyze a larger collection of isolates from regions with and without human influence in order to determine whether this ST is endemic/prevalent to this area or it was introduced. This is particularly important since we have previously determined that antibiotic-resistant E. coli isolates in Antarctica are more frequent in regions with a significant anthropic influence (Rabbia et al., 2016). In the case of A154, it was collected from the same region of the study mentioned above, thus the local microbiome and resistome are probably affected by human activity. However, since the soil from which the isolate was recovered corresponded to surface soil, which is determined by the influence of organic matter from animals, it is possible that it could be carried by migrating birds (Segawa et al., 2013), which might be playing an important role in the genetic contamination of this region.

Moreover, no plasmid replicons were detected by PlasmidFinder and PATRIC. However, we detected a large plasmid of ca. 280 kb which was not detected in silico from WGS (data not shown). Moreover, the plasmid could not be transferred by conjugation and neither cured by temperature. The latter could be explained by the presence of the type II toxinantitoxin systems RelB/DinJ (accession number PSD36295.1) and RelE/ParE (accession number PSD36025.1), which were detected from WGS. Interestingly, the RelE/ParE genes were located adjacent to strA and strB genes (**Figure 2**), which suggests that both are contained in the same plasmid. The toxin-antitoxin systems are defined as plasmid maintenance systems present in almost all free-living bacteria (Kang et al., 2018), which could confer stability against curing agents. Conjugation experiments under different conditions should be performed in order to determine the ability of the plasmid to be mobilized.

Remarkably, a large diversity of IS-family transposases was found on the genomes of A154. Specifically, its IS content was composed by fifteen copies of IS/IS1182 transposase, twelve of IS6, 11 of IS30, eight of IS4, four of ISL3, two each of IS3-like and IS5-like, and a single copy each of IS982, IS200/IS605, ISNCY, IS481, and IS1. IS-elements are capable of mobilizing and controlling the expression of ARGs, among other functions, therefore the isolate has an important potential to acquire/disseminate and over-express resistance determinants (Siguier et al., 2014). Notably, the blaPER−2; aph(3′ )-VIa; strA; strB; tet(B) were identified in the plasmid extract obtained from alkaline lysis, and are also located in the same region of the genome in which diverse ISs were also located (**Figure 1**). This suggests that they could has been incorporated by A154 by horizontal-gene transfer mediated by plasmids and/or ISelements. As shown in **Figure 2**, the plasmid-encoded ARGs were located in different contigs from WGS, in which also several IS-elements were located, reaffirming the potential role of these mobile-genetic elements in their mobilization. The contig in which aph(3′ )-VIa is located, was not represented since it was contained in a short contig, which harbored only two genes.

An important limitation of our study is the Illumina platform generates short reads, making it difficult to determine the entire sequence and structure of the plasmid contained by A154 (Roosaare et al., 2018). In consequence, the use of this sequencing technology to predict plasmids (i.e., by PlasmidFinder) is limited, being only possible to search for the incompatibility groups detected in the genome which are deposited on the databases. A potential solution for this problem, is to isolate the plasmid and sequence it or use new sequencing technologies, such as Oxford Nanopore Technology (ONT) (Lemon et al., 2017).

In conclusion, our results represent the first description of A. radioresistens in Antarctica containing several ARGs, in an isolate recovered from a region under both human and animal influences, thus these factors could be contributing to the introduction of ARGs in Antarctica. These findings contribute to better understand the knowledge of the biology of Acinetobacter as well as to add more data about the content of ARGs in the region.

#### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

# AUTHOR CONTRIBUTIONS

AO-C, PG-M, PH, and JW contributed to the acquisition, analysis, and interpretation of the data. AO-C, CC, PG-M, and MQ-A made important contributions to the design of the work. HS, MD-Y, HB-T, LV, and GG-R provided approval for publication of the content and contributed drafting the work and critically revisiting the manuscript.

#### FUNDING

This work was funded by the post-doctoral project N◦ 3150286 from The National Fund for Scientific and Technological Development (FONDECYT) and the Chilean Antarctic Institute (INACH) project number RT\_06\_12.

#### ACKNOWLEDGMENTS

The authors highly appreciate the support from the Chilean National Commission for Scientific and Technological Research (CONICYT), the Chilean Antarctic Institute (INACH), and the Millennium Science Initiative of the Ministry of Economy, Development and Tourism, Chile.

# REFERENCES

strains in the Americas. Antimicrob. Agents Chemother. 50, 3222–3224. doi: 10.1128/AAC.00284-06

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Opazo-Capurro, Higgins, Wille, Seifert, Cigarroa, González-Muñoz, Quezada-Aguiluz, Domínguez-Yévenes, Bello-Toledo, Vergara and González-Rocha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The First Isolation and Molecular Characterization of Shiga Toxin-Producing Virulent Multi-Drug Resistant Atypical Enteropathogenic *Escherichia coli* O177 Serogroup From South African Cattle

#### *Edited by:*

Patrícia Poeta, University of Trás-os-Montes and Alto Douro, Portugal

#### *Reviewed by:*

Indranil Samanta, West Bengal University of Animal and Fishery Sciences, India Farhad Safarpoor Dehkordi, University of Tehran, Iran

#### *\*Correspondence:*

Collins Njie Ateba<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology

*Received:* 17 June 2019 *Accepted:* 10 September 2019 *Published:* 24 September 2019

#### *Citation:*

Montso PK, Mlambo V and Ateba CN (2019) The First Isolation and Molecular Characterization of Shiga Toxin-Producing Virulent Multi-Drug Resistant Atypical Enteropathogenic Escherichia coli O177 Serogroup From South African Cattle. Front. Cell. Infect. Microbiol. 9:333. doi: 10.3389/fcimb.2019.00333 Peter Kotsoana Montso1 , Victor Mlambo2 and Collins Njie Ateba1 \*

1 Bacteriophage Therapy and Phage Bio-control Laboratory, Department of Microbiology, Faculty of Natural and Agricultural Sciences, North-West University, Mmabatho, South Africa, 2 Faculty of Agriculture and Natural Sciences School of Agricultural Sciences, University of Mpumalanga, Nelspruit, South Africa

Atypical enteropathogenic E. coli (aEPEC) is a group of diarrhoeagenic Escherichia coli with high diversity of serogroups, which lack the bundle-forming pili (BFP) and genes encoding for shiga toxins. The aim of this study was to isolate, identify and determine virulence and antibiotic resistance profiles of aEPEC O177 strains from cattle feces. A total of 780 samples were collected from beef and dairy cattle and analyzed for the presence of E. coli O177. One thousand two hundred and seventy-two (1272) presumptive isolates were obtained and 915 were confirmed as E. coli species. Three hundred and seventy-six isolates were positively confirmed as E. coli O177 through amplification of rmlB and wzy gene sequences using multiplex PCR. None of these isolates harbored bfpA gene. A larger proportion (12.74%) of the isolates harbored hlyA gene while 11.20, 9.07, 7.25, 2.60, and 0.63% possessed stx2, stx1, eaeA, stx2a, and stx2d, respectively. Most of E. coli O177 isolates carried stx2/hlyA (9.74%). Furthermore, 7.40% of the isolates harbored stx1/stx2 while 7.09% possessed stx1/stx2/hlyA genes. Only one isolate harbored stx1/stx2/hly/eaeA/stx2a/stx2d while 5.11% of the isolates harbored all the four major virulence genes stx1/stx2/hlyA/eaeA, simultaneously. Further analysis revealed that the isolates displayed varied antimicrobial resistance to erythromycin (63.84%), ampicillin (21.54%), tetracycline (13.37%), streptomycin (17.01%), kanamycin (2.42%), chloramphenicol (1.97%), and norfloxacin (1.40%). Moreover, 20.7% of the isolates exhibited different phenotypic multi-drug resistance patterns. All 73 isolates harbored at least one antimicrobial resistance gene. The aadA, streA, streB, erm, and tetA resistance genes were detected separately and/or concurrently. In conclusion, our findings indicate that environmental isolates of aEPEC O177 strains obtained from cattle in South Africa harbored virulence and antimicrobial resistance gene determinants similar to those reported in other shiga-toxin producing E. coli strains and suggest that these determinants may contribute to the virulence of the isolates.

Keywords: atypical enteropathogenic *E. coli* (aEPEC), bundle-forming pili (BFP), *E. coli* O177, virulence factors, antimicrobial resistance, shiga-toxins, diarrhoeagenic *E. coli*

#### INTRODUCTION

Enteropathogenic E. coli (EPEC) is a group of diarrhoeagenic E. coli that is reported to cause high morbidity and mortality in humans, especially in immune-compromised subjects, elderly individuals and young children. EPEC are characterized by the presence of intimin (eae) genes coupled with the absence of the stx genes (Martins et al., 2016; Alonso et al., 2017). The eae gene is responsible for attaching and effacing (A/E) lesions on the intestinal epithelial cell of the host (Kaper et al., 2004; Martins et al., 2016; Malik et al., 2017). Based on the presence or absence of the EPEC adherence factor (EAF) plasmid, EPEC is subdivided into two groups that include typical Enteropathogenic E. coli (tEPEC) and atypical Enteropathogenic E. coli (aEPEC) (Trabulsi et al., 2002; Alonso et al., 2017). The tEPEC strains possess EAF plasmid, which encodes a bundle forming pili (bfpA) while aEPEC strains lack the bfpA gene (Canizalez-Roman et al., 2013; Malik et al., 2017). It is on these bases that the virulence potentials of aEPEC is poorly understood and highly questioned. Despite the fact that tEPEC have been most often associated disease complications in humans, it is only recently that aEPEC been reported to cause diseases in both animals and humans (Malik et al., 2017). This may account for the reason why previous studies that have been documented worldwide and in the study area have focused on EHEC, especially E. coli O157 and non-O157 strains that received great attention due to its high pathogenicity (Ateba and Bezuidenhout, 2008; Ateba and Mbewe, 2011; Iwu et al., 2016; Jajarmi et al., 2017; Toro et al., 2018). The findings of most of these studies were in agreement with previous reports indicating that EHEC strains possessed stx, stx2, hlyA, and eaeA gene determinants that enhance their potential to cause infections such as diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans (Farrokh et al., 2013). However, to the best of our knowledge there is currently no report on the occurrence of EPEC, and aEPEC in particular among South African foodproducing animals. This therefore creates a knowledge gap on the virulence profiles of aEPEC strains in the area. Despite the fact that aEPEC strains were known to be less pathogenic when compared to EHEC counterparts, data from some recent studies have revealed the presence of virulence determinants in aEPEC strains thus making them to start receiving attention as pathogens of severe clinical significance in humans (Beutin et al., 2005; Bibbal et al., 2017) as well as in food-producing animals, especially sheep and wildlife (Otero et al., 2013; Álvarez-Suárez et al., 2016; Martins et al., 2016). In this study, we expand our previous investigations on aEPEC in the area by determining the occurrence of aEPEC O177 strains in cattle and also provide an overview of the putative virulence and antimicrobial resistance profiles of the isolates. This was motivated from the fact that cattle are the primary reservoir of both EHEC (stx+) and aEPEC (stx−) strains, thus providing opportunities for horizontal transfer of virulence and antibiotic resistant genes between species and/or strains living in the same ecological niches (Bibbal et al., 2017).

#### MATERIALS AND METHODS

#### Samples Collection and Isolation of *E. coli* O177 Serogroup

A total of 780 cattle fecal samples were collected from eight commercial dairy and beef cattle farms in the North West Province, South Africa. Ethical clearance was obtained from the North West University Research Ethics Committee prior to the commencement of the study. Sampling was done between January 2017 and July 2017. The selection of the farms was based on the production system; either intensive, semi-intensive, and/or extensive farming system in the study area. The selection of all the three systems was based on the fact their management system is different. Furthermore, this was to avoid biasness in terms of selecting the farms. Ethical procedures such as restraining the animals using proper facilities and equipment were enforced during the collection of the samples. Fecal samples were collected directly from the rectum of individual animals using sterile arm-length gloves and in order to avoid duplication of sampling, the cattle were looked in their respective handling pens immediately after collection. Samples were placed in sterile sample collection bottles, labeled appropriately and immediately transported on ice to the Antimicrobial Resistance and Phage Biocontrol Laboratory (AREPHABREG), in the Department of Microbiology, North-West University for microbial analysis. Data on antibiotic type and treatment history were collected for the purpose of understanding antibiotic exposure histories of isolates from the study population.

Approximately, 2 g of fecal samples were dissolved in 10 mL of 10% (w/v) saline solution and homogenized. Aliquots of 5 µL were transferred into 10 mL buffered peptone water (Oxoid Ltd., Basingstok, Hampshire, UK). Ten-fold serial dilutions were prepared and aliquots of 100 µL from each dilution was spread-plated on Rainbow agarO157 plates (Kase et al., 2015) obtained from Biolog Inc., USA. The plates were incubated aerobically at 37◦C for 24 h. Colonies with purple and pink colors were randomly picked and purified by streaking on sorbitol MacConkey agar (Merck, S.A) supplemented with 1 mg/L potassium tellurite (Merck, SA) and incubated aerobically at 37◦C for 24 h. Pure isolates were preserved in 20 % (v/v) glycerol (Merck, SA) and stored at −80◦C for future use.

# Genomic DNA Extraction From Presumptive Isolates

Overnight culture for each sample was prepared and genomic DNA was extracted from the cultures using the Zymo Research Genomic DNATM-Tissue MiniPrep Kit (Biolab, South Africa) following the manufacturer's instructions. DNA concentration and purity was determined using the NanoDrop Lite 1,000 spectrophotometer (model: Thermo Fisher Scientific, USA) and the DNA was stored at −80◦C until further analysis by PCR.

#### Identification of *E. coli* O177 Serogroup Using Multiplex PCR Assay

A singleplex PCR assay targeting the uidA E. coli O177 specific gene sequence was performed using a previous protocol (Anbazhagan et al., 2011). Escherichia coli O177 serogroup-specific primer pairs were designed using the Primer3 software (this study), based on rmlB and wzy gene sequences that encode for dTDP-glucose 4, 6-dehydratase, and O-antigen polymerase, respectively (Ye et al., 2012). The specificity of the PCR primers was tested using National Center for Biotechnology Information/Primer-Basic Alignment Search Tool (NCBI/Primer-BLAST) (https://www.ncbi.nlm.nih. gov/tools/primer-blast/). The oligonucleotides were synthesized and supplied by Inqaba Biotechnical Industry Ltd., Pretoria, South Africa. The newly developed multiplex PCR protocol was empirically validated for its specificity, sensitivity reproducibility, and robustness. PCR assays was performed to amplify rmlB and wzy gene fragments and the primer sequences, targeted genes, amplicon sizes as well as the PCR conditions are listed in **Table 1**. The PCR reactions constituted 12.5 µL of 2X DreamTaq Green Master Mix, 0.5µM of each primer and 1 µL of template DNA. The volume of the reaction mixture was adjusted to 25 µL with RNase-nuclease free PCR water. A non-DNA template (nucleasefree water) reaction tube served as a negative control while a DNA sample from E. coli O177 (isolated during the preliminary study) was used as positive control. All the PCR reagents used were Fermentas USA products supplied by Inqaba Biotec Industry Ltd., Sunnyside, Pretoria, South Africa. Amplifications were performed using DNA thermal cycler (model- Bio-Rad C1000 TouchTM Thermal Cycler, Singapore). All the PCR products were held at 4◦C until gel electrophoresis.

# Detection of Virulence Genes in *E. coli* O177 Isolates

E. coli O177 isolates were subjected to a bfp gene PCR analysis in order to screen for characters of aEPEC strains. A highly virulent environmental E. coli O157:H7 isolate (bfp+) previously isolated from our research group (AREPHABREG) was used as positive control. The positive control E. coli O157:H7 possessed the stx1, stx2, eaeA, and hlyA genes. In addition, DNA extracted from an E. coli (ATCC 98222) that is a non-pathogenic strain was included in each PCR run as a negative control. Isolates that were positive for the bfp gene were further screened for the presence of an array of STEC virulence genes that included eaeA, hlyA, stx1, stx2, and stx2 variants (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, stx2g ) using previously described protocols (Paton and Paton, 1998; He et al., 2012). Primer sequences, target genes, amplicon sizes as well as PCR cycling conditions for the different genes are listed in **Table 1**. All the PCR reactions were prepared as 25 µL standard volumes comprising 12.5 µL of 2X DreamTaq Green Master Mix, 0.5µM of each primer, 1 µL of template DNA and RNasenuclease free PCR water. Amplifications were performed using DNA thermal cycler (model- Bio-Rad C1000 TouchTM Thermal Cycler, Singapore). All the PCR products were held at 4◦C until they were separated by electrophoresis.

### Antimicrobial Susceptibility Test

The antimicrobial susceptibility profile of the isolates was determined using the Kirby-Bauer disc diffusion technique (Bauer et al., 1966), making use of antibiotic discs obtained from Mast Diagnostics, UK. The antibiotics using in this study comprised Ampicillin (AMP), 10 µg; Chloramphenicol (C), 30 µg; Erythromycin (E), 15 µg; Kanamycin (K), 30 µg; Norfloxacin (NOR), 10 µg; Streptomycin (S), 10 µg and Tetracycline (TE), 30 µg. Some of these antimicrobial agents were selected due to the fact that they are widely used as prophylactics in both beef and dairy cattle farming in South Africa. Plates were incubated aerobically at 37◦C for 24 h and antibiotic growth inhibition zone diameter data were compared with standard reference values in order to classify the isolates as sensitive, intermediate resistance or resistant to a particular antibiotic (Clinical Laboratory Standards Institute, 2016). Escherichia coli ATCC 25922 was used as a reference for quality control in antimicrobial susceptibility test.

# Detection of Genetic Determinants for Antibiotic Resistance Genes by PCR

All confirmed E. coli O177 isolates that were resistant to three or more antibiotics were designated multi-drug resistant isolates and were screened for the presence of the tetA, tetW, aadA, strA, strB, ampC, cmlA, ermB, and kan antibiotic resistance determinants. Primer sequences, target genes, amplicon sizes as well as PCR cycling conditions for the different genes are listed in **Table 2**. All the PCR reactions were prepared as 25 µL standard volumes comprising 12.5 µL of 2X DreamTaq Green Master Mix, 0.5µM of each primer, 1 µL of template DNA and RNasenuclease free PCR water. Amplifications were performed using DNA thermal cycler (model- Bio-Rad C1000 TouchTM Thermal Cycler, Singapore). All the PCR products were kept at 4◦C and later resolved by electrophoresis.

# Agarose Gel Electrophoresis

All PCR amplicons were resolved by electrophoresis on a 2% (w/v) agarose gel containing 0.001µg/mL ethidium bromide. A 100 bp DNA molecular weight DNA marker (Fermentas, USA) was included in each gel and used to confirm the sizes of the amplicons. A ChemiDoc Imaging System (Bio-Rad ChemiDocTM MP Imaging System, UK) was used to capture the images using Gene Snap software, version 6.0022.

#### Sequence Analysis

The amplified rmlB and wzy gene fragments were sequenced by Inqaba Biotec, Pretoria, South Africa, and sequences TABLE 1 | Oligonucleotide primers used for amplification of the various targeted genes in E. coli O177 serogroup.

were subjected to a Blast Search Tool (https://blast.ncbi.nlm. nih.gov/Blast.cgi) in order to confirm the identities of the isolates (Altschul et al., 1997). Nucleotide sequence data were further deposited into GenBank using the BankIt NCBI webbased sequence submission tool (https://www.ncbi.nlm.nih.gov/ genbank/) in order to obtain their accession numbers.

#### Statistical Analysis

Measured parameters were tested for normality using the NORMAL option in the Proc Univariate statement prior to analysis of variance. Data on the proportions of samples positive for E. coli O177 serogroup were analyzed for effect of farming system using the general linear models (GLM) procedures of SAS

(2010) according to the following statistical model:

$$Y\_{i\dot{\jmath}} = \mu + F\_{\dot{\imath}} + E\_{i\dot{\jmath}}$$

where, Yij, response variable, µ, overall mean, Fi , farming system (intensive, semi-intensive and extensive) effect and Eij, random error associated with observation ij, assumed to be normally and independently distributed. Data on number of isolates carrying virulent genes, number of antibiotic resistant isolates, number of isolates carrying antibiotic resistance genes, and number of multidrug resistant isolates were square root-transformed before statistical analysis using the GLM procedures (SAS, 2010). For all statistical tests, significance was declared at p < 0.05.

#### RESULTS

#### Identification of *E. coli* O177 Serogroup Using Multiplex PCR Analysis

A total of 780 cattle fecal samples were analyzed for the presence of E. coli O177 using Rainbow agarO157 and a total of 1,272 non-repetitive presumptive isolates were obtained. Amplification of the uidA E. coli genus-specific gene sequence revealed that a large proportion (915; 71.93%) of the isolates were successfully identified as E. coli isolates. All the 915 E. coli isolates were further screened for the presence of E. coli O177 serogroup specific rmlB and wzy gene fragments using multiplex PCR analysis. Out of 915 isolates screened, 376 (41.09%) were identified as E. coli O177 serogroup and **Figure 1** indicates a 2% (w/v) agarose gel of representative rmlB and wzy gene fragments amplified in the study. No significant (p > 0.05) differences were observed on the occurrence of E. coli O177 serogroup across intensive, semi-intensive, and extensive animal production systems. The occurrence of E. coli O177 serogroup was 33.3 ± 14.43, 33.3 ± 14.43, and 50.0 ± 14.18% in intensive semi-intensive extensive farming systems, respectively.

#### Detection of Virulence Genotypes in *E. coli* O177 Isolates

A total of 376 atypical E. coli O177 isolates that were positively confirmed by PCR were screened for the presence of five virulence genes; bfpA, eaeA, hlyA, stx1, and stx2, using PCR analysis. None of the isolates possessed the bfpA gene fragment and thus were classified as aEPEC strains. On the contrary, all the other four virulence genes (eaeA, hlyA, stx1, and stx2) were successfully detected in this study. Generally, 350 isolates harbored these virulence genes. There were significant differences (p < 0.001) in terms of occurrence of various virulence genes in E. coli O177 isolates. The hlyA (12.74%) and stx2 (11.20%) were the most commonly detected genes (p < 0.001), followed by stx1 (9.07%), and eaeA (7.25%), **Figure 2**. Two stx2 subtypes namely; the stx2a and stx2d, were detected among the isolates. A few of the isolates (2.60%) harbored stx2a, while 0.63%

possessed stx2dgene. Some of the isolates carried a combination of the genes detected. The majority (9.74%) of the isolates carried a combination of hlyA/stx2 while 7.40, 7.09, 6.80, and 5.55% possessed stx1/stx2, stx1/stx2/hlyA, hlyA/eaeA, and stx2/hlyA/eaeA, respectively (p < 0.001). There was no difference (p > 0.05) between the occurrences of stx1/eaeA (6.35%) and stx1/stx2/hlyA/eaeA (5.11%) in E. coli O177 isolates. Only one isolate possessed stx1/stx2/stx2a/stx2d/hlyA/eaeA. Despite the fact that none of the isolates possessed the stx2 subtypes stx2b, stx2c, stx2e, stx2f, and stx2g, the virulence gene profiles of these aEPEC strains especially the isolate with the stx2 subtypes was a cause for concern.

#### Antimicrobial Resistance Profiles

Antimicrobial susceptibility tests of the 376 isolates revealed varied antimicrobial resistance profiles against all the seven antibiotics tested. Most of the E. coli O177 isolates were resistant to erythromycin (63.84%) compared to other antibiotics (p < 0.001), **Figure 3**. No difference (p > 0.05) was observed in E. coli O177 resistance to ampicillin (21.54%), tetracycline (13.37%), streptomycin (17.01%), kanamycin (2.42%), chloramphenicol (1.97%), and norfloxacin (1.40%). The isolates showed resistance to at least two antibiotics tested. Most of the isolates (20.74%) exhibited high level of resistance to three or more antibiotics, **Figure 4** (p < 0.001).

#### Proportion of Isolates With Antimicrobial Resistance Genes

A total of 73 E. coli O177 isolates harbored either one or more different antimicrobial resistance genes investigated, however, only five of the ten genes were detected in the isolates. Resistance genes associated with streptomycin and erythromycin were the most frequently detected. Genes aadA (22.86%), strA (19.85%), strB (19.38%) ermB (19.68%), and tetA (18.23%) were detected. However, there was no significant differences in the occurrence of these genes in E. coli O177 isolates. No isolates possessed the tetW gene. Some isolates harbored a combination of three or more antibiotic resistance genes. Most (24%) of the isolates carried a combination of the aadA, strA, and strB genes while a smaller proportion 11% isolates possessed all the five antimicrobial resistance genes (aadA/strA/strB/ermB/tetA) (p < 0.05).

### Sequence Identifier and Accession Numbers

Sequence data analysis of the rmlB and wzy gene fragments amplified from chromosomal DNA of isolates revealed a high percentage similarity (97%) sequence homology to E. coli serogroup O177. The nucleotide sequences for Seq1, Seq2, and Seq3 were assigned GenBank Accession numbers; MH389799, MH389800, and MH389801, respectively.

# DISCUSSION

In this study, the occurrence of aEPEC, E. coli O177 serogroup in cattle was investigated. Although the virulence profiles of aEPEC strains is poorly understood, recent reports suggest increased incidence of human infections caused by aEPEC strains worldwide (Ingle et al., 2016; Martins et al., 2016). Cattle are primary reservoir of E. coli species and contaminated meat as well as their associated food products have been implicated in foodborne infections in humans (Ateba and Mbewe, 2011). Studies have reported the occurrence of aEPEC strain in food-producing animals, foods of animal origin and farm environments (Horcajo et al., 2012; Otero et al., 2013; Ghanbarpour et al., 2017). Escherichia coli was successfully isolated from cattle feces using Rainbow agar O157. The identities of the isolates were confirmed as E. coli through amplification of uidA gene fragments. The rmlB and wzy O-antigen gene clusters specific for E. coli O177 were successfully amplified through Multiplex PCR analysis. The PCR assay was highly specific and reproducible for the detection of E. coli O177 serogroup from cattle feces. The occurrence of E. coli O177 in cattle was higher (41.09 %) in this study when compared to a previous report which detected this serogroup in ovine (Martins et al., 2016). Despite this, there were no significant (p > 0.05) differences on the occurrence of E. coli O177 serogroup across all the three farming systems.

Given the fact that aEPEC strains are known to lack medically important virulence factors, their ability to cause diseases particularly in humans is not yet well-understood (Ingle et al., 2016). Previous reports indicated that aEPEC strains do not harbor stx and bfpA genes. However, recent reports have revealed that aEPEC strains especially those from ruminants, harbor

virulence gene profiles similar to those of the EHEC strains (Horcajo et al., 2012). In addition, some studies have reported that aEPEC strains may represent LEE-positive Shiga toxinproducing E. coli (STEC) that have lost the toxin-encoding prophage, or tEPEC that have lost the genes for BFP indicating the potential to have evolved from other pathogenic E. coli strains (Tennant et al., 2009). A motivation was the fact that previous studies conducted in the study area revealed a large proportion of virulent EHEC strains (Ateba and Bezuidenhout, 2008; Ateba and Mbewe, 2011, 2013, 2014). Despite the fact that we were unable at this stage to determine if aEPEC isolated from humans in South African cattle fall into either of these categories, an assessment of the bfpA and other primary STEC virulence genes (eaeA, hlyA, stx1, stx2, and stx2 variants) was performed. Data of the shiga toxin subtypes among non-O157 STEC serogroups in cattle may provide an indication of the potential health risks these aEPEC isolates may cause humans. The bfpA gene was not detected in all the E. coli isolates analyzed and thus confirmed that the isolates

belonged to aEPEC group, emerging diarrheagenic pathogens in both developing and developed countries (Ingle et al., 2016; González et al., 2017).

All the major virulence genes (stx1, stx2, hlyA, and eaeA) were detected in this study. Similar findings were reported by Momtaz et al. (2013a) and Momtaz et al. (2013b). Generally, the majority of E. coli O177 isolates possessed virulence genes. The hlyA was the most frequently detected virulence gene in this study. These results are similar to those reported in other studies (Ateba and Bezuidenhout, 2008; Ateba and Mbewe, 2011; Momtaz et al., 2013c), which described high prevalence of hlyA gene in E. coli O157 isolated from cattle, beef, vegetable and water intended for human consumption. The high prevalence of hlyA gene may be attributed to the fact that hlyA is a plasmid encoded gene and as a result, it may be transferred between strains of the same species (Ateba and Mbewe, 2011). The hlyA gene encodes α-hemolysin, a toxin that lyses mammalian erythrocytes (Toro et al., 2018).

Another observation was that the E. coli O177 isolates possessed stx genes. These genes are considered as primary virulence genes in shiga toxin-producing E. coli and their presence in aEPEC strains, especially E. coli O177 serogroup raises a serious public health concern. The stx2 was the most prevalent gene detected in STEC/EHEC (Ateba and Mbewe, 2011; Rantsiou et al., 2012). Despite the fact that low frequency of stx2 gene was observed in this study, the occurrence of this gene was significantly higher than stx1 (p < 0.001). This observation was similar to the findings of the previous studies (Beutin et al., 2005; Jajarmi et al., 2017; Toro et al., 2018). Interestingly, stx2 positive isolates also harbored stx2a and stx2d gene subtypes. However, the other stx2 subtypes were not detected. Despite this, the presence of stx2, stx2a, and stx2d genes in E. coli O177 serogroup is a serious concern. Strains harboring these genes have been implicated in hemolytic colitis and hemorrhagic uremic syndrome infections in humans (Farrokh et al., 2013). It is also reported that a strain harboring stx2 gene is more virulent than a strain carrying either stx1 or both stx1 and stx2 (Farrokh et al., 2013; Toro et al., 2018). Moreover, stx2a and stx2d subtypes are associated with severe HC and HUS infections in human (Farrokh et al., 2013; Cha et al., 2018). Therefore, the presence of stx2gene and its subtypes in E. coli O177 may increase the spectrum of infections in humans and thus creating a public health concern.

The stx1 and eaeA genes were also detected but at low levels compared to hlyA and stx2 gene fragments. This was in contrast with the previous studies, which reported high occurrence of stx1 and eaeA genes in E. coli isolated from poultry meat and humans (Momtaz et al., 2013b; Hemmatinezhad et al., 2015). Despite low detection of these genes, especially E. coli O177 serogroup, these findings cannot be overemphasized. The strain carrying stx1 may cause diarrhea in immunocompromised individuals (Farrokh et al., 2013). In addition, the eaeA gene encodes for the outer membrane which mediates adherence between the STEC and/or EPEC and intestinal epithelial cells (Kaper et al., 2004; Farrokh et al., 2013). Although eaeA was detected in this study, this gene was not detected in some isolates. This was surprising because all the isolates were negative for bfpA gene and thus classified as aEPEC strains. These results differ significantly with other studies where the eaeA gene has been detected in aEPEC strains (Trabulsi et al., 2002; Beutin et al., 2005; Otero et al., 2013; Martins et al., 2016; Ghanbarpour et al., 2017; González et al., 2017). The possible explanation for this observation could be that the isolates may have lost the gene during sub-culturing process (Karch et al., 1992). Given that the strains carrying eaeA gene are considered potentially pathogenic, absence of this gene in some of the isolates in this study should not be underestimated. The eaeA-negative isolates may use other genes such as saa, aidA, agn43, ehaA, or iha to adhere to the epithelial cell of the host (Toro et al., 2018). However, the presence of these genes was not investigated in this study.

The co-expression of stx1, stx2, hlyA, and eaeA genes may increase the pathogenicity of E. coli strains (Jajarmi et al., 2017). It was observed that E. coli O177 isolates possessed combinations of virulence genes. There were twelve different combinations of virulence genes detected in this study. The most frequently detected combinations were stx2/hlyA (9.74 %), stx1/stx2 (7.40%), stx1/stx2/hlyA (7.09%), and hlyA/eaeA (6.80%). Only 5.11% of the isolates harbored all the four genes (eaeA/hlyA/stx1/stx2). It was also remarkable that one isolate possessed all the virulence (stx1/stx2/hlyA/eaeA) genes and two stx2 (stx2a and stx2d genes) subtypes. Simultaneous detection of these virulence genes in E. coli O177 serogroup in cattle pose a public health concern. Furthermore, these findings are similar to those reported in E. coli O26 in a previous study (Jajarmi et al., 2017; Ranjbar et al., 2017). The combinations of virulence genes in this study are higher than those reported by the previous studies (Ateba and Mbewe, 2011; Jajarmi et al., 2017; Toro et al., 2018). However, there was low occurrence of combination of stx2 subtypes as compared to a previous study (Jajarmi et al., 2017).

Antimicrobial agents primarily play a vital role in the lives of both humans and animals worldwide (Hudson et al., 2017). South Africa, is one of the countries where the usage of antimicrobial agents in animal production is very high (Hudson et al., 2017). In addition, the overuse of antimicrobial agents in food-producing animals, often without either professional consultation or supervision, has resulted in the emergence of antimicrobial resistant bacteria and antibiotic resistant genes in human and animal pathogens (Qiao et al., 2017). Moreover, previous investigations in the study area revealed the presence of antimicrobial resistant gene determinants in E. coli O157 isolated from cattle, beef, pig, pork, vegetables, and water intended for human consumption (Ateba and Bezuidenhout, 2008). Despite this, farmers continue to use antimicrobial agents to maximize production and this presents severe public health challenges.

In this study, E. coli O177 isolates revealed phenotypic resistance against all the seven classes of antimicrobial agents tested. In contrary to the previous study which reported high prevalence of resistance against ampicillin (100%), gentamicine (100%), and tetracycline (96.87%) Ranjbar et al. (2018), this study revealed resistance against erythromycin (63.84%), ampicillin (21.54%), tetracycline (13.37%), streptomycin (17.01%), and kanamycine (2.42%). These disparities could be attributed to geographical location and the sample type. Furthermore, 20.74% of the isolates were resistant to at least three and antimicrobial agents tested. These results were similarly to those reported in the previous study (Ranjbar et al., 2017). Antimicrobial resistance genes encoding for three antibiotics (erythromycin, streptomycin, and tetracycline) were detected. It has been reported that antibiotic resistance is common among E. coli isolates obtained from animals and food of animal origin due to frequent use of antibiotics in animals (Ryu et al., 2012). Generally, all isolates harbored antibiotic resistance genes. It is also worth mentioning that the same isolates harbored STEC virulence genes and previous studies have shown that isolates with similar genetic determinants may pose severe complications in humans (Ateba and Bezuidenhout, 2008; Hemeg, 2018). The occurrence of antibiotic resistance genes detected in this study was higher as compared to the previous study (Ryu et al., 2012). This could be due to the relatively higher usage of antimicrobial agents in the intensive livestock farming system where the isolates were obtained.

Antibiotic resistance genes associated with some of the antibiotics tested in this study were detected. The aadA, strA, and strB were the most frequently detected genes. However, there were significant differences among all the genes. Most of the isolates possessed aadA (22.86%), strA (19.85%), and strB (19.38%) genes that code for resistance to streptomycin and similar observations have been reported in other studies (Srinivasan et al., 2007; Shahrani et al., 2014; Bibbal et al., 2017). Despite the fact that tet genes are commonly found in E. coli from cattle due to the high usage of tetracycline in dairy and feedlot cattle, the occurrence of tet resistance genes in E. coli O177 isolates was low. Only the tetA gene was detected individually or in combination with other genes. However, in a previous report (Olowe et al., 2013), the proportion of tetA (43.8%) was higher than tetB (32.0%) and a combination of both tetA and tetB (4.4%) among clinical E. coli isolates. In addition, Ranjbar et al. (2018) reported tetA (76.56%), tetB (20.31%), cat1 (18.75%) and cmlA (1.50%) in E. coli O157 and non-O157 serogroups from milk. Another study reported high detection of tetA and tetB in E. coli isolated from urinary infection patients, ruminants and donkey raw milk (Momtaz et al., 2012, 2013c). Similar to a previous report (Olowe et al., 2013), none of the isolates in this study harbored the tetW, amp, cmlA, and kan genes.

In conclusion, to the best of our knowledge this is the first study to report the occurrence of E. coli O177 strain in cattle, especially in South Africa. In this study, the search for virulence determinants in aEPEC clearly revealed that a majority of strains harbored DNA sequences that encode known virulenceassociated determinants of other pathogenic E. coli such as O157 and non-O157 strains. In addition, E. coli O177 strains from this study displayed high levels of resistance to aminoglycoside antibiotic group thus have the potential to pose a public health concern to humans. Indeed, the fact that all aEPEC strains in this study expressed a variety of tEPEC virulence determinants supports the case for continued efforts to conduct a large scale study designed to determine the phylogenetic homology among aEPEC isolates from different sources. Furthermore, whole genome sequencing of E. coli O177 is required in order to understand the complete pathogenic determinants of this serogroup.

#### DATA AVAILABILITY STATEMENT

The datasets generated for this study can be found in the NCBI.

# AUTHOR CONTRIBUTIONS

VM and CA contributed to project conception and contributed reagents, materials, and analysis tools. PM, VM, and CA designed the experiments. PM performed the experiments. All authors

#### REFERENCES

contributed to manuscript revision and read and approved the submitted version.

#### FUNDING

This research was supported financially by the National Research Foundation (Grant No. 112543) and the North-West University Postgraduate Bursary.

### ACKNOWLEDGMENTS

The authors wish to thank Mr. BJ Morapedi and Dr. A Kumar for their assistance with this project.

city and slaughterhouse wastewater. Int. J. Hyg. Environ. Health 221, 355–363. doi: 10.1016/j.ijheh.2017.12.009

by repeated acquisition of LEE pathogenicity island variants. Nat Microbiol. 1:15010. doi: 10.1038/nmicrobiol.2015.10

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Montso, Mlambo and Ateba. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Characterization of a Novel Conjugative Plasmid in *Edwardsiella piscicida* Strain MS-18-199

Hossam Abdelhamed1 \*, Reshma Ramachandran1 , Ozan Ozdemir 1 , Geoffrey Waldbieser 2 and Mark L. Lawrence1

*1 College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, United States, 2 Warmwater Aquaculture Research Unit, Thad Cochran National Warmwater Aquaculture Center (USDA-ARS), Stoneville, MS, United States*

*Edwardsiella piscicida* is a pathogenic bacterium responsible for significant losses in important wild and cultured fish species. *E. piscicida* strain MS-18-199 recovered from a diseased hybrid catfish from East Mississippi and showed resistance to florfenicol, chloramphenicol, oxytetracycline, doxycycline, erythromycin, tetracycline, azitromycin, spectinomycin, sulfonamide, and bacitracin. To explore the mechanisms of resistance in *E. piscicida* strain MS-18-199, genomic DNA was extracted and subjected to whole genome sequencing (WGS) using a combination of long (Oxford Nanopore) and short (Illumina) reads. The genome of strain MS-18-199 revealed a novel plasmid named pEPMS-18199. The 117,448 bp plasmid contains several antimicrobial resistance (AMR) elements/genes, including florfenicol efflux pump (*floR*), tetracycline efflux pump (*tetA*), tetracycline repressor protein (*tetR*), sulfonamide resistance (*sul2*), aminoglycoside O-phosphotransferase *aph(6)-*Id (*strB*), and aminoglycoside O-phosphotransferase *aph(3)-*Ib (*strA*). Two genes, *arsA* and *arsD*, that encode protein components related to transport/resistance to arsenic were also found in pEPMS-18199. In addition, pEPMS-18199 carried twelve conjugative transfer genes (*tra*), eight transposases and insertion elements, two plasmid stability proteins, two replication proteins, and three partitioning proteins (*par* system). Results from mobilization and stability experiments revealed that pEPMS-18199 is highly stable in the host cell and could be transferred to *Escherichia coli* and *Edwardsiella ictaluri* by conjugation. To our knowledge, this is the first detection of a multidrug resistance (MDR) conjugative plasmid in *E. piscicida* in the United States. Careful tracking of this plasmid in the aquaculture system is warranted. Knowledge regarding the molecular mechanisms of AMR in aquaculture is important for antimicrobial stewardship.

Keywords: antimicrobial resistance, mobile genetic element, *Edwardsiella*, aquaculture, genome sequence

# INTRODUCTION

Edwardsiella piscicida, a member of family Enterobacteriaceae, is a motile Gram negative and facultative anaerobic bacterium (Ewing et al., 1965). The description of E. piscicida (previously identified as Edwardsiella tarda) resulted from a reclassification of diverse isolates obtained from diseased fish (Abayneh et al., 2013). Besides its importance as a pathogen of aquatic species, E. piscicida has been isolated from a wide range of hosts (including birds, reptiles, and humans)

#### *Edited by:*

*Xiao-Peng Xiong, Sanford-Burnham Institute for Medical Research, United States*

#### *Reviewed by:*

*Zhijie Xia, Sanford Burnham Prebys Medical Discovery Institute, United States Jingting Yu, Salk Institute for Biological Studies, United States*

*\*Correspondence: Hossam Abdelhamed<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology*

*Received: 31 July 2019 Accepted: 12 November 2019 Published: 27 November 2019*

#### *Citation:*

*Abdelhamed H, Ramachandran R, Ozdemir O, Waldbieser G and Lawrence ML (2019) Characterization of a Novel Conjugative Plasmid in Edwardsiella piscicida Strain MS-18-199. Front. Cell. Infect. Microbiol. 9:404. doi: 10.3389/fcimb.2019.00404* from a broad geographical range and ecological niches (Camus et al., 2016; Buján et al., 2018b). At present, E. piscicida is the etiologic agent of edwardsiellosis and is responsible for high mortality rates in several fish species (more than 20 fish species as hosts) (Abayneh et al., 2013; Camus et al., 2016; Loch et al., 2017; Buján et al., 2018b). Common clinical signs associated with edwardsiellosis include exophthalmia, abdominal distension, skin hemorrhages, mild to moderate dermal ulcerations, discolorations on the fish surface, and erratic swimming (Buján et al., 2018b).

Commercial production of channel catfish (Ictalurus punctatus) and channel catfish × blue catfish (I. furcatus) hybrids dominates aquaculture in the United States accounting for \$360 million in 2018, and the majority of production occurs in Mississippi (USDA, 2019). Loss to disease and feed cost are the major factors that influence profitable catfish aquaculture (Wagner et al., 2002). Approximately 45% of inventory losses on catfish farms are attributable to infectious diseases, of which 60% are associated with single or mixed bacterial infections (Hawke and Khoo, 2004). Edwardsiella species continue to be the bacterial pathogen of primary concern in catfish aquaculture in the southeastern United States (Hawke et al., 1981, 2013). There is a trend toward increased incidence and prevalence of E. piscicida septicemia in US catfish aquaculture (Griffin et al., 2019). The recent reclassification and review of archival data indicated that E. piscicida is more problematic in catfish aquaculture than E. tarda (Buján et al., 2018b; Griffin et al., 2019). Moreover, recent genotypic analysis of bacterial isolates historically classified as E. tarda concluded that many isolates previously classified as E. tarda actually belong to the species E. piscicida (Reichley et al., 2017; Buján et al., 2018a).

Because no definite vaccine is available, antimicrobial (AM) medicated-feeds are the current available strategy to control the bacterial diseases caused by E. piscicida and other fish pathogens (Reichley et al., 2017; Buján et al., 2018b). Terramycin (oxytetracycline), Romet-30 (sulfadimethoxine-ormetoprim), and Aquaflor (florfenicol) are three AM agents approved by the FDA for use in medicated-feed to control bacterial infections in cultured fish (Bowker et al., 2010). Although AM use is highly restricted and strongly regulated in the United States aquaculture, a multidrug resistant (MDR) strain of E. piscicida strain MS-18-199 (unpublished data) has been recovered from moribund hybrid catfish submitted to Aquatic Diagnostic Laboratories at College of Veterinary Medicine Mississippi State University (CVM/MSU). The mechanisms that drive resistance in this isolate is unclear. Here, we describe the genetic feature of a large conjugative plasmid identified in E. piscicida. Moreover, we evaluated plasmid mobility and stability. Characterizing a plasmid carrying resistance determinants, especially in clinical isolates, will increase our understanding of the flow of resistance.

#### MATERIALS AND METHODS

#### Bacterial Isolates and Growth Condition

Edwardsiella piscicida strain MS-18-199 was isolated from moribund hybrid catfish clinical case presented to the Fish Diagnostic Laboratory at CVM/MSU. The diseased fish exhibited large ulceration on the dorsal head with bone erosion and scant yellow ascites. The internal examination of catfish revealed moderate granulomatous hepatitis. The initial presumptive diagnosis based on clinical signs was E. piscicida infection. Pure colonies with identical morphology were isolated from posterior kidney on Mueller Hinton agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 5% defibrinated sheep blood (HemoStat Laboratories, Dixon, CA, USA) at 30◦C. A stock culture was stored at −80◦C in 20% glycerol. The isolate was confirmed phenotypically as E. piscicida infection. The API 20E profile of E. piscicida strain MS-18-199 is present in **Supplementary Table 1**. Escherichia coli J53Azr (resistant to sodium azide) (Martinez-Martinez et al., 1998), E. ictaluri 93-146 with pAKgfplux1 (ampicillin resistant) (Karsi and Lawrence, 2007), and Aeromonas hydrophila ML09-119 (ampicillin resistant) (Tekedar et al., 2013b) were used as a recipient for conjugation. Edwardsiella species were cultured in brain heart infusion (BHI) media (Difco, Sparks, MD, USA) and incubated at 30◦C with orbital shaking (250 rpm). E. coli and A. hydrophila strains were cultured in Luria– Bertani (LB) agar and broth (Becton Dickinson) at 30◦C and 37◦C, respectively.

# Antimicrobial Susceptibility

The susceptibilities of E. piscicida strain MS-18-199 to 23 AMs were determined using Kirby-Bauer disk diffusion assays according to CDC guidelines, as described by the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2010).  Bacterial strains were reported as susceptible, intermediate, or resistant based on inhibition zone size as published in the CLSI standard (CLSI, 2010).

#### DNA Isolation

A single colony of E. piscicida strain MS-18-199 was grown overnight at 30◦C in BHI. Overnight culture was pelleted by centrifugation, and genomic DNA (gDNA) isolated using standard ethanol precipitation following phenol: chloroform: isoamyl alcohol (24:25:1 v/v; AppliChem) treatment, as previously described (Psifidi et al., 2010; Green and Sambrook, 2017). The quality of extracted gDNA was assessed using an Agilent 2100 Bioanalyzer (High sensitivity DNA chip), and a Nanodrop spectrophotometer. Genomic DNA with an ultraviolet absorbance 260/280 of ∼ 1.8 and absorbance 260/230 of 2.0–2.2 was used for library preparation.

# Whole Genome Sequence of *E. piscicida* Strain MS-18-199

The genome of E. piscicida strain MS-18-199 was sequenced using long and short read technologies. Long reads were produced on a GridION sequencer (Oxford Nanopore Technologies, Oxford, UK) from 400 ng genomic DNA using the RAD004 kit on a v9.4.1 flow cell. The nanopore sequences were filtered to an average quality of 10 (90% accuracy) with 100 bp cropped from each end and minimum length of 700 bp (De Coster et al., 2018) to produce 2.4 Gb of sequence from 185,481 reads ranging from 700 bp to 191 kb (average length 13.4 kb). A genomic DNA library was prepared using the Nextera XT DNA kit (Illumina Inc., San Diego, CA, USA) and 150 bp paired reads were sequenced on the Illumina MiSeq4000 Platform (Novogene Corporation, Sacramento, CA). The Illumina sequence was filtered using Trimmomatic v0.38 (Bolger et al., 2014) using the parameters "LEADING:30 TRAILING:30 SLIDINGWINDOW:4:30 MINLEN:50" to produce 1.6 Gb sequence in 5.5 M paired reads.

# Genome Assembly

The nanopore sequence was assembled de novo [Canu v1.7; Koren et al., 2017] into two contigs of 3.9 Mb and 230 kb. The linear contigs were manually trimmed at the sequence overlap within the putative circular genome and plasmid, then sequence from the 3′ end of each contig (1 Mb and 100 kb, respectively) was cut and moved to the 5′ end of each respective contig. The filtered nanopore reads were mapped to these contigs and visualized using the IGV viewer (Robinson et al., 2011) to validate the sequence contiguity and therefore the circular nature of the genome and plasmid. The consensus sequences were corrected using four iterations of Nanopolish v.10.2 (Loman et al., 2015) using the filtered Nanopore sequences, then corrected with two iterations of Pilon v1.2.2 (Walker et al., 2014) using the filtered Illumina reads.

# Genome Annotation and Function Analysis of Strain MS-18-199

The assembled genome was submitted to the National Center for Biotechnology Information (NCBI) database (http://www. ncbi.nlm.nih.gov/) prokaryotic genome annotation pipeline. Antimicrobial resistance (AMR) genes were automatically annotated as resistance genes at the NCBI. Each annotated resistant protein was searched against NCBI database using the best match from a blastn search (NCBI BLAST v2.2.27+). Further confirmation was performed by blast search against the Antibiotic Resistance Database (ARDB) (http://ardb.cbcb.umd. edu/) (Balabanov et al., 2012) and Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/) (McArthur et al., 2013). The NCBI BLAST results were used to confirm the annotation of insertion sequence, replication, and conjugative proteins. Gene function was predicted using the Gene Rapid Annotation Subsystem Technology (RAST) server (http://rast.nmpdr.org/) (Overbeek et al., 2014).

# Conjugation Experiment

Plasmid mobility was evaluated by conjugation experiments on filter paper placed on agar plates as previously described (Abdelhamed et al., 2018a). E. piscicida strain MS-18-199 was used as a donor strain, and E. coli J53, E. ictaluri, and A. hydrophila ML09-119 were used as recipient strains. Briefly, overnight cultures of donor and recipient cells were diluted and incubated at 30◦C with shaking until they reached logarithmic phase. Bacteria were harvested by centrifugation at 12,000 rpm, mixed into 1:1 ratio, placed onto filter paper on agar plate, and incubated at 30◦C overnight. After mating, bacteria were collected, washed, diluted, and plated on BHI plates containing sodium azide (100µg/ml) or ampicillin (100µg/ml) for counter selection, and florfenicol (30µg/ml) and tetracycline (30µg/ml) to select for resistance. The transconjugant colonies were counted and the mobilization efficiency was estimated as the number of transconjugants CFU (colony-forming unit) per recipient CFU (Zeng et al., 2015). The resistance phenotypes of the transconjugant colonies were evaluated using the disc diffusion assay as described in the above section. Further confirmation of plasmid carriage in the transconjugant colonies was performed by PCR amplification of florfenicol resistance using FlorR\_F (CTGATGGCTCCTTTCGACAT) and FlorR\_R (AGACGACT GGCGACTTATCG) primers.

# Plasmid Stability Assays

A stability test of the plasmid in E. piscicida strain MS-18-199 was conducted as previously described (Moleres et al., 2015). Briefly, a single colony of E. piscicida strain MS-18-199 harboring the plasmid was inoculated in BHI without antibiotic and grown overnight at 30◦C. This culture was passaged by serial 1:100 dilutions in 5 ml BHI on consecutive days for up to 20 days. In each subculture step, bacteria were plated on BHI agar, and the proportion of plasmid-containing cells was deduced by replica inoculation of 100 colonies on BHI broth and BHI broth containing florfenicol. The number of colonies carrying the plasmid was calculated as the percentage of florfenicol-resistant colonies in every subculture/total number of colonies replicated each subculture (Moleres et al., 2015).

# Plasmid Copy Number

Plasmid copy numbers in E. piscicida strain MS-18-199 were determined using quantitative real-time PCR (qRT-PCR) as previously described (Botts et al., 2017). Briefly, total genomic DNA (gDNA) was extracted from early stationary phase cells using the Genomic DNA Mini Kit (IBI Scientific), as per manufacturer's instructions. The gDNA was quantified then serially diluted two-fold and used as the qRT-PCR template. qRT-PCR was performed in triplicate using SYBR Green Real-time PCR master mix (Roche Diagnostic GmbH, Mannheim, Germany). Chromosomal and plasmid DNA were quantified using primers targeting L-asparaginase 1 gene of E. piscicida strain MS-18-199 (locus tag: EVK84\_00005; FP: ACCGATACCATGGCGTTTAC; RP: CCAGATAGAGGGCG TTTAACAG), which exists on the chromosome as a single copy, and the single-copy floR gene encoded on the plasmid TABLE 1 | Antibiotic resistance patterns of *Edwardsiella piscicida* strain MS-18-199.

(locus tag: EVK84\_18305; FP: TGATCGTGACAACCCGTTTC; RP: GATGAAGGTGAGGAATGACGG).

# RESULTS

#### Resistance Phenotypes of *E. piscicida* Strain MS-18-199

The susceptibility pattern of E. piscicida strain MS-18-199 to 23 AM agents are shown in **Table 1**. Strain MS-18-199 was resistant to florfenicol, chloramphenicol, oxytetracycline, doxycycline, erythromycin, tetracycline, azitromycin, spectinomycin, sulfamethoxazole, and bacitracin.

### Basic Data of *E. piscicida* Strain MS-18-199 Genome

We conducted whole genome sequencing and assembly to gain further insight on the molecular mechanism of resistance of E. piscicida strain MS-18-199. The genome of strain MS-18-199 consisted of one circular chromosome and one circular plasmid, and both sequences have been submitted to GenBank under accession numbers CP035668.1 and CP035669.1, respectively. The 3,905,600 bp genome contained 59.47% GC content and 3,758 genes, including 3,631 coding sequences (CDS), 83 pseudogenes, and 127 RNAs [including 25 rRNAs (9, 8, and 8 for 5S, 16S, and 23S, respectively), 97 tRNAs, and 5 ncRNAs]. The complete genome of E. piscicida strain MS-18-199 shared 99.40% average nucleotide identity (ANI) with Edwardsiella piscicida C07-08 (Tekedar et al., 2013a), and 99.35% with Edwardsiella piscicida isolate S11-285 (Reichley et al., 2016).

#### Identification of pEPMS-18199 Plasmid in *E. piscicida* Strain MS-18-199

A circular plasmid was identified from the assembly of long sequencing reads from E. piscicida strain MS-18-199. The plasmid pEPMS-18199 is 117,448 bp with an average 47.34% GC content, ∼12% lower than the genome. Genome annotation revealed 124 open reading frames (ORFs), of which five were associated with plasmid DNA replication and partition, 12 with DNA transfer, 13 with DNA-restriction and site-specific DNA methylation, eight with family transposase, eight with antimicrobial resistance, and 44 with unknown function "hypothetical protein" (**Figure 1A**). A BLASTN search revealed pEPMS-18199 was nearly identical (99.97 % identity, ANI 99.94%, and 95% coverage) to 127,046 bp plasmid ET080813 (CP006665.1) isolated from E. anguillarum in China (Shao et al., 2015).

Six AMR genes were identified in the pEPMS-18199 sequence, including major facilitator superfamily (MFS) antibiotic efflux pump (floR), tetracycline repressor protein (tetR), tetracycline resistance protein class A (tetA), sulfonamideresistant dihydropteroate synthase (sul2), aminoglycoside O-phosphotransferase aph(6)-Id, and aminoglycoside Ophosphotransferase aph(3′′)-Ib (strA) (**Table 2**). Two genes corresponding to proteins predicted to be involved in arsenic resistance were also identified in the pEPMS-18199 sequence; arsenite efflux transporter metallochaperone arsD and arsenical pump-driving ATPase arsA. All the AMR genes were clustered in one region between 9,052 and 28,696 of pEPMS-18199. This region was flanked upstream and downstream with transposable elements (**Figure 1B**).

The pEPMS-18199 plasmid contains twelve conjugative transfer genes clustered in two regions, designated as Tra1 and Tra2. The Tra1 region is located between sequence coordinates 40,209 and 47,900 and consists of three proteins (TraFHG). The Tra2 region is located between 72,240 and 98,771, and consists of nine proteins (TraI, TraL, TraKBVA, and TraCWU). The pEPMS-18199 plasmid encodes eight transposases and insertion sequences belonging to Tn3-like element TnAs1 family transposase, IS6-like element IS26 family transposase, IS91-like element ISVsa3 family transposase, IS110 family transposase, two Tn3 family transposase, IS6-like element IS26 family transposase, and IS5-like element ISKpn26 family transposase. The plasmid also harbors plasmid stability protein (ParM/StbA family protein) and two plasmid replication proteins. Furthermore, the pEPMS-18199 plasmid encodes additional maintenance proteins including three chromosome partitioning proteins (ParB, ParA, and ParB/RepB family protein).

General Annotation for pEPMS-18199 Plasmid and *E. piscicida* Strain MS-18-199 Genome

and conjugative transfer genes (red). (B) Antimicrobial resistant cassette regions in pEPMS-18199 plasmid.

The genomic features of pEPMS-18199 plasmid annotated using RAST revealed that pEPMS-18199 contains 13 proteins encoding for membrane transport, one protein encoding for nucleosides and nucleotides, one protein encoding for protein metabolism, four proteins encoding DNA metabolism, one protein encoding respiration, and three proteins encoding stress response (**Figure 2A**).

The genomic features of chromosome annotated using RAST revealed that E. piscicida strain MS-18-199 genome contains 3,551 coding sequences and 497 subsystems (**Figure 2B**). The most represented subsystem features are cofactors, vitamins, prosthetic croups, pigments (294 genes), cell wall and capsule (191), virulence, disease and defense (72), potassium metabolism (30), phages, prophages, transposable elements, plasmids (43), iron acquisition and metabolism (46), membrane transport (99), RNA metabolism (230), nucleosides and nucleotides (113), protein metabolism (277) fatty acids, lipids, and isoprenoids (121), amino acids and derivatives (359), phosphorus metabolism (33), and carbohydrates (384).

#### Mobilization and Stability of the pEPMS-18199 Plasmid

Successful conjugation mating experiments indicated that the pEPMS-18199 plasmid was capable of being transferred from E. piscicida to E. coli and E. ictaluri with an average efficiency of 6.86 × 10−5 and 6.13 × 10−5 CFU/recipient, respectively. Furthermore, E. coli and E. ictaluri transconjugants showed the

same pattern of resistance as donor strain. Also, PCRs confirmed the presence of plasmid in transconjugants. Conversely, pEPMS-18199 could not be transferred from E. piscicida to A. hydrophila.