# ANTIMICROBIAL RESISTANCE AS A GLOBAL PUBLIC HEALTH PROBLEM: HOW CAN WE ADDRESS IT?

EDITED BY : Ilana L. B. C. Camargo, Leonardo Neves de Andrade, Thierry Naas, Luciene Andrade Da Rocha Minarini, Raffaele Zarrilli, Eliana De Gregorio and Filipa Grosso PUBLISHED IN : Frontiers in Microbiology, Frontiers in Public Health and Frontiers in Medicine

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ISSN 1664-8714 ISBN 978-2-88966-284-5 DOI 10.3389/978-2-88966-284-5

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# ANTIMICROBIAL RESISTANCE AS A GLOBAL PUBLIC HEALTH PROBLEM: HOW CAN WE ADDRESS IT?

Topic Editors:

Ilana L. B. C. Camargo, University of Sao Paulo, Brazil Leonardo Neves de Andrade, University of São Paulo, Brazil Thierry Naas, Assistance Publique Hopitaux De Paris, France Luciene Andrade Da Rocha Minarini, Federal University of São Paulo, Brazil Raffaele Zarrilli, University of Naples Federico II, Italy Eliana De Gregorio, University of Naples Federico II, Italy Filipa Grosso, University of Porto, Portugal

Citation: Camargo, I. L. B. C., De Andrade, L. N., Naas, T., Da Rocha Minarini, L. A., Zarrilli, R., De Gregorio, E., Grosso, F., eds. (2020). Antimicrobial Resistance As a Global Public Health Problem: How Can We Address It?. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-284-5

# Table of Contents

## *08 Editorial: Antimicrobial Resistance as a Global Public Health Problem: How Can We Address It?*

Luciene Andrade Da Rocha Minarini, Leonardo Neves de Andrade, Eliana De Gregorio, Filipa Grosso, Thierry Naas, Raffaele Zarrilli and Ilana L. B. C. Camargo

*14 Counter Clinical Prognoses of Patients With Bloodstream Infections Between Causative* Acinetobacter baumannii *Clones ST191 and ST451 Belonging to the International Clonal Lineage II*

Eun-Jeong Yoon, Dokyun Kim, Hyukmin Lee, Hye Sun Lee, Jong Hee Shin, Young Uh, Kyeong Seob Shin, Young Ah Kim, Yoon Soo Park, Jeong Hwan Shin and Seok Hoon Jeong

*23 Synergistic Microbicidal Effect of Auranofin and Antibiotics Against Planktonic and Biofilm-Encased* S. aureus *and* E. faecalis

Pengfei She, Linying Zhou, Shijia Li, Yiqing Liu, Lanlan Xu, Lihua Chen, Zhen Luo and Yong Wu

*35 Genomic Characterization of Prevalent* mcr-1, mcr-4, *and* mcr-5 Escherichia coli *Within Swine Enteric Colibacillosis in Spain* Isidro García-Meniño, Dafne Díaz-Jiménez, Vanesa García, María de Toro,

Saskia C. Flament-Simon, Jorge Blanco and Azucena Mora

*49* In vitro *Activity and Heteroresistance of Omadacycline Against Clinical*  Staphylococcus aureus *Isolates From China Reveal the Impact of Omadacycline Susceptibility by Branched-Chain Amino Acid Transport System II Carrier Protein, Na/Pi Cotransporter Family Protein, and Fibronectin-Binding Protein*

Bing Bai, Zhiwei Lin, Zhangya Pu, Guangjian Xu, Fan Zhang, Zhong Chen, Xiang Sun, Jinxin Zheng, Peiyu Li, Qiwen Deng and Zhijian Yu

*61 Increasing Frequencies of Antibiotic Resistant Non-typhoidal* Salmonella *Infections in Michigan and Risk Factors for Disease* Sanjana Mukherjee, Chase M. Anderson, Rebekah E. Mosci, Duane W. Newton,

Paul Lephart, Hossein Salimnia, Walid Khalife, James. T. Rudrik and Shannon D. Manning

*73 Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections*

Shelley B. Gibson, Sabrina I. Green, Carmen Gu Liu, Keiko C. Salazar, Justin R. Clark, Austen L. Terwilliger, Heidi B. Kaplan, Anthony W. Maresso, Barbara W. Trautner and Robert F. Ramig

*90 Characterization of Lytic Bacteriophages Infecting Multidrug-Resistant Shiga Toxigenic Atypical* Escherichia coli *O177 Strains Isolated From Cattle Feces*

Peter Kotsoana Montso, Victor Mlambo and Collins Njie Ateba

*103 Is Meropenem as a Monotherapy Truly Incompetent for Meropenem-Nonsusceptible Bacterial Strains? A Pharmacokinetic/Pharmacodynamic Modeling With Monte Carlo Simulation* Xiangqing Song, Yi Wu, Lizhi Cao, Dunwu Yao and Minghui Long

## *118 Multiple* Klebsiella pneumoniae *KPC Clones Contribute to an Extended Hospital Outbreak*

Carolina Ferrari, Marta Corbella, Stefano Gaiarsa, Francesco Comandatore, Erika Scaltriti, Claudio Bandi, Patrizia Cambieri, Piero Marone and Davide Sassera

*129 Over-Expression of IS*Aba1*-Linked Intrinsic and Exogenously Acquired OXA Type Carbapenem-Hydrolyzing-Class D-ß-Lactamase-Encoding Genes is Key Mechanism Underlying Carbapenem Resistance in*  Acinetobacter baumannii

Marcus Ho-yin Wong, Bill Kwan-wai Chan, Edward Wai-chi Chan and Sheng Chen

*138 Antibiotic Susceptibility, Virulence Pattern, and Typing of* Staphylococcus aureus *Strains Isolated From Variety of Infections in India*

Shifu Aggarwal, Smrutiti Jena, Sasmita Panda, Savitri Sharma, Benu Dhawan, Gopal Nath, N. P. Singh, Kinshuk Chandra Nayak and Durg Vijai Singh

Divakar Sharma, Anjali Garg, Manish Kumar, Faraz Rashid and Asad U. Khan

Elodie Olivares, Stéphanie Badel-Berchoux, Christian Provot, Gilles Prévost, Thierry Bernardi and François Jehl

*211 Colistin Resistance Gene* mcr-1 *Mediates Cell Permeability and Resistance to Hydrophobic Antibiotics*

Baiyuan Li, Fang Yin, Xuanyu Zhao, Yunxue Guo, Weiquan Wang, Pengxia Wang, Honghui Zhu, Yeshi Yin and Xiaoxue Wang

*218 CRISPR-Cas System in Antibiotic Resistance Plasmids in* Klebsiella pneumoniae

Muhammad Kamruzzaman and Jonathan R. Iredell

*231 Linezolid and Rifampicin Combination to Combat* cfr*-Positive Multidrug-Resistant MRSA in Murine Models of Bacteremia and Skin and Skin Structure Infection*

Yu-Feng Zhou, Liang Li, Meng-Ting Tao, Jian Sun, Xiao-Ping Liao, Ya-Hong Liu and Yan Q. Xiong

Zhichang Liu, Dun Deng, Huijie Lu, Jian Sun, Luchao Lv, Shuhong Li, Guanghui Peng, Xianyong Ma, Jiazhou Li, Zhenming Li, Ting Rong and Gang Wang

Yufei Lin, Xiaohong Dong, Jiao Wu, Dawei Rao, Lihua Zhang, Yousef Faraj and Kun Yang

*355 Molecular Analysis of Selected Resistance Determinants in Diarrheal Fecal Samples Collected From Kolkata, India Reveals an Abundance of Resistance Genes and the Potential Role of the Microbiota in Its Dissemination*

Rituparna De, Asish Kumar Mukhopadhyay and Shanta Dutta

Alexandra Vasilakopoulou, Polyxeni Karakosta, Sophia Vourli, Aikaterini Tarpatzi, Paraskevi Varda, Maria Kostoula, Anastasia Antoniadou and Spyros Pournaras

*386 Inactivation of Glutamine Synthetase-Coding Gene* glnA *Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in* Salmonella enterica *Serovar Typhi*

Ana R. Millanao, Aracely Y. Mora, Claudia P. Saavedra, Nicolás A. Villagra, Guido C. Mora and Alejandro A. Hidalgo

*396 Confronting Tigecycline-Resistant* Acinetobacter baumannii *via Immunization Against Conserved Resistance Determinants* Ming-Hsien Chiang, Ya-Sung Yang, Jun-Ren Sun, Yung-Chih Wang,

Shu-Chen Kuo, Yi-Tzu Lee, Yi-Ping Chuang and Te-Li Chen *409 Prevalence, Characterization, and Drug Resistance of* Staphylococcus

Aureus *in Feces From Pediatric Patients in Guangzhou, China* Xiaolan Ai, Fei Gao, Shuwen Yao, Bingshao Liang, Jialiang Mai, Zhile Xiong, Xiantang Chen, Zhuwei Liang, Hongling Yang, Zhiying Ou, Sitang Gong, Yan Long and Zhenwen Zhou

Thaysa Leite Tagliaferri, Natália Rocha Guimarães, Marcella de Paula Martins Pereira, Liza Figueiredo Felicori Vilela, Hans-Peter Horz, Simone Gonçalves dos Santos and Tiago Antônio de Oliveira Mendes

*457 Characterization of KPC-Producing* Serratia marcescens *in an Intensive Care Unit of a Brazilian Tertiary Hospital*

Roumayne L. Ferreira, Graziela S. Rezende, Marcelo Silva Folhas Damas, Mariana Oliveira-Silva, André Pitondo-Silva, Márcia C. A. Brito, Eduardo Leonardecz, Fabiana R. de Góes, Emeline Boni Campanini, Iran Malavazi, Anderson F. da Cunha and Maria-Cristina da Silva Pranchevicius

*471 Prediction of Antimicrobial Resistance in Gram-Negative Bacteria From Whole-Genome Sequencing Data*

Pieter-Jan Van Camp, David B. Haslam and Aleksey Porollo

Zahid Hayat Mahmud, Mir Himayet Kabir, Sobur Ali, M. Moniruzzaman, Khan Mohammad Imran, Tanvir Noor Nafiz, Md. Shafiqul Islam, Arif Hussain, Syed Adnan Ibna Hakim, Martin Worth, Dilruba Ahmed, Dara Johnston and Niyaz Ahmed

*533 Molecular Mechanisms and Epidemiology of Fosfomycin Resistance in*  Staphylococcus aureus *Isolated From Patients at a Teaching Hospital in China*

Wenya Xu, Tao Chen, Huihui Wang, Weiliang Zeng, Qing Wu, Kaihang Yu, Ye Xu, Xiucai Zhang and Tieli Zhou

*543 Alternative Therapeutic Options to Antibiotics for the Treatment of Urinary Tract Infections*

Paul Loubet, Jérémy Ranfaing, Aurélien Dinh, Catherine Dunyach-Remy, Louis Bernard, Franck Bruyère, Jean-Philippe Lavigne and Albert Sotto

*561 Coexistence of the Oxazolidinone Resistance–Associated Genes* cfr *and*  optrA *in* Enterococcus faecalis *From a Healthy Piglet in Brazil*

Lara M. Almeida, Anthony Gaca, Paulo M. Bispo, François Lebreton, Jose T. Saavedra, Rafael A. Silva, Irinaldo D. Basílio-Júnior, Felipe M. Zorzi, Pedro H. Filsner, Andrea M. Moreno and Michael S. Gilmore

# Editorial: Antimicrobial Resistance as a Global Public Health Problem: How Can We Address It?

Luciene Andrade Da Rocha Minarini 1 , Leonardo Neves de Andrade2 , Eliana De Gregorio3 , Filipa Grosso4 , Thierry Naas 5 , Raffaele Zarrilli 6 and Ilana L. B. C. Camargo7 \*

1 Department of Pharmaceutical Sciences, Federal University of São Paulo, Diadema, Brazil, 2 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Brazil, 3 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy, 4 Faculty of Pharmacy, University of Porto, Porto, Portugal, 5 Assistance Publique Hopitaux De Paris, Paris, France, 6 Department of Public Health, University of Naples Federico II, Naples, Italy, 7 Laboratory of Molecular Epidemiology and Microbiology (LEMiMo), São Carlos Institute of Physics, University of São Paulo, São Carlos, Brazil

Keywords: antimicrobial resistance, CRISPR-Cas, oxazolidinone-resistant *Enterococcus faecalis*, carbapenem-resistant *A. baumannii*, colistin-resistant *E. coli*, antibiotic-resistant non-typhoidal Salmonella, MRSA, VRE

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

#### *Edited by:*

Marc Jean Struelens, Université Libre de Bruxelles, Belgium

> *Reviewed by:* Sergey Eremin, World Health Organization, Switzerland

*\*Correspondence:* Ilana L. B. C. Camargo<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Infectious Diseases - Surveillance, Prevention and Treatment, a section of the journal Frontiers in Public Health

*Received:* 30 September 2020 *Accepted:* 21 October 2020 *Published:* 12 November 2020

#### *Citation:*

Minarini LADR, Andrade LNd, De Gregorio E, Grosso F, Naas T, Zarrilli R and Camargo ILBC (2020) Editorial: Antimicrobial Resistance as a Global Public Health Problem: How Can We Address It? Front. Public Health 8:612844. doi: 10.3389/fpubh.2020.612844

#### **Antimicrobial Resistance as a Global Public Health Problem: How Can We Address It?**

Misuse of antibiotics in agriculture, food production, and especially among humans and animals is the predominant factor in the emergence and spread of antimicrobial resistance, which might further lead to deaths from infections worldwide (1). More specifically, in the European Union, attributable deaths due to antimicrobial-resistant microorganisms were estimated to be 33,110 per year (2). At the same time, it is now easier to isolate and characterize antimicrobial-resistant bacteria in clinical settings or the environment (1). In 2017, the WHO described the most critical multidrug-resistant bacteria for which novel therapeutics are urgently needed (3). Without surprise, they belonged to the already known group, ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) (4), which causes most of the healthcare-associated infections nowadays.

On the top of the WHO list are the critical priorities: carbapenem-resistant A. baumannii, P. aeruginosa, and Enterobacteriaceae, plus ESBL-producing Enterobacteriaceae (3). As high priorities are vancomycin-resistant E. faecium, Staphylococcus aureus (methicillin-resistant, vancomycinintermediate and resistant), clarithromycin-resistant Helicobacter pylori, fluoroquinolone-resistant Campylobacter spp., fluoroquinolone-resistant Salmonellae, Neisseria gonorrhoeae (cephalosporinresistant, fluoroquinolone-resistant) (3). The third part of the WHO list ranked as medium priority consists of penicillin-non-susceptible Streptococcus pneumoniae, ampicillin-resistant Haemophilus influenzae, and fluoroquinolone-resistant Shigella spp. (3). In addition to the hospital (patients and healthcare workers) (5–8), bacteria of this list can also disseminate through the community (causing infections in humans or pets) (9–11), food chain (10, 12–14), and environment (15, 16).

Overuse of antibiotics prescribed to humans and animals plus the improper use in agriculture can select resistant bacteria that emerge after a spontaneous mutation or after acquiring resistance genes through horizontal transfer mediated by mobile genetic elements (17). This selection can occur in both the human and non-human gastrointestinal tracts as well as in contaminated environments. Once they become resistant to antibiotics, clonal bacterial cells are selected in the microbial population and disseminates its daughter cells. Alternatively, plasmids carrying resistance genes are transferred to other bacteria through conjugation or transformation (17). In Switzerland, a recent study on freshwater samples from rivers, inland canals, and streams showed environmental dissemination of high-risk carbapenemase-producing Enterobacteriaceae with plasmids composed of elements identical to those of resistance plasmids retrieved from clinical and veterinary isolates locally and worldwide (15). The authors concluded that these plasmids carrying carbapenemase genes replicate and evolve pollutants of river ecosystems and represent a threat to public health and environmental integrity (15). ESBL-producing Enterobacteriaceae were isolated from a Tunisian semi-industrial pilot plant with biological treatment (WWPP) and its receiving river with high rates of CTX-M-15 and high genetic diversity (16). Therefore, the water treatment and appropriate use of antibiotics may represent important infection control measures to avoid antimicrobial resistance spread in hospitals and communities (1, 18).

Bacteria may grow planktonically but most often form biofilms in nature or human body infections (19). These biofilms are common in the hospital environment, especially in polymeric clinical devices, such as catheters and cardiac pacemakers. The inhibition of biofilm growth is of great importance in infection control and treatment of healthcare-associated infections owing to their inherent tolerance and "resistance" to antimicrobial therapies (19). In addition, biofilms constitute a great challenge in clinical settings since there is no "gold standard" available to reveal the presence of microbial biofilms.

The prevention of bacterial resistance spread is a significant challenge among healthcare professionals, especially in hospital settings. Surveillance of clones and lineages spread in a hospital environment or among patients, and the knowledge of their susceptibility to antimicrobials are crucial data to initiate a proper empiric treatment of hospital-acquired infections and infection prevention and control.

In many cases, precision medicine is the key to therapy since different microbes are prone to different remedies. New antimicrobials might seem more effective and promising, but some traditional compounds, such as beta-lactamase or efflux pump inhibitors that could prevent enzymatic action on known antibiotics or efflux pump activity, respectively, are also beneficial.

This Research Topic harbors 48 published manuscripts, including original research, review, opinion, and brief research reports addressing these subjects. The 443 involved authors focused on tackling antimicrobial resistance in bacteria. According to Merlin, although the increasing occurrence of antibiotic resistance among bacteria due to the antimicrobial agent consumption has been recognized for decades, only recently, the seriousness of the situation has been considered by international, national, and local health agencies. Some recommendations were defined in order to educate and improve the practices of health professionals and consumers. Most recommendations proposed coordinated actions in public health and veterinary/agricultural domains, limiting the inappropriate exposure of bacteria to antibiotics, in order to delay the natural evolution toward resistance and its propagation in the downstream environment, in a One Health context.

The chapters of this Research Topic show that it is crucial to pay attention to antimicrobial resistance in microorganisms from wild animals, pets, livestock, food, and the environment, in addition to those isolated from humans, to understand how antimicrobial resistance spreads. Lin et al. used a genomic approach to study the horizontal transfer of colistin resistance between polluted rivers and wild birds. Metadata analysis of mcr-1- positive multidrug-resistant E. coli strains, which were isolated from the environment of egret habitat (polluted river) and egret feces, showed highly homologous mcr-1-bearing IncI2 plasmids among isolates from different regions along the East Asian-Australian Flyway, suggesting that migratory birds may mediate the intercontinental transportation of colistin resistance.

Mahmud et al. call the attention to the prevalence and characterization of MDR and ESBL-producing E. coli in drinking water samples collected from Rohingya camps, Bangladesh. The authors described that 66 out of 384 E. coli isolates in this study were ESBL-producers, of which 71% (47/66) were MDR. Sixtyfour percent (42/66) of the ESBL-producing E. coli carried one to seven plasmids ranging from 1 to 103 MDa. Only large plasmids with antibiotic resistance properties were found transferrable via conjugation. This study shows that drinking water samples could foster exposure and dissemination of MDR, ESBL-producing, and pathogenic E. coli lineages, posing a health risk to the people residing in the densely populated camps of Bangladesh.

Chen et al. investigated the antimicrobial resistance trends and characteristics of ESBL-producing E. coli isolates from pets and whether this correlates with antibiotic usage in the clinic. The authors observed high rates of resistance to β-lactams and fluoroquinolones and resistance to >3 antibiotic classes. ESBL-encoding genes blaCTX−M (n = 44, 34.65%) and plasmidmediated quinolone resistance genes qnrB (n = 119, 93.70%) were most common in 36 PFGEs types and 28 different sequence types (STs), including ST405 (7, 15.9%), ST131 (3, 6.8%), ST73, ST101, ST372, and ST827 (2, 4.5% each). Additionally, blaNDM−5 was detected in three isolates (n = 3, 2.36%, two ST101 and one ST405), and mcr-1 was also observed in three colistin-resistant E. coli (ST6316, ST405, and ST46).

García-Meniño et al. performed molecular characterization of 35 prevalent strains of colistin-resistant E. coli from swine enteric colibacillosis in Spain. The resistome analysis showed six different mcr variant genes. In seven of them, the authors observed an association with ESBL. The summative presence of mechanisms associated with high-level resistance to quinolone/fluoroquinolones and colistin could confer adaptive advantages to prevalent pig E. coli strains. The authors also reported the co-occurrence of double colistin-resistance mechanisms in a significant number of E. coli isolates.

Mukherjee et al. observed increasing frequencies of antibioticresistant non-typhoidal Salmonella (NTS) infections in Michigan, which is not part of the FoodNet surveillance system in the USA. A total of 198 isolates, from 2011 to 2014, belonged to 35 different serovars with the predominance of Enteritidis (36.9%), followed by Typhimurium (19.5%), and Newport (9.7%). A total of 30 (15.2%) NTS isolates were resistant to ≥1 antibiotic, and 15 (7.5%) were resistant to ≥3 antimicrobial classes. They demonstrated the importance of surveillance, resistance frequency monitoring, and identification of risk factors that can aid in the development of new prevention strategies.

Almeida et al. described the coexistence of oxazolidinone resistance genes cfr and optrA in a MDR E. faecalis isolate belonging to ST29, obtained from a healthy piglet in Brazil in the context of surveillance study. Whole-genome sequencing revealed that the cfr gene was in a transposon-like structure of 7,759 nucleotides flanked by IS1216E and capable of excising and circularizing, distinguishing it from known genetic contexts for cfr in Enterococcus spp., while optrA was in an Inc18 broad-hostrange plasmid of >58 kb. Both genes were able to conjugate. This study highlights the need for monitoring the use of antibiotics in the Brazilian swine production system to control the spread and proliferation of antibiotic resistance.

This Research Topic also presents studies reporting the mechanisms of resistance and lineages of bacteria of clinical importance. Carbapenem-resistant A. baumannii (CRAB) often causes fatal infections among seriously ill patients. Wong et al. used whole-genome sequencing, gene expression studies, and enzyme kinetics analyses to investigate the underlying carbapenem resistance mechanisms in 14 clinical A. baumannii. A large majority of isolates belonged to the International Clone II (IC-II), ST208. CRAB harbored blaOXA−23/blaOXA−72, or overexpression of the blaOXA−51 gene upon ISAba1 insertion, and had strong carbapenem-degrading activities. Over-production of carbapenem-hydrolyzing-class D-ß-lactamases (CHDLs) is the key mechanism of carbapenem resistance in the isolates studied.

Fosfomycin exhibits significant antimicrobial activity against a broad spectrum of pathogens, including S. aureus. Although widely described in gram-negative bacteria, fosfomycin's resistance mechanism is rarely reported among gram-positive bacteria (Xu et al.). The molecular mechanisms of 11 fosfomycinresistant S. aureus isolates from a Teaching hospital in China were determined. The authors showed that the mutations in the uhpT, glpT, and murA genes and the overexpression of the efflux pump gene tet38 under a subinhibitory concentration of fosfomycin might play a major role in conferring fosfomycin resistance in these isolates. In contrast, none of the resistant strains carried the fosA, fosB, fosC, fosD, and fosX genes, indicating that these genes might not be the primary factors mediating the resistance of S. aureus against fosfomycin. Furthermore, MLST analysis identified ST5708, a novel sequence type of S. aureus (Xu et al.).

The possible transference of resistance genes among different species and the consequences that these genes may cause in the cells were covered in this Research Topic, for instance, in the study by Xiang et al., which described the presence of blaNDM−1 in 54 out of 1,735 carbapenem-non-susceptible strains, including K. pneumoniae, A. baumannii, and E. coli. After analyzing the blaNDM−1 gene and its genetic environment, the authors hypothesized that, due to the presence of the ble and tnpA genes, blaNDM−1 originates from A. baumannii, which is retained in K. pneumoniae over a long period by transposition of mobile elements.

Mobile genetic elements harboring resistance genes, in certain cases, may reduce bacterial fitness. In this Research Topic, Li et al. investigated the physiological function of the mcr-1 gene, which encodes an LPS-modifying enzyme, in E. coli K-12. The removal of the mcr-1 gene not only reduced resistance to colistin, but also increased cell viability under high osmotic stress conditions and led to increased resistance to hydrophobic antibiotics. Increased expression of mcr-1 also resulted in a decreased growth rate and changed the cellular morphology of E. coli (Li et al.).

Sharma et al. used a proteomic approach to study the growth and fitness of a carbapenem-resistant NDM-4-producing K. pneumoniae clinical isolate under meropenem stress. The authors showed the downregulation of flagellar, fimbriae, and pili proteins by mass spectrometry and system biology analysis of networking pathways. Sharma et al. suggested that these proteins might be used as targets for the development of novel therapeutics against antimicrobial-resistant K. pneumoniae infections.

This Research Topic also brings the search and analysis of the CRISPR-Cas system, a microbial adaptive immune system involved in defense against different types of mobile genetic elements, in whole plasmid and chromosomal sequences of multidrug-resistant K. pneumoniae from GenBank (Kamruzzaman and Iredell). The authors found a putative CRISPR-Cas system in 44 plasmids from Klebsiella species and identified an identical system in three plasmids from other Enterobacteriaceae, with CRISPR spacers targeting different plasmids and chromosome sequences. Additionally, the authors showed that plasmid type IV CRISPR may depend on the chromosomal type I-E CRISPRs for their competence (Kamruzzaman and Iredell). Different aspects are involved in the acquisition and transference of resistance determinants, and the presence of CRISPR-Cas in plasmids just showed us we still have a lot to understand.

In this Research Topic, several manuscripts described the diversity of the most important pathogenic bacterial species and their susceptibility profiles. For instance, in India, the antibiotic susceptibility and typing of 109 S. aureus strains isolated from a variety of infections showed that all S. aureus isolates were multidrug-resistant, virulent, and diverse irrespective of sources and place of isolation being assigned to 46 spa-types, 33 STs and eight clonal complexes (Aggarwal et al.).

Ai et al. studied the prevalence, characterization, and drug resistance of S. aureus fecal carriage among pediatric patient feces in Southern China and found that the fecal carriage rates were 20.0% for S. aureus and 4.5% for methicillin-resistant S. aureus (MRSA). Among the 76 STs, the authors found 25 new STs, and the most prevalent were ST188, ST6, and ST15 for methicillin-sensitive S. aureus (MSSA) and ST59 and ST45 for MRSA. The authors described high rates of penicillin, erythromycin, and clindamycin resistance among the isolates. However, CC59 (ST338 and ST59) and CC45 exhibited different antibiotic resistance patterns. Ai et al. indicated an urgency for strengthening the surveillance programs in China that could contribute to S. aureus infection prevention and treatment.

Bispo et al. analyzed the population structure of ocular MRSA isolates using a combination of MLST analysis, SCCmec typing, and detection of the panton-valentine leukocidin (PVL) coding gene. The 68 isolates (2014–2016) belonged to 14 different sequence types (STs) that grouped within two predominant clonal complexes: CC8 (47.0%) and CC5 (41.2%). Although the isolates were associated with lineages with community and hospital origins, the authors suggested that the niche specialization could act as a driver for the community structure observed.

In Iran, Goudarzi et al. conducted a study from August 2013 to July 2018 to investigate the prevalence and molecular epidemiology of inducible macrolide-lincosamide streptogramin B (MLSB) resistance in S. aureus. The prevalence of inducible MLSB (iMLSB) S. aureus increased from 7.5 to 21.7% during the study period. The authors observed a replacement of the CC22, predominant in 2013–2014, by CC8 in 2017–2018. Goudarzi et al. described for the first time the temporal shifts of iMLSB S. aureus isolates in Iran that identify predominant clones and treatment options for iMLSB S. aureus–related infections.

Vasilakopoulou et al. described the prevalence and risk factors of antimicrobial resistance by studying the gastrointestinal carriage of vancomycin-resistant enterococci (VRE) and carbapenem-resistant gram-negative bacteria (CRGN) in the fecal flora of the inpatients of a tertiary university hospital in Greece. The authors found high prevalence rates for VRE and CRGN carriage. Prolonged hospitalization and age were independent risk factors for VRE carriage, while CRGN carriage was associated with an increased risk of acquiring a resistant pathogen, prolonged hospital stay, and increased mortality.

In South Korea, Yoon et al. showed that most (84.5%) of the A. baumannii blood isolates belonged to the international clonal lineage II (ICLII), and 89.5% of the isolates were either multidrug- or extensively drug-resistant. The patient's old age, the sequential organ failure assessment score, and causative A. baumannii ST191 belonging to ICLII were risk factors for 30-day mortality (Yoon et al.).

Ferrari et al. identified the circulation of three distinct genome clusters of carbapenem-resistant KPC-producing K. pneumoniae in an intensive care unit in a hospital in Northern Italy. The authors described two clusters, one linked to ST512 and one to ST258; the ST258 isolates were also colistin-resistant and responsible for a high number of clinical infections (Ferrari et al.).

Ferreira et al. characterized KPC-producing Serratia marcescens strains isolated during an outbreak involving patients hospitalized in the intensive care unit (ICU) and neonatal intensive-care-unit (NIUC) of a Brazilian tertiary hospital. All isolates carried KPC-carbapenemase (blaKPC) and extendedspectrum beta-lactamase blaTEM genes. In addition to the MDR profiles exhibited by almost 25% of isolates, 98.2% of the isolates had the genes codifying to the pore-forming toxin (ShlA), phospholipase with hemolytic and cytolytic activities (PhlA), flagellar transcriptional regulator (FlhD), and positive regulator of prodigiosin and serratamolide production (PigP).

Due to increasing resistance to multiple antibiotics classes, the search for molecules displaying potent activity against multidrug-resistant (MDR) bacteria is mandatory. Additionally, the discovery of new therapeutic approaches and/or new pathways to be inhibited is an open door for a directed search for bioactive compounds. As an example, Barra et al. discussed the possibilities, using an in silico-based analysis, of enzyme inhibition of two new possible druggable biosynthesis pathways present in most bacteria, fungi, and plants but incomplete in humans: the thiamine (vitamin B1) and the pyridoxal 5′ phosphate and its vitamers (vitamin B6).

Loubet et al. discussed alternative therapies and preventative strategies for urinary tract infections caused by multidrugresistant uropathogens, focusing on the following phases of the pathogenesis: colonization, adherence of pathogens to uroepithelial cell receptors, and invasion. In this review, the authors discussed vaccines, small compounds, nutraceuticals, immunomodulating agents, probiotics, and bacteriophages, highlighting the challenges each of these approaches face Loubet et al.

Zhang et al. investigated the effects of pharmacological doses of copper, an essential microelement for animals, on the microbial communities in the hindgut, and the antimicrobial resistance profiles of E. coli in weaned piglets. The pharmacological dose of copper affected the composition of the microbial community (affected the microbial metabolic functions of energy metabolism, protein metabolism, and amino acid biosynthesis), increased antimicrobial resistance (to chloramphenicol and ciprofloxacin) of intestinal E. coli, and was most likely harmful to the health of piglets at the early stage after weaning (Zhang et al.).

The new anti-biofilm or antimicrobial agents' analysis, as well as their mode of action, were assessed in some studies. Murugan et al. described that arborine and skimmianine compounds from ethyl acetate extract of Gycosmis pentaphylla harbor a high antibacterial activity against MDR S. aureus. Wang et al. showed that the flavone glycoside baicalin inhibits biofilm formation and the quorum-sensing system in Staphylococcus saprophyticus, thus suggesting that it might be a potential therapeutic agent for S. saprophyticus biofilm-associated infections.

She et al. described the potent antimicrobial activity of auranofin against MRSA/MSSA and E. faecalis, both planktonic cells and biofilms, with minimum inhibitory concentrations ranging from 0.125 to 0.5 mg/L. Auranofin, in combination with linezolid or fosfomycin, showed synergistic antimicrobial activities against MSSA and MRSA both in vitro and in a mouse infection model. There was also a synergistic effect with chloramphenicol against E. faecalis.  Therefore, they showed that the combination of auranofin with linezolid, fosfomycin, and chloramphenicol provides a synergistic microbicidal effect in vitro and in vivo, which rapidly enhances antimicrobial activity.

Lytic bacteriophage therapy is a potential antibacterial therapy. Montso et al. characterized lytic E. coli O177-specific bacteriophages isolated from cattle feces. A total of 31 lytic E. coli O177-specific bacteriophages were isolated and 71% of these phage isolates produced large plaques on 0.3% soft agar. Selected phage isolates had a similar morphotype (an icosahedral head and a contractile tail) and were classified under the order Caudovirales, Myoviridae family. Montso et al. demonstrated that lytic E. coli O177-specific bacteriophages isolated from cattle feces are highly stable and can infect different E. coli strains.

Among the papers, the authors analyzed new antimicrobials and different compounds against MDR bacteria. Cefoselis, a fourth-generation cephalosporin, was evaluated by Cheng et al. against bacterial pathogens in China. The authors observed that cefoselis showed good activity against non-ESBL-producing E. coli, K. pneumoniae, and P. mirabilis, MSSA, and was also potent against Enterobacteriaceae, P. aeruginosa, and Streptococcus.

Omadacycline, a new tetracycline-class broad-spectrum aminomethylcycline, exhibited excellent in vitro activity against both MRSA and MSSA clinical isolates (Bai et al.). Omadacycline susceptibility in S. aureus may be affected by efflux pump proteins (i.e., a branched-chain amino acid transport system II carrier protein and a Na/Pi cotransporter family protein) (Bai et al.).

Zheng et al. compared the effects of radezolid and linezolid on planktonic and biofilm cells of E. faecalis. After analyzing 302 E. faecalis, they observed that the MIC50/MIC90 values of radezolid (0.25/0.50 mg/L) were eight-fold lower than those of linezolid (2/4 mg/L). They observed that the radezolid MICs against the high-level linezolid-resistant isolates (linezolid MICs ≥64 mg/L) increased to ≥4 mg/L with mutations in the four copies of the V domain of the 23S rRNA gene. In addition, they showed the involvement of OG1RF\_12220 (mdlB2, multidrug ABC superfamily ATP-binding cassette transporter) in the alteration of radezolid and linezolid susceptibility. Zheng et al. described that Radezolid inhibited E. faecalis biofilm formation to a greater extent than linezolid. Therefore, radezolid is more effective than linezolid against E. faecalis.

Hao et al. showed a potent in vitro activity of apramycin, an antibiotic with good activity against a range of multidrugresistant pathogens, against clinical carbapenem-resistant, and hypervirulent K. pneumoniae (CR-hvKp) along with carbapenem-resistant non-hvKp (CR-non-hvKp) isolates, including those resistant to amikacin or gentamicin.

Dong et al. demonstrated that ebselen, an organo-selenium with well-characterized toxicology and pharmacology, has high bactericidal activity against multidrug-resistant (MDR) S. aureus based on taking TrxR as a major target and disruption of the

## REFERENCES

redox microenvironment. The authors showed that ebselen is an effective topical antibacterial agent in animal model of MDR S. aureus LT-1 skin infection, reducing the bacterial load and the expression of pro-inflammatory cytokines.

Murugan et al. focused on the isolation and structural characterization of bioactive compounds from the ethyl acetate extract of Gycosmis pentaphylla. The isolated compounds identified as arborine and skimmianine have high bactericidal effects against MDR S. aureus clinical isolates. These compounds induced intracellular molecular imbalance and cell membrane disturbances that caused cell death of all MDR S. aureus strains and the reference standard S. aureus MTCC-96 strain.

A bottleneck in clinical microbiology, the incubation time necessary to the susceptibility test is ready for the antibiotic decision by the clinician is something to overcome. Genomics and technology can work together to improve the turnaround time in the clinical laboratory. Liu et al. used 2 genotype-based machine learning methods named Support Vector Machine (SVM) and Set Covering Machine (SCM) to predict the resistance to tetracycline, ampicillin, sulfisoxazole, trimethoprim, and enrofloxacin. The correlation results between the phenotype and the model predictions of the five antimicrobial agents indicated that both SVM and SCM models could significantly discern the resistant isolates of the sensitive isolates and could be potential tools in antimicrobial resistance surveillance and clinical diagnosis in veterinary medicine.

Resistance mechanisms, surveillance of antimicrobialresistant bacteria, and new drug discovery studies offer different views of the antimicrobial resistance, which became a public health problem. There are more articles dedicated to the community focused on antimicrobial resistance in this Research Topic, and we hope you enjoy it.

## AUTHOR CONTRIBUTIONS

All authors wrote about the manuscripts they edited in the Research Topic. IC also compiled and edited the complete text.

## ACKNOWLEDGMENTS

We are grateful to all reviewers who put their efforts during the COVID-19 pandemic to analyze all the manuscripts.

tertiary hospital. Revista da Sociedade Brasileira de Medicina Tropical. (2016) 49:51–6. doi: 10.1590/0037-8682-0284-2015

Enterobacteriaceae in rivers in Switzerland. Environ Pollut. (2020) 265(Pt B):115081. doi: 10.1016/j.envpol.2020.115081

**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 © 2020 Minarini, Andrade, De Gregorio, Grosso, Naas, Zarrilli and Camargo. 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.

# Counter Clinical Prognoses of Patients With Bloodstream Infections Between Causative *Acinetobacter baumannii* Clones ST191 and ST451 Belonging to the International Clonal Lineage II

#### *Edited by:*

*Marc Jean Struelens, European Centre for Disease Prevention and Control (ECDC), Sweden*

#### *Reviewed by:*

*Kwan Soo Ko, Sungkyunkwan University School of Medicine, South Korea Raffaele Zarrilli, University of Naples Federico II, Italy*

> *\*Correspondence: Seok Hoon Jeong<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Infectious Diseases - Surveillance, Prevention and Treatment, a section of the journal Frontiers in Public Health*

> *Received: 15 April 2019 Accepted: 02 August 2019 Published: 16 August 2019*

#### *Citation:*

*Yoon E-J, Kim D, Lee H, Lee HS, Shin JH, Uh Y, Shin KS, Kim YA, Park YS, Shin JH and Jeong SH (2019) Counter Clinical Prognoses of Patients With Bloodstream Infections Between Causative Acinetobacter baumannii Clones ST191 and ST451 Belonging to the International Clonal Lineage II. Front. Public Health 7:233. doi: 10.3389/fpubh.2019.00233* Eun-Jeong Yoon1 , Dokyun Kim1 , Hyukmin Lee1 , Hye Sun Lee2 , Jong Hee Shin3 , Young Uh4 , Kyeong Seob Shin5 , Young Ah Kim6 , Yoon Soo Park 7 , Jeong Hwan Shin8 and Seok Hoon Jeong1 \*

*1 Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, South Korea, 2 Biostatistics Collaboration Unit, Yonsei University College of Medicine, Seoul, South Korea, 3 Department of Laboratory Medicine, Chonnam National University Medical School, Gwangju, South Korea, 4 Department of Laboratory Medicine, Yonsei University Wonju College of Medicine, Wonju-si, South Korea, 5 Department of Laboratory Medicine, Chungbuk National University College of Medicine, Cheongju-si, South Korea, 6 Department of Laboratory Medicine, National Health Insurance Service Ilsan Hospital, Goyang-si, South Korea, 7 Department of Internal Medicine, National Health Insurance Service Ilsan Hospital, Goyang-si, South Korea, 8 Department of Laboratory Medicine and Paik Institute for Clinical Research, Inje University College of Medicine, Busan, South Korea*

This study was conducted to evaluate the possible clinical and bacteriologic features associated with 30-day mortality from *Acinetobacter baumannii (A. baumannii)* bloodstream infections (BSIs). We conducted a prospective, multicenter, observational study of 181 entire episodes of *A. baumannii* BSI from six general hospitals between May 2016 and April 2017 in South Korea. Cox proportional-hazards regression model was used to estimate risks of the primary endpoint, i.e., all-cause mortality within 30 days from the initial blood culture. Most (84.5%) of the *A. baumannii* blood isolates belonged to the international clonal lineage II (ICLII) and 89.5% of the isolates were either multidrug- or extensively-drug resistant. We identified three risk factors including the old age of patient {hazard ratio, 1.033; [95% Confidential Interval (CI), 1.010–1.056]}, the sequential organ failure assessment score [1.133 (1.041–1.233)], and causative *A. baumannii* sequence type (ST) 191 belonging to ICLII [1.918 (1.073–3.430)], and three protective factors including causative *A. baumannii* ST451 belonging to ICLII [0.228 (0.078–0.672)], platelet count [0.996 (0.993–0.999)], and definitive therapy within 72 h [0.255 (0.125–0.519)]. Differing 30-day mortality rate in the dominant ICLII was observed by ST, which was much high in ST191 and low in ST451 and it was likely associated with the molecular traits, rather than the drug resistance.

Keywords: *Acinetobacter baumannii*, bloodstream infection, international clonal lineage II, ST191, ST451

## INTRODUCTION

Acinetobacter baumannii is dominant in clinical settings primarily targeting immunocompromised patients in intensive care units (ICUs) and has the potential to cause healthcareassociated infections (1). Infections caused by A. baumannii have repeatedly been reported to have grave clinical outcomes (2, 3). A. baumannii has an epidemic potential of emerging diseases because of its ability to persist in hospital environments by resisting desiccation and disinfectants (1). A propensity toward rapid acquisition of foreign DNA, including antimicrobial resistance determinants and virulence determinants, also contributes to its effective dissemination (4).

Previous studies have demonstrated that A. baumannii infections are accompanied by serious morbidity and mortality associated with a high sequential organ failure assessment (SOFA) score (5), comorbidities (6), immunosuppression (3), and antimicrobial treatment failure, mainly due to the multidrug resistance (MDR) of the causative A. baumannii (5, 7, 8). The rates of resistance to the major anti-Acinetobacter agents are increasing (9). In particular in South Korea, predominance of the multidrug-resistant international clonal lineage II (ICLII) in hospital settings is of great concern (10–12).

This study was performed to evaluate the possible clinical and bacteriologic features associated with 30-day mortality from A. baumannii bloodstream infection (BSI) for the entire 1-year incidence of A. baumannii BSI cases of multiple general hospitals in South Korea.

## METHODS

## Study Design

A prospective, multicenter, observational study was designed to monitor the entire A. baumannii BSI episodes between May 2016 and April 2017 occurring in six general hospitals with 715–1,050 beds participating in a national surveillance study of antimicrobial resistance in South Korea (13).  The A. baumannii BSI cases were detected through reviewing daily blood culture results. One authorized investigator at each hospital reviewed patients' medical records, all of the raw data were recorded in a pre-formatted spreadsheet, and the datasheet was submitted via email to an analysis center without monitoring. Prognoses of patients were followed for at least 30 days and the cases of transfer and hospital discharge were recorded as they were. The Charlson comorbidity index (14) and the SOFA score (15) were calculated in the analysis center using the collected raw data for the day of initial blood culture. For the sequential isolation of A. baumannii from one patient, the first isolate was used for analyses and the ensuing isolates were discarded. Among a total of 67,803 patients subjected to blood culture, A. baumannii isolates were recovered from blood specimens of 188 patients, and following the elimination of 7 episodes of death on the day of the initial blood culture, 181 cases were included in the analysis. The microbiological assessment was carried out in the analysis center.

## Definitions

Laboratory-confirmed A. baumannii BSI was defined as an A. baumannii-positive blood culture from one or more blood specimens (16). Hospital-originated (HO) infection was specified by ≥2 calendar days of hospitalization at the day of initial blood culture, including the time spent at a previous health care facility before transfer. The 30-day mortality was defined as all-cause mortality within 30 days from the initial blood culture. Non-susceptibility (NS) to a drug class was defined as NS to at least one drug in a class and resistance phenotypes were categorized following Magiorakos et al. (17): MDR (NS to three or more drug classes), extensively drug resistance (XDR, NS to at least one agent in all but one or two drug classes), and pan-drug resistance (NS to at least one agent in all drug classes). An empirical antimicrobial therapy was defined as an initial antimicrobial treatment before the susceptibility testing results for the causative pathogen. A definitive antimicrobial therapy was defined as prescribing a revised antimicrobial regimen based on the antimicrobial susceptibility testing results and, if the revised regimen was administered after 72 h, it was deemed to be a delayed definitive therapy. Adequate antimicrobial therapy must include at least one antimicrobial agent active in vitro and both the dosage and route of administration should meet current medical standards.

## Microbiological Assessment

Bacterial species were initially identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry using a Bruker BiotyperTM system (Billerica, MA, USA) and the detailed species of Acinetobacter spp. were confirmed by PCR and sequencing the rpoB gene. For A. baumannii, multilocus sequence typing was followed through the Oxford scheme (18). Antimicrobial susceptibility was tested following the Clinical and Laboratory Standards Institute (CLSI) guidelines (19). Antimicrobial susceptibilities to 13 anti-Acinetobacter agents belonging to seven drug classes were tested by the disk diffusion method. For carbapenem-resistant isolates, the blaOXA−23 gene and the ISAba1-blaOXA−51−like were identified by PCR and direct sequencing (20). Colistin MICs were determined by the broth microdilution method (21). Twenty-six virulence genes associated with eight virulence characteristics (22) were investigated by real-time PCR using gene-specific primers (23) and a KAPA SYBR Fast quantitative PCR master mix (Kapa biosystems, Inc., Wilmington, MA, USA). The resistance islands AbaR and AbGRI1 were specified by PCR using a set of primers, comMtr3\_145-126F (5′ - CAAGCGGTTGGCGTAAGACT-3′ ) and reverse primers tniB\_546-525R (5′ -CAAGTACATGCTCTGCAAGATG-3 ′ ) only for the full tniB gene and tniB\_37-16R (5′ - CAGACTCAATTCATTGCTGAGG-3′ ) for both the full and the truncated tniB genes.

## Statistical Analysis

Statistical analyses were performed in SPSS statistics (version 23, IBM Corp., Armonk, NY, USA). Data are expressed as the mean ± standard deviation if no special description is indicated. Categorical variables were compared by Pearson's X 2 test, and continuous variables were compared by an independent two sample t-test. Associations between 30-day mortality and risk factors were evaluated using univariable and multivariable Cox proportional hazard regression models. The proportional hazards assumption was evaluated by including an interaction term between variables, and variables were natural-log transformed by follow-up time and by log-minus-log survival plots. For multivariable analyses, variables with a P value <0.1 in univariable analyses were considered to include any possible variables. Multicollinearity between variables was diagnosed by calculating the variance inflation factors, and a multivariable analysis was conducted by the backward stepwise method. All tests of significance were two-tailed; P values <0.05 were considered to be significant.

## RESULTS

## Characteristics of Enrolled Patients

A total of 181 laboratory-confirmed A. baumannii BSI cases were enrolled from the six participating hospitals and the number of cases was ranged from 22 to 52 by hospital (**Figure 1**). Among patients with A. baumannii BSIs, the sex ratio of male vs. female was 115:66, and their mean age was 67.7 ± 14.3 years (**Table 1**). The mean Charlson comorbidity index score was 4.1 ± 2.2. Solid tumors (26.5%, 48/181), diabetes mellitus (23.2%, n = 42), cerebrovascular diseases (18.2%, n = 33), kidney diseases (13.8%, n = 25), and chronic pulmonary diseases (6.6%, n = 12) were the five most observed underlying diseases and dementia (n = 9) and chronic liver diseases (n = 7) were followed. Of the total, 88.4% (n = 160) of cases were HO infections, 95.0% (n = 172) were inpatients, and 68.0% of those (117/172) stayed in ICUs. The SOFA score had a mean value of 5.9 ± 3.5. Secondary BSIs were identified in 44.2% (80/181) of cases, and the majority (86.3%, 69/80) were originated from a pulmonary tract infection.

## The BSI-Causative *A. baumannii* Isolates Sequence Types

The majority of the A. baumannii blood isolates belonged to ICLII (84.5%, 153/181) and the ICLII mostly comprised sequence type (ST) 191 (allele numbers of gltA-gyrB-gdhB-recA-cpn60-gpirpoD, 1-3-3-2-2-94-3; 47.7%, 73/153), ST784 (1-3-3-2-2-107-3; 20.3%, n = 31), and ST451 (1-3-3-2-2-142-3; 19.0%, n = 29). The 30-day mortality rate of patients with A. baumannii ICLII BSIs was 1.83-fold higher than that with A. baumannii BSIs by non-ICLII strains (45.8 vs. 25.0%); and among the ICLII BSIs, the 30-day mortality rate with ST191 BSIs was the highest and, the lowest for ST451 BSIs (60.3 vs. 17.2%).

## Antimicrobial Resistance

Majority of the A. baumannii blood isolates (89.5%, 162/181) were either MDR or XDR, and the proportion was markedly greater in A. baumannii ICLII (98.7%, 151/153; 18 MDR and 133 XDR) than in non-ICLII (39.3%, 11/28; 5 MDR, and 6 XDR). All of the tested isolates were susceptible to colistin, and no pandrug resistant isolate was observed. All but one carbapenem-NS A. baumannii harbored the blaOXA−23 gene (99.4%, 159/160) and the remaining one carried an insertion sequence ISAba1 upstream from the blaOXA−51−like gene. Two thirds and onequarter of A. baumannii possessed AbGRI1 (63.0%, 114/181) and AbaR (26.5%, 48/181), respectively.

## Virulence Factors

Factors for adhesion and membrane integrity, biofilm formation, carbohydrate metabolism, quorum sensing, and siderophore

FIGURE 1 | Participating sentinel hospitals and collected *Acinetobacter baumannii* blood isolates. Locations of the hospitals (in gray) and covering parts (in gray patterns) are indicated in a map of the South Korean peninsula, respectively, and the associated table represent information regarding blood isolates from each hospital including demographic information from 2016. HO, hospital-origin; ICLII, international clonal lineage II; ST, sequence type.

TABLE 1 | Thirty day mortality-associated factors for patients with an *Acinetobacter baumannii* BSI.

*(Continued)*

#### TABLE 1 | Continued

\**The percentage was calculated from the total number of AB-BSI.*

*† All-cause mortality within 30 days from the initial blood culture.*

*‡P value is either from Pearson's X2 test for categorical data or from the t-test for continuous data. Bonferroni corrected significance level from 32 hypotheses was considered to be P* < *0.0016, and the significant P values are indicated in bold.*

*§Cox proportional hazards regression model was applied to calculate the hazard ratio (HR) and the 95% confidence interval (CI).*

*¶Underlying disease and primary site of infection could be multiple by patient.*

*#Ulcer (n* = *2), connective tissue disease (n* = *2), congestive cardiac insufficiency (n* = *2), and leukemia (n* = *2) are included.*

*††Multilocus sequence typing profiles are indicated with the combination of alleles (gltA-gyrB-gdhB-recA-cpn60-gpi-rpoD).*

*SD, standard deviation; ICLII, the international clonal lineage II.*

were detected in A. baumannii blood isolates (**Table 1**). The acinetobactin-associated basD (98.9%, 179/181), the glycokinase gntK (98.9%, n = 179), the adhesion-associated smpA (97.8%, n = 177), and the biofilm formation-associated pga genes (95.6%, n = 173) were harbored by most of the A. baumannii blood isolates, while the capsule protein mviM (3.9%, n = 7), the polysaccharide exporter epsA (5.0%, n = 9), lipopolysaccharide vipA (8.8%, n = 16), and the type IV pili pilA (9.9%, n = 18) genes were occasionally identified. Among the virulence factors tested, the pga gene was significantly associated with 30 day death and the adhesion-associated porB had a tendency of 30-day mortality-associated.

A different frequency of each gene by ST was occasionally identified (**Table 2**). The psaB gene, formerly the pab2 gene (24), was identified in all but one (96.6%, 28/29) ST451 isolates.

### Antimicrobial Treatment

The majority (95.0%, 172/181) of patients with A. baumannii BSIs received an empirical antimicrobial therapy with anti-Acinetobacter drugs and 28.5% (49/172) of those were adequate (**Table 3**). The other nine patients received either not-for-Acinetobacter drugs (n = 7) or none due to refusal of treatment (n = 2). Among the 132 patients who received an inadequate empirical therapy, 37.1% (n = 49) of patients were treated with a definitive antimicrobial therapy within 72 h and 20.4% (10/49) of those patients met death within 30 days, much less than that of patients who received a delayed definitive therapy (57.9%, 48/83).

## Risk Factors for 30-Day Mortality

Risk and protective factors associated with the 30-day mortality in patients with A. baumannii BSIs were searched for among the variables of P <0.1 through a Cox-regression univariable analysis. Most of the variables were omitted by stepwise selection procedure, and each three of risk factors and protective factors associated with the 30-day mortality in patients with A. baumannii BSIs were identified through a Cox-regression multivariable analysis (**Table 4**): the age of patients of hazard ratio (95% CI), 1.033 (1.010–1.056); the SOFA score of 1.133 (1.041–1.233); and the BSI-causative A. baumannii ST191 of 1.918 (1.073–3.430). Three protective factors were also identified: the platelet count of 0.996 (0.993–0.999), the BSI-causative A. baumannii ST451 of 0.228 (0.078–0.672), and the definitive therapy within 72 h of 0.255 (0.125–0.519).

## DISCUSSION

During the surveillance study period, the overall incidence of A. baumannii was the third-highest (1.1/10,000 patient-days) BSIcausative Gram-negative bacteria followed by Escherichia coli and Klebsiella pneumoniae. Besides, the incidence was strikingly higher in ICUs (6.6/10,000 patient-days) than in general wards (0.4/10,000 patient-days) (25). The 30-day mortality rate of patients with A. baumannii BSIs is pretty varied by study depending on the subjected patients, and we observed 42.5% of 30-day mortality for entire A. baumannii BSI cases.

Among the variables studied, the old age and the high SOFA score were identified as the patient-associated risk factors. Those were reasonable hazardous factors since both factors pronounced the severity of illness directly or indirectly. A patient-associated protective factor, platelet counts in peripheral blood reflecting the patients' condition also made sense. As well, a treatmentassociated protective factor, a proper definitive therapy within 72 h was comprehensible.

Curiously among the causative-pathogen-associated variables, the A. baumannii ST191 was a risk factor, while the ST451, another ICLII member, was a protective factor. A. baumannii

#### TABLE 2 | Characteristics of the *A. baumannii* ICLII blood isolates.

\**P value is either from Pearson's X2 test for categorical data or from the t-test for continuous data. The Bonferroni corrected significance level from 33 hypotheses was considered to be P* < *0.0015, and the significant P values are indicated with asterisks.*

ICLII, is a well-known multidrug resistant clone (26, 27). The treatment options for patients infected by the clone are often limited to the very last resorts, such as carbapenems, tigecycline, minocycline, and polymyxins. For the infections cause by multidrug-resistant A. baumannii, the use of combination antimicrobial therapy is recommended (28) and two thirds (66.3%, 120/181) of the patients were treated empirically by antimicrobial combinations. Carbapenem resistance is already Yoon et al. *Acinetobacter baumannii* Bloodstream Infection

common in ICLII, and the remaining choices are always risky because tigecycline (29) and minocycline (30) have a bacteriostatic activity, which requires a combinational treatment; colistin has a severe dose-dependent neuro- and nephrotoxicity (31); and the colistin resistance is easily developed by prior use of the drug (32). The importance of an adequate antimicrobial therapy (33), including the proper use of colistin (34), has been addressed to diminish the 30-day mortality rate caused by A.

\**Patients received drugs not-for-Acinetobacter (n* = *7) or none due to refusal of treatment (n* = *2).*

baumannii infections. The present results confirmed the notion through a significant protective effect of the definitive therapy including colistin. Among ICLII members, ST191 was the most and both ST784 and ST451 are recently emerging ICLII clones (27, 32). In this study, ST784 and ST451 were the second and third most ICLII clones and the clones were evenly recovered from the six hospitals (**Figure 2**).

In this study, all but two (98.7%) ICLII blood isolates, one ST191 and the other ST357, exhibited MDR/XDR. Since drug resistance is one of the noted hazardous factors obstructing an adequate antimicrobial therapy (7, 8), similar mortality rates were presumed for BSIs caused by any STs belonging to the ICLII. However, a marked difference in 30-day mortality was observed in the three dominant STs: 60.3% in ST191 BSIs, 45.2% in ST784 BSIs, and 17.2% in ST451 BSIs. Curiously, the least proportion of patients with ST451 BSIs (10.3%) were treated by an adequate empirical therapy (**Table 2**), indicating that the drug resistance and the relevant adequacy of treatment were not related to the 30-day mortality rate at least to BSIs caused by ST191 and ST451. ST451 was more associated with a primary pulmonary tract infection (58.6%) than ST191 (32.9%), however the primary site of infection

TABLE 4 | Risk factors associated with the 30-day mortality in patients with *A. baumannii* BSI.

\**VIF, variance inflation factor. The collinearity was diagnosed for the variables including the multivariate analysis.*

*† A Cox-regression multivariate analysis was conducted by the backward method using the Wald model.*

FIGURE 2 | Mortality of BSI patients by causative pathogen. (A) Mortality of patients with BSIs by *A. baumannii*, (B) comparison of mortality of BSI patients by ICLII and by non-ICLII *A. baumannii*, (C) comparison of mortality of BSI patients by ST191, ST451, and other STs belonging to CC91. The Kaplan-Meier plot illustrated survival in patients with AB-BSI infected with the indicated bacterial clone. Differences between groups were compared by the Log-Rank test of the Mantel-Cox model, and the *P* values are presented. ICLII, international clonal lineage II; ST, sequence type.

was insignificantly associated with a 30-day death (**Table 1**). Similarly, ST451 harbored the Tn6019-associated AbaR (82.8%) more frequently than the Tn6022-associated AbGRI1 (17.2%) differently from ST191 (2.7 vs. 94.5%), however no difference was expected since both may harbor the blaOXA−23 gene (12). The lipooligosaccharide biosynthesis-associated psaB gene was harbored strikingly more by ST451 than by ST191 (96.6 vs. 2.7%). The psaB gene is a part of the oligosaccharide gene clusters generating an ordinary side-branch sugar Gal, differing from the A. baumannii ST191 often carries the gene cluster producing FucNAc sugar composing the oligosaccharide (24). Since the lipooligosaccharides of pathogenic bacteria have long been recognized as a major virulence factor (35), the difference in oligosaccharides between the two clones is possibly associated with the 30-day mortality rate and the detailed impact should be further studied.

This study should be considered with the following caveats in mind. First, the enrolled cases had a lack of external validation due to a distinct epidemiology of A. baumannii clinical isolates in South Korea in terms of high carbapenem resistance, mostly conferred by OXA-23. Second, analyses of bacterial lipooligosaccharides differed by the clones are remained for further study. However, to the best of our knowledge, this is the first notification of the counter clinical prognoses of the patients with a BSI by dominant ICLII clones, drawing an attention to a further follow-up for the clonal exchange and expansion of ICLII clones in clinical settings.

This delicate prognosis assessment combined with an evaluation of the molecular epidemiology for the causative A. baumannii blood isolates confirmed (i) the predominance of ICLII in clinical settings, (ii) the hazardousness of antimicrobial resistance and the importance of an adequate antimicrobial therapy, and finally, (iii) we highlighted

## REFERENCES

the differing 30-day mortality rates in patients with BSIs between causative multidrug-resistant A. baumannii clones ST191 and ST451 belonging ICLII probably due to the differing lipooligosaccharides.

## 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.

## ETHICS STATEMENT

The research, which has no involvement of human subject but the clinical isolates, does meet the exempt category without approval from Ethics Committee on Human Research of the Health Ministry in South Korea and the study design has not been reviewed by the committee.

## AUTHOR CONTRIBUTIONS

SJ conceived the study. E-JY and SJ designed the study. E-JY, HSL, and SJ analyzed the data. E-JY and SJ wrote the manuscript. DK, HL, JongS, YU, KS, YK, YP, JeongS, and SJ collected the clinical data and bacterial isolates.

## FUNDING

This study was supported by the Research Programme funded by the Korea Centers for Disease Control and Prevention (2017E4400101#). We would like to thank to Yena Oh for technical support for microbiological experiments.

on clinical outcomes. Antimicrob Agents Chemother. (2013) 57:6270–5. doi: 10.1128/AAC.01520-13

May 2016 to April 2017: first one-year report from Kor-GLASS. Euro Surveill. (2018) 23:1800047. doi: 10.2807/1560-7917.ES.2<IP_ADDRESS> 00047

**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 Yoon, Kim, Lee, Lee, Shin, Uh, Shin, Kim, Park, Shin and Jeong. 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.

# Synergistic Microbicidal Effect of Auranofin and Antibiotics Against Planktonic and Biofilm-Encased S. aureus and E. faecalis

Pengfei She, Linying Zhou, Shijia Li, Yiqing Liu, Lanlan Xu, Lihua Chen, Zhen Luo and Yong Wu\*

Department of Clinical Laboratory, The Third Xiangya Hospital of Central South University, Changsha, China

#### Edited by:

Ilana L. B. C. Camargo, University of São Paulo, Brazil

#### Reviewed by:

Mariusz Stanislaw Grinholc, Intercollegiate Faculty of Biotechnology of University of Gdansk and Medical University ´ of Gdansk, Poland ´ Airat R. Kayumov, Kazan Federal University, Russia

> \*Correspondence: Yong Wu<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 12 July 2019 Accepted: 11 October 2019 Published: 24 October 2019

#### Citation:

She P, Zhou L, Li S, Liu Y, Xu L, Chen L, Luo Z and Wu Y (2019) Synergistic Microbicidal Effect of Auranofin and Antibiotics Against Planktonic and Biofilm-Encased S. aureus and E. faecalis. Front. Microbiol. 10:2453. doi: 10.3389/fmicb.2019.02453 Methicillin-resistant/susceptible Staphylococcus aureus (MRSA/MSSA) and Enterococcus faecalis strains are often found in community- and hospital-acquired infections. The single use of conventional antibiotics hardly completely kills the bacterial cells of interest, especially in the form of biofilms. Thus, drug repurposing and antimicrobial combination are promising ways to solve this problem. Antimicrobial susceptibility assays against cocci in a suspension and in a biofilm mode of growth were performed with broth microdilution methods. Checkerboard assays and the cutaneous mouse infection model were used to examine the activity of auranofin and conventional antibiotics alone and in combination. In the present study, auranofin possesses potent antimicrobial activities against both planktonic cells and biofilms with minimum inhibitory concentrations ranging 0.125–0.5 mg/L. Auranofin in combination with linezolid or fosfomycin showed synergistic antimicrobial activities against S. aureus MSSA and MRSA both in vitro and in vivo. Similarly, auranofin also behaved synergistic effect with chloramphenicol against E. faecalis. Additionally, auranofin improved the antibiofilm efficacy of chloramphenicol and linezolid, even on the biofilms grown on a catheter surface. Though, S. epidermidis showed significant susceptibility to AF treatment, no synergistic antimicrobial effects were observed with antibiotics we tested. In all, the use of a combination of auranofin with linezolid, fosfomycin, and chloramphenicol can provide a synergistic microbicidal effect in vitro and in vivo, which rapidly enhances antimicrobial activity and may help prevent or delay the emergence of resistance.

Keywords: auranofin, biofilm, combination therapy, subcutaneous abscess model, Staphylococcus aureus, Enterococcus faecalis

## INTRODUCTION

Staphylococcus aureus and Enterococcus faecalis have been known to be responsible for most of healthcare- and nosocomial-associated infections. S. aureus could cause polymicrobial infections with many pathogens, such as enterococcus (Reyes et al., 2010; Hayakawa et al., 2013), Pseudomonas aeruginosa (Radlinski et al., 2017; Alves et al., 2018),

Peptostreptococcus anaerobius (Yamagishi et al., 2017), Streptococcus pyogenes (Gilmer et al., 2013), and even Candida species (Nash et al., 2015; Todd et al., 2019), which are hard to be eradicated and finally led to a striking mortality rate. According to the report by the SENTRY antimicrobial surveillance program (North America), the main pathogens isolated from skin as well as soft tissue infections (SSTIs) now include 45.9% S. aureus and 8.2% Enterococcus sp. (Rennie et al., 2003). SSTIs abscesses, for instance, create fluid, pus-filled pockets infiltrated by bacteria as well as inflammatory cells, and are frequently extremely resilient to conventional antibiotic therapy (Ki and Rotstein, 2008). In addition, abscesses are the utmost common sign for high-dose, recurrent and long-term intravenous broad-spectrum antibiotic administration (Ramakrishnan et al., 2015).

Biofilms are a widespread problem in healthcare facilities and hospitals. Indeed, the United States National Institutes of Health reported that 80% of chronic infections are related to biofilms (Monroe, 2007). The attachment of S. aureus, S. epidermidis, and E. faecalis onto tissues or the surface of medical apparatuses contributes to the pathogenesis of infection (Archer, 1998). The bacterial cells living in a biofilm are responsible for a number of chronic infections and become resilient to antibiotics as well as host-defense mechanisms (Gomes et al., 2009).

Recently, many studies have been conducted to address the repurposing of FDA-approved drugs as new antimicrobial agents. Auranofin (AF) is a gold-containing compound and prescribed for the treatment of rheumatoid arthritis (Glennas et al., 1997). The study of AF for its antimicrobial effects and inhibition of biofilm formation is an attractive possible treatment approach (Natsis and Cohen, 2018). Researchers found its antimicrobial efficacy against cocci (including Staphylococcus sp. and E. faecalis) and Mycobacterium tuberculosis. AF employs its effects via a distinctive process comprising the prevention of TrxR, and it maintains action against current antibiotic-resistant strains (Cassetta et al., 2014; Harbut et al., 2015; Fuchs et al., 2016). In addition, AF compared with most of the conservative medications available might be an appropriate feature in the fight against a dynamic as well as quickly altering microbial community such as biofilms.

AF shows good antimicrobial effects on cocci and AF in combination with topical antibiotics (mupirocin, retapamulin, and fusidic acid) exhibits additive antimicrobial activity against MRSA (Thangamani et al., 2016). However, to the best of our knowledge, there is no research reporting combinationary therapy with conventional systemic administration associated antibiotics in a subcutaneous abscess infections model. In the present study, we showed the antimicrobial and antibiofilm activities of AF alone or in combination with conventional antibiotics against S. aureus and E. faecalis strains in vitro and in vivo.

## MATERIALS AND METHODS

## Bacterial Strains

AF and antibiotics [fosfomycin (FOF); ciprofloxacin (CIP); tetracycline (TET); linezolid (LZD); chloramphenicol (CHL); levofloxacin (LVX); teicoplanin (TEC); clindamycin hydrochloride (CLI) hydro; gentamicin (GEN); vancomycin (VAN)] were purchased from the MedChemExpress company (Monmouth Junction, NJ, United States). E. faecalis ATCC 29212, S. aureus ATCC25923 and ATCC29213 were kindly provided by Juncai Luo (Tiandiren Biotech, Changsha, China).

TABLE 1 | Antimicrobial susceptibility testing of AF and VAN toward bacterial strains (mg/L).

aMethicillin resistant Staphylococcus aureus (MRSA). bBiofilm formation positive strains determined by CV staining method.

S. epidermidis RP62A and ATCC 12228 were given by Di Qu (Shanghai Medical College of Fudan University), S. aureus ATCC43300 (MRSA), Newman, and RJ-2 were given by Min Li (Renji Hospital, Shanghai Jiao Tong University School of Medicine). Other clinical strains were isolated from the wound secretion or sputum of inpatients at the Third Xiangya Hospital of Central South University. Staphylococcus spp. were grown in tryptic soy broth (TSB) broth medium (Solarbio, Shanghai, China), and E. faecalis was grown in brain heart infusion (BHI) broth medium (Solarbio, Shanghai, China) at 37◦C.

## Susceptibility Testing of Planktonic Bacteria

Bacterial strains were cultured in cationic corrected Mueller– Hinton (MH) broth (BD/Difco, United States). Susceptibility

tests were performed by twofold regular broth microdilution of the test compounds, as recommended by the Clinical and Laboratory Standards Institute (CLSI) (Harbut et al., 2015). After 16–18 h of incubation at 37◦C, the nominal concentration necessary to stop the development of test bacteria was defined as the MIC, and the minimum bactericide concentration (MBC) was identified depending on the lowermost concentration of antimicrobials that killed 99.9% of the test bacteria by spreading the bacterial culture out onto a suitable agar plate (CLSI, 2005).

## Susceptibility Testing of Biofilms

For S. aureus biofilm determination. The culture was grown overnight in TSB and successively diluted 1:50 in TSB to achieve an absorbance at 630 nm of ∼0.1. Two hundred microliter aliquots of the diluted culture were added to every well of a microtiter plate and incubated at 37◦C for 24 h. For E. faecalis biofilm determination, bacterial suspensions (18 µL) from overnight cultures were mixed with 162 µL of BHI in the wells, and biofilms were allowed to form on the plates for 24 h (Lee et al., 2012).

Following the incubation, the contents were removed and rinsed, 50 µL of medium and 50 µL of the specified drug were added to every well, and incubated at 37◦C for 24 h. Then, the contents were removed and the remaining biofilms were determined by crystal violet (CV), XTT staining or live cell count as follows:

MBEC30/MBEC50/MBEC70 were defined as the minimal concentration of the particular antimicrobial's ability to inhibit 30/50/70% growth of the biofilms, respectively, compared to the control group (Gomes et al., 2009).

(3) Biofilm viable count (Mataraci and Dosler, 2012). One hundred microliters of 1 × PBS was aliquoted into each well, and the contents were scrapped and mixed thoroughly with pipette tips. A sample volume of 100 µL was plated onto blood agar and successively diluted with a saline solution before plating onto additional agar plates.

## Checkerboard Assays for Planktonic Bacteria

The impacts of individual antibiotics and in combination with AF were evaluated using the broth microdilution checkerboard technique (Odds, 2003; Flamm et al., 2019). Each microtiter well-comprising the designated combination of antibiotics was inoculated with an overnight culture diluted to provide an absolute concentration of ∼5 × 105 CFU/ml. Following incubation, the optimal fractional inhibitory concentration index (FICI) was measured as the minimal inhibitory concentration of the combination divided by that of the single antibiotic (Odds, 2003): FICI ≤ 0.5 designates synergy; 0.5 < FICI ≤ 4.0 designates no interaction; FICI > 4.0 designates antagonism.

## Checkerboard Assays for Preformed Biofilms

The preparation of overnight biofilms was the same as explained earlier in this study. The biofilms were rinsed, twofold sequential dilutions of antibiotics and AF in a 96-well microtiter plate were prepared, and 100 µL of these mixtures were added to the biofilms. Concentration ranges, as recognized with susceptibility testing, were utilized for the antibiotics as well as the AF. Following an incubation for 24 h at 37◦C, the medium containing antimicrobials was removed, and 100 µL of XTT with PSM was

TABLE 3 | The antibacterial activity and combined effects of AF and selected antibiotics alone or in combination against MRSA and other clinical isolates.

staining. Overnight cultures of strains were diluted with AF to the designated concentrations. After 24 h incubation, planktonic cells were removed and stained with 0.25% CV. (B) Biofilm eradication by AF detected by XTT staining. Biofilms grown for 24 h were treated with AF at the designated concentrations. After incubation, planktonic cells were removed and stained with a solution of XTT/PMS. The red dashed line indicates 50% of the biofilm biomass of the control group. (C) Live cell counts. Twenty-four hour biofilms were treated with AF at a concentration of MBEC50 for 24 h, and serial dilutions and plate counts were performed to determine the live cells in the biofilms (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).

added as described above (Koppen et al., 2019). The MBEC50 values were quantified.

## Antibiofilm Effect of AF on Catheters

To study the efficacy of AF combined with antibiotics against biofilms on catheters, overnight cultures of the biofilm-forming strains were diluted 1:40 in TSB (S. aureus) or BHI (E. faecalis) containing 5% rabbit plasma. Catheter (Jerry infusion set, Shandong, China) pieces (1 cm in size) were cut, divided into two halves, and added to the culture. Next, they were incubated at 37◦C for 24 h. Afterward, the catheters were removed and washed. The biofilms on catheters were challenged with AF alone or in combination with antibiotics for 24 h. The catheters were scratched by an inoculation loop and sonicated for 15 min. Then,

TABLE 4 | Activity of AF in combination with conventional antibiotic against preformed biofilms (mg/L).

the samples were vortexed carefully and plated on blood agar plates (Nair et al., 2016).

## Confocal Laser Scanning Microscopy (CLSM)

The above-treated bacteria were cultured on glass cover slides and incubated with 10 µL of 1000-fold diluted SYTO9 fluorescent staining solution and propidium iodide at a ratio of 1:1 (vol/vol) for 15 min in the dark. After rinsing, the stained biofilm was examined with a CLSM (Zeiss LSM 800, Jena, Germany) (Nair et al., 2016).

## Cutaneous Mouse Infection Model

Seven-week-old female mice CD-1 were purchased from Hunan Slake Jingda Experimental Animal, Co., Ltd. (Hunan, China). They weighed approximately 25 ± 3 g at the time of the experiments.

The high bacterial load abscess infection model was performed as defined earlier with slight adaptations (Pletzer et al., 2018). Before the injection, bacterial cells were rinsed resuspended in 1 × PBS. An injection of bacterial suspension was given to the dorsum to achieve the concentrations to generate reproducible abscesses and bacterial counts: S. aureus, 1 × 108 CFU/mice; and E. faecalis, 1 × 109 CFU/mice. Antimicrobial administration was given directly into the subcutaneous space of the infected area at 1 h post-infection. The development of the infection was observed every day. Abscesses were determined on day 2 using a caliper. Skin abscesses were removed (comprising all accrued pus) and regimented in sterile PBS by an automatic tissue homogenizer (Servicebio KZ-II, Wuhan, China). Bacterial counts were quantified by serial dilution. For histopathological analyses, hematoxylin and eosin (H&E) staining was performed.

## Statistical Analysis

Statistical evaluations were performed using GraphPad Prism 7.0. Checkerboard methods were performed at least in biological duplicates, and other experiments were performed in triplicate.

cells, red). (A) S. aureus LZB1, AF 8 mg/L, LZD 16 mg/L. (B) E. faecalis ATCC29212, AF 2 mg/L, CHL 8 mg/L. Scale bar: 40 µm.

## RESULTS

## Determination of the Susceptibility of Planktonic Cells

The MICs of AF and VAN against type strains and clinical isolates of S. aureus (MSSA/MRSA), S. epidermidis, and E. faecalis were 0.125–2 mg/L. The MBCs against S. aureus and S. epidermidis were 0.5–4 and 1–8 mg/L for AF and VAN, respectively. And the susceptibility of AF against MRSA and MSSA strains showed no difference. However, the MBCs of E. faecalis were > 32 mg/L for both AF and VAN (**Table 1**). In all, the strains we tested were more sensitive to the AF treatment than the VAN treatment.

## Synergistic Effect Between AF and Antibiotics Against Planktonic Cells

The synergistic effects of AF were investigated with some conventional systemic antibiotics (antibiotics with MIC values greater than 256 mg/L were excluded). The results of the combination screening assay are presented in **Table 2**. Synergistic interactions between AF and FOF (FICI = 0.375) or LZD (FICI = 0.375) were observed against S. aureus LZB1. For E. faecalis ATCC29212, synergistic interactions were observed between AF and CHL (FICI = 0.375). But no interactions between AF and antibiotics were observed against S. epidermidis RP62A (FICI > 0.5). Combinations with the lowest FICI values were selected for other representative strains. As shown in **Table 3**, the combination of AF and CHL still showed synergistic effect against the E. faecalis clinical isolates; combinations of AF + LZD/FOF still showed synergistic effects against S. aureus ATCC43300 (MRSA) and most of the clinical isolates, except for strain SA1435 which showed no interaction between AF and FOF with FICI of 0.5.

## Determination of the Susceptibility of Biofilms

Staphylococcus aureus LZB1 and E. faecalis ATCC29212 were selected to test the antibiofilm activities of AF due to their strong biofilm formation abilities (Kart et al., 2017). AF showed strong biofilm inhibitory effects against S. aureus, and E. faecalis at concentrations of 0.125 and 1 mg/L (p < 0.05), respectively, in a dose-dependent manner (**Figure 1A**), which were very close to its MICs, indicating that the biofilm inhibitory effect of AF could be mainly due to its bacteriostatic or bactericidal activity by targeting thiol-redox homeostasis (Harbut et al., 2015). Because biofilm formation strongly increased the antimicrobial resistance to AF, the lowest concentrations needed to eradicate preformed biofilms were up to 4 and 2 mg/L for S. aureus and E. faecalis, respectively (**Figure 1B**). MBEC50 was selected to detect the time kill efficacy of AF against biofilms. AF showed significant biofilm killing activity against these strains in a time-dependent manner. Compared to the control group, AF reduced the live biofilm cells of S. aureus from (1.38 ± 0.29) × 109 CFU/ml to (1.13 ± 0.90) × 107 CFU/ml (p < 0.001). Although statistical significance was only observed at 8 h after treatment (p < 0.05), AF killed E. faecalis ATCC29212 biofilm cells throughout the 24 h period (**Figure 1C**). In addition, AF could also effectively eradicate clinical isolates with low MBEC50 values (**Supplementary Table S2**).

## Synergistic Effect Between AF and Antibiotics Against Biofilms

Antibiotics that showed a synergistic effect on planktonic cells were tested against preformed biofilms in combination with AF (**Table 4**). AF significantly promoted the antibiofilm efficacy of CHL against E. faecalis ATCC29212 (4- and 8-fold decrease of MBEC50 for CHL and AF, respectively). Meanwhile, AF also increased the antibiofilm activity of LZD against S. aureus LZB1 and exhibited a 2- and > 8-fold decrease of MBEC50 for AF and LZD, respectively, but showed no interaction with FOF (**Supplementary Figure S1**). Similar observations were made by visualization of AF and/or antibiotic-treated biofilms by CLSM, when AF used in combination with FOF (S. aureus LZB1, **Figure 2A**) or CHL (E. faecalis ATCC29212, **Figure 2B**), the live cells in the biofilms were significantly reduced, although some intact patches of biofilm could still be visualized.

To simulate the in vivo conditions for biofilm formation in device-associated infections, we allowed strains to form biofilms on the surfaces of catheters. Treatment of biofilms with AF and in combination with antibiotics led to their synergistic eradication (**Figure 3**). A single dose of AF or antibiotics only showed moderate antibiofilm effects; however, combination treatment led to a 4.96- and 1.95-log reduction in CFUs for S. aureus (AF + LZD, **Figure 3A**) and E. faecalis (AF + CHL, **Figure 3B**), respectively, confirming that AF possesses antibiotic-promoting activity against preformed biofilms on catheters.

## Therapeutic Efficacy of AF Combined With Conventional Antibiotics in vivo

To optimize the treatment strategy, antimicrobials were chosen based on their moderate in vivo pharmacodynamics (**Figure 4**), and the concentrations used in the present study were equal or less than those empirically tested in vivo (CHL, 10 mg/kg; LZD, 60 mg/kg; and FOF, 100 mg/kg) (Shibl, 1982; Guo et al., 2013; Zykov et al., 2018) to determine an appropriate concentration that reduces abscess sizes just enough to observe the synergy between the AF and the antibiotics (**Figure 4**, red dashed line). A significant reduction in the mean bacterial load was observed for each combined treatment condition compared with the control (receiving DMSO or Tween-80) or single dose group.

Except for FOF, which reduced the abscess area of S. aureus LZB1 57.38 mm2 , single use of AF (0.5 mg/kg), LZD (5 mg/kg) or FOF (20 mg/kg) showed no statistical significance in reducing abscess area or bacterial loads of S. aureus LZB1 (MSSA) (**Figure 5A**) and ATCC43300 (MRSA) (**Figure 5B**) infections; however, AF combined with LZD significantly decreased the abscess area and reduced the bacterial load for 4.51- (S. aureus LZB1, p < 0.01) and 2.45-fold log10 (S. aureus ATCC43300, p < 0.001). Similarly, AF or CHL could not inhibit the abscess growth of E. faecalis ATCC29212 individually, but when combined, the area of abscess was reduced by 74.14 mm2 (p < 0.01). Single use of AF or CHL had no impact on bacterial load; however, combined therapy reduced the bacterial load by 0.61-fold log10 (p < 0.01) (**Figure 5C**). For in vivo observations, the abscesses caused by S. aureus were more obvious than those caused by E. faecalis. The ulcers were formed when infected with S. aureus LZB1 or ATCC43300 (**Figures 6A,B**); however, infection with a high load of E. faecalis ATCC29212 (**Figure 6C**) only caused subcutaneous lumps. In accordance with the in vitro observations, the representative pictures of abscesses and histological examinations showed that single use of AF, LZD, or FOF showed no/moderate activity against infections caused by S. aureus LZB1 (**Figure 6A**) or ATCC43300 (**Figure 6B**), and extensive inflammation with leukocyte infiltration emerged; however, drug combination (AF + LZD or AF + FOF) significantly reduced the size and inflammation of the abscesses,

which even eventually disappeared. The single use of AF or CHL had no influence on the abscesses caused by E. faecalis ATCC29212, the drug combination significantly diminished the abscess size and inflammation infiltration (**Figure 6C**). These important observations highlight that antimicrobial monotherapies are often ineffective when bacteria form highdensity infections for S. aureus and E. faecalis. In addition, drug combinations could significantly improve the efficacy.

## DISCUSSION

In the present study, the antimicrobial activity of AF was assessed against a panel of type strains and clinical isolates of Staphylococcus spp. and E. faecalis. In accordance with the previous studies reported by Cassetta et al. (2014), Harbut et al. (2015), Fuchs et al. (2016), the MICs for S. epidermidis, S. aureus (including MSSA and MRSA), and E. faecalis were 0.125–0.5 mg/L, which showed more susceptibility than with VAN treatment, with MICs ranging from 0.5 to 2 mg/L.

Drug combination is a promising way to improve the efficacy of drugs and reduce side effects and cytotoxicity. In our study, highly synergistic interactions between AF and CHL were observed against E. faecalis. CHL is a broad-spectrum antibiotic against many gram-positive/negative bacteria (Civljak et al., 2014). However, CHL is an old antimicrobial agent that is rarely used today mainly due to its most significant adverse effect of dose-related bone marrow suppression, according a meta-analysis by Eliakim-Raz et al. (2015), CHL is as safe a treatment alternatives as short antibiotic courses. In this way, drug combination could significantly diminish the dose required but achieve better antimicrobial efficacy, so that AF combined with CHL could be a better choice than CHL used alone in clinical therapy. Moreover, E. faecalis has shown many different metabolic responses from anaerobic to aerobic circumstances; these main metabolic cascades are related to the response to nutrients and may change the susceptibility of this bacterium to bactericidal drugs (Portela et al., 2014). However, even in the anaerobic condition, AF still showed a highly synergistic effect with CHL (**Supplementary Table S1**).

Highly synergistic interactions between AF and LZD/FOF were also observed against MSSA or MRSA strains of S. aureus. LZD has a wide spectrum of action against the mainstream of common gram-positive cocci. However, due to the development

of resistance to antibiotics as well as their unwanted side effects, combination therapy has evolved as an imperative novel treatment approach (Yang et al., 2018). FOF by itself has a bactericidal impact both in vitro as well as in vivo. Nevertheless, MRSA can easily develop tolerance, making utilization of FOF unattainable for medical situations (Roussos et al., 2009). FOF displays positive synergistic impacts on MRSA and its biofilms when utilized in combination with other antibiotics (Shi et al., 2014). Similarly, in our study, AF showed an excellent synergistic effect with FOF against MSSA and MRSA planktonic cells. In addition, different clinical isolates showed outcomes of different drug combinations, which indicates the importance of an in vitro synergistic test before clinical use.

Biofilms are easily formed on indwelling medical apparatus surfaces (Francolini and Donelli, 2010). During implantation of catheters, tissue damage might occur due to the buildup of platelets as well as fibrin at the suture site as well as on the devices. Microbial cells have enhanced capability to colonize these sites (Jamal et al., 2018). The formation of biofilms increases the antibiotic resistance and leads to persistent infections posing major healthcare challenges. AF showed modest biofilm inhibitory and eradicating effects against Staphylococcus aureus and E. faecalis both in type strains and clinical isolates with high values of MBEC70. Thus, our favorable outcomes of synergy among AF and antibiotics encouraged us to inspect the action of drug combinations against biofilms. AF combined with CHL showed synergistic antibiofilm effects for E. faecalis on cover slides or infusion catheters (**Supplementary Figure S1**), AF significantly improved the antibiofilm effects of LZD against S. aureus. As numerous antibiotics have diverse antibacterial mechanisms and several bacteria have diverse resistance mechanisms, to entirely eliminate the whole biofilmbacteria is a difficult challenge. Combination therapy comprising two or more antibiotics with diverse bactericidal mechanisms could synergistically eliminate biofilms (Simoes, 2011).

A high bacterial load-containing abscess model has rarely been studied for AF efficacy evaluation. In our in vivo subcutaneous abscess model study, single use of antimicrobials showed an extremely modest effect on abscess area or bacterial load. However, AF combined with LZD or FOF synergistically inhibited abscess and inflammation formation and reduced the bacterial load for both MSSA and MRSA strains. The safety of AF in in vivo animal studies and clinical use is welldocumented. AF is widely used in clinical settings for longterm treatment at the daily dosage at 6 mg/day, and a average blood concentration of 3.5 µM (∼2.38 µg/ml, which is far beyond the value of MICs) is reached in 12 weeks. Besides, the effectiveness and safety of AF at an dose of 12 mg/day is under Phase II clinical trial (Harbut et al., 2015). As reported by Aguinagalde et al. (2015), the dosage of AF used for murine model even reach to 10 mg/kg due to its safe toxicity profile and well-known pharmacokinetic/pharmacodynamic characteristics.

Similarly, the safety of antibiotics of FOF and LZD is wellstudied and documented. As reported by Pachón-Ibáñez et al. (2011) and Guo et al. (2013), the dosages of FOF and LZD used are reached to 100 and 60 mg/kg in murine models, respectively. And the FOF and LZD used in our study are only 20 and 5 mg/kg, respectively. Therefore, the inflammation caused in our animal models is not caused by the antimicrobials we used. In all, the combination therapy of AF plus LZD/FOF might be an effective option for treating patients with S. aureus-related subcutaneous abscess infection. Similarly, AF combined with CHL also showed synergistic antibacterial effects on E. faecalis abscesses and partially reduced inflammation formation.

## CONCLUSION

The present study provides a valuable effect of antimicrobial combination therapy against cocci in subcutaneous abscess infections. This type of synergistic combination of two medications is likely preferred in clinical situations. The rationality of the outcomes should be validated by future clinical trials.

## DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

## ETHICS STATEMENT

Ethical approval was obtained from the Animal Ethics Committee (certificate number 2017-S139), the Third Xiangya Hospital, Central South University, China.

## REFERENCES

## AUTHOR CONTRIBUTIONS

PS and YW designed and performed the experiments and wrote the manuscript. PS, YW, and LZ performed the experiments and data collection. SL, YL, LC, and ZL performed the experiments and revised the manuscript. All authors read and approved the final manuscript.

## FUNDING

This study was funded by the Hunan Provincial Natural Science Fund, China (Grant No. 2019JJ80029).

## ACKNOWLEDGMENTS

We are particularly grateful to the staff of the Department of Clinical Laboratory of the Third Xiangya Hospital of Central South University, Changsha, China.

## SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.02453/full#supplementary-material

FIGURE S1 | Representative CLSM images of biofilm eradication by AF and FOF mono-/combination treatment. S. aureus LZB1 biofilms on the surfaces of cover slides were treated with AF (16 mg/L) and/or FOF (128 mg/L) as described in Section "Materials and Methods," the stained with the SYTO9/PI fluorescent dye mixture.

TABLE S1 | The combinational antibacterial activities of AF and different antibiotics against E. faecalis ATCC29212 in anaerobic conditions.

TABLE S2 | Biofilm eradication activities of AF against other type and clinical strains (mg/L).

rheumatoid arthritis. Br. J. Rheumatol. 36, 870–877. doi: 10.1093/rheumatology/ 36.8.870

**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 She, Zhou, Li, Liu, Xu, Chen, Luo and Wu. 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.

# Genomic Characterization of Prevalent mcr-1, mcr-4, and mcr-5 Escherichia coli Within Swine Enteric Colibacillosis in Spain

Isidro García-Meniño1† , Dafne Díaz-Jiménez1† , Vanesa García1‡ , María de Toro2 , Saskia C. Flament-Simon1 , Jorge Blanco1 and Azucena Mora1 \*

1 Laboratorio de Referencia de Escherichia coli, Departamento de Microbiología y Parasitología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain, 2 Plataforma de Genómica y Bioinformática, Centro de Investigación Biomédica de La Rioja, Logroño, Spain

#### Edited by:

Luciene Andrade Da Rocha Minarini, Federal University of São Paulo, Brazil

#### Reviewed by:

Darren Trott, The University of Adelaide, Australia Maite Muniesa, University of Barcelona, Spain

> \*Correspondence: Azucena Mora<EMAIL_ADDRESS>
†These authors have contributed equally to this work

### ‡Present address:

Vanesa García, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark

#### Specialty section:

This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology

Received: 26 August 2019 Accepted: 15 October 2019 Published: 01 November 2019

#### Citation:

García-Meniño I, Díaz-Jiménez D, García V, de Toro M, Flament-Simon SC, Blanco J and Mora A (2019) Genomic Characterization of Prevalent mcr-1, mcr-4, and mcr-5 Escherichia coli Within Swine Enteric Colibacillosis in Spain. Front. Microbiol. 10:2469. doi: 10.3389/fmicb.2019.02469 Antimicrobial agents are crucial for the treatment of many bacterial diseases in pigs, however, the massive use of critically important antibiotics such as colistin, fluoroquinolones and 3rd–4th-generation cephalosporins often selects for co-resistance. Based on a comprehensive characterization of 35 colistin-resistant Escherichia coli from swine enteric colibacillosis, belonging to prevalent Spanish lineages, the aims of the present study were to investigate the characteristics of E. coli clones successfully spread in swine and to assess the correlation of the in vitro results with in silico predictions from WGS data. The resistome analysis showed six different mcr variants: mcr-1.1; mcr-1.10; mcr-4.1; mcr-4.2; mcr-4.5; and mcr-5.1. Additionally, blaCTX-M-14, blaCTX-M-32 and blaSHV-12 genes were present in seven genomes. PlasmidFinder revealed that mcr-1.1 genes located mainly on IncHI2 and IncX4 types, and mcr-4 on ColE10-like plasmids. Twenty-eight genomes showed a gyrA S83L substitution, and 12 of those 28 harbored double-serine mutations gyrA S83L and parC S80I, correlating with in vitro quinolone-resistances. Notably, 16 of the 35 mcrbearing genomes showed mutations in the PmrA (S39I) and PmrB (V161G) proteins. The summative presence of mechanisms, associated with high-level of resistance to quinolones/fluoroquinolones and colistin, could be conferring adaptive advantages to prevalent pig E. coli lineages, such as the ST10-A (CH11-24), as presumed for ST131. SerotypeFinder allowed the H-antigen identification of in vitro non-mobile (HNM) isolates, revealing that 15 of the 21 HNM E. coli analyzed were H39. Since the H39 is associated with the most prevalent O antigens worldwide within swine colibacillosis, such as O108 and O157, it would be probably playing a role in porcine colibacillosis to be considered as a valuable subunit antigen in the formulation of a broadly protective Enterotoxigenic E. coli (ETEC) vaccine. Our data show common features with other European countries in relation to a prevalent clonal group (CC10), serotypes (O108:H39, O138:H10, O139:H1, O141:H4), high plasmid content within the isolates and mcr location, which would support global alternatives to the use of antibiotics in pigs. Here, we report for first time a rare finding so far, which is the co-occurrence of double colistin-resistance mechanisms in a significant number of E. coli isolates.

Keywords: Escherichia coli, colistin, mcr, ESBL, fluoroquinolones, ST10, colibacillosis, swine

## INTRODUCTION

fmicb-10-02469 October 31, 2019 Time: 18:39 # 2

Multidrug-resistant Enterobacteriaceae, such as Escherichia coli, represent a threat to both human and veterinary health. E. coli has a great capacity to accumulate resistance genes, mostly through horizontal gene transfer. The major problematic mechanisms correspond to the acquisition of genes coding for extended-spectrum beta-lactamases (ESBL), carbapenemases, 16S rRNA methylases, plasmid-mediated quinolone resistance (PMQR) and mcr genes conferring resistance to polymyxins (Poirel et al., 2018).

Colistin has been widely used in Spain for the control of neonatal and post-weaning diarrhoea (PWD) in pigs caused by certain E. coli pathotypes: Enterotoxigenic E. coli (ETEC), defined by the presence of genes encoding enterotoxins (eltA, and/or estA, and/or estB); atypical Enteropathogenic E. coli (aEPEC), carriers of eae but negative for bfpA (aEPEC); Shiga toxin–producing E. coli (STEC), positive for stx2e; STEC/ETEC, positive for both shiga toxin type 2e and enterotoxin-encoding genes (stx2e and estB and/or estA) (García-Meniño et al., 2018). PWD results in significant economic losses for the pig industry due to costs derived of treatment and handling, decreased weight gain, and mortality. These circumstances have promoted the use and abuse of antibiotics in intensive farming (Luppi, 2017; Rhouma et al., 2017). However, specific regulations have been set up in Europe due to the concern that colistin resistance could be transmitted from food-production animals to humans which makes necessary the investigation of sustainable alternatives to antimicrobials (EUROPEAN COMMISSION, 2018).

In Spain, the rates of antibiotic resistance in pig farming were recently analyzed in a collection of 499 E. coli isolates from 179 outbreaks of enteric colibacillosis occurred during a period of 10 years (2006–2016) (García et al., 2018; García-Meniño et al., 2018). The results revealed a prevalence of colistin-resistant E. coli implicated in PWD in Spanish farms as high as 76.9% within 186 ETEC, STEC and STEC/ETEC isolates. Besides, PCR and sequencing identified the presence of mcr-4 in 102 isolates, mcr-1 in 37 isolates and mcr-5 in five isolates. Interestingly, almost all mcr-4 isolates belonged to the clonal group ST10- A (CH11-24) (García et al., 2018), which was shown to be highly present (more than 50%) within the mcr-1 diarrheagenic isolates of a second study (García-Meniño et al., 2018). Both studies reinforced other countries' findings that the pig industry is an important reservoir of colistin-resistant E. coli, as well as being carriers of other additional risk genes such as blaESBL genes (García et al., 2018; García-Meniño et al., 2018; Magistrali et al., 2018; Manageiro et al., 2019). Based on reported evidences (Beyrouthy et al., 2017; Gilrane et al., 2017), there is great concern about the in vivo acquisition of mcr- and blaESBL-bearing plasmids by human E. coli isolates following treatment with colistin, or via animal transmission through direct contact or via food chain. Particular attention is given to those named as high-risk clones of (ESBL)-producing bacteria, worldwide spread within humans and animals, including Escherichia coli sequence types ST10, ST131, ST405, and ST648 (Mathers et al., 2015; Sellera and Lincopan, 2019).

The aims of this study were (i) the characterization of resistances and plasmid profiles of successfully spread mcr-1, mcr-4, and mcr-5 E. coli in Spanish pig farming; (ii) the assessment of WGS-based approaches for the characterization of pathogenic E. coli, through the correlation of the in vitro results with in silico predictions using the bioinformatics tools of the Center for Genomic Epidemiology (CGE).

## MATERIALS AND METHODS

## E. coli Collection

Thirty-five swine E. coli, positive by PCR for mcr-genes, were fully sequenced. Specifically, the 35 E. coli were selected from 499 diarrheagenic isolates of different geographic areas of Spain (2006–2016) (García et al., 2018; García-Meniño et al., 2018), taking into account the results of prevalence and significant association observed between pathotypes, presence of mcr and certain serogroups. In brief, the serogroups O108, O138, O141, O149, O157 were found significantly associated with ETEC; serogroups O26, O49, O80, O111 with aEPEC; serogroups O138 and O141 with STEC/ETEC; serogroup O139 with STEC; and serogroups O2, O15, O26, O45, O111, O138, O141, O157 with mcr-positive isolates (García-Meniño et al., 2018). Therefore, the collection analyzed here included 27 ETEC isolates (of serogroups O7, O8, O15, O45, O108, O138, O141, O149, O157, ONT); four STEC (O2, O139); three STEC/ETEC (O138 and O141) and one aEPEC (O111). The 35 representative isolates were carriers of the three mcr-types (mcr-1, mcr-4, and mcr-5) detected so far in our E. coli collection of porcine origin. Conventional pheno- and geno-typing was performed to complete classical characterization of serotypes, phylogroups, pathotypes and resistance profiles.

## Conventional Typing

The H antigen was established for motile isolates by serotyping using H1 to H56 antisera, while non-motile isolates (HNM) were analyzed by PCR to determine their flagellar genes as described elsewhere (García-Meniño et al., 2018). The phylogroup was assigned by means of the quadruplex PCR of Clermont et al. (2013). Antimicrobial susceptibility was determined by minimal inhibitory concentrations (MICs) using the MicroScan WalkAway R -automated system (Siemens Healthcare Diagnostics, Berkeley, CA, United States) according to the manufacturer's instructions for: amikacin, ampicillin-sulbactam, aztreonam, cefepime, ceftazidime, ciprofloxacin, colistin, fosfomycin, gentamicin, imipenem, levofloxacin, meropenem, minocycline, nitrofurantoin, piperacillin-tazobactam, ticarcillin, tigecycline, and tobramycin. Additionally, resistance to ampicillin, amoxicillin/clavulanate, cefazolin, cefotaxime, cefoxitin, cefuroxime, chloramphenicol, doxycycline, nalidixic acid and trimethoprim-sulfamethoxazole was determined by disk (Becton Dickinson, Sparks, MD, United States) diffusion assays. All results were interpreted according to the CLSI break points (Clinical and Laboratory Standards Institute, 2019). Genetic identification of the ESBLs was performed by PCR using

the TEM, SHV, CTX-M-1 and CTX-M-9 group-specific primers followed by amplicon sequencing (García-Meniño et al., 2018).

## Whole Genome Sequencing (WGS) and Sequence Analysis

The libraries for sequencing were prepared following the instructions provided by the TruSeq Illumina PCR-Free protocol. Mechanical DNA fragmentation was performed with Covaris E220, and the final quality of the libraries assessed with Fragment Analyzer (Std. Sens. NGS Fragment Analysis kit 1- 6000 bp). Lastly, the libraries were sequenced in an Illumina HiSeq1500, obtaining 100–150 bp paired-end reads which were trimmed (Trim Galore 0.5.0) and filtered according to quality criteria (FastQC 0.11.7). The reconstruction of the genomes and plasmids in the genomes was carried out using the methodology PLAsmid Constellation NETwork (PLACNETw)1 (Lanza et al., 2014). The assembled contigs, with genomic size ranging between 4.9 and 5.9 Mbp (mean size 5.5 Mbp), were analyzed using the bioinformatics tools of the Center for Genomic Epidemiology (CGE)2 for the presence of antibiotic resistance (ResFinder V2.1.), virulence genes (VirulenceFinder v1.5.), plasmid replicon types (PlasmidFinder 1.3./PMLST 1.4.), and identification of clonotypes (CHTyper 1.0), sequence types (MLST 2.0) and serotypes (SerotypeFinder 2.0). All the CGE predictions were called applying a select threshold for identification and a minimum length of 95 and 80%, respectively. Phylogroups were predicted using the ClermonTyping tool at the iame-research center web3 . The mcr gene location was determined using PlasmidFinder/ResFinder prediction, together with PLACNETw references, and automatic annotation with Prokka v1.13 (Seemann, 2014).

## RESULTS AND DISCUSSION

The phenotypic and genotypic traits of the 35 mcr-positive E. coli of swine origin, as well as their resistome and mobilome are summarized in **Table 1**. ResFinder confirmed that all genomes were mcr carriers. Likewise, VirulenceFinder predicted the acquired virulence genes encoding for the enterotoxins (sta1, stb, itcA), for fimbriae (fedF, k88), verotoxin (stx2) and intimin (eae), correlating in all cases with the pathotype assignation previously determined by PCR (García et al., 2018; García-Meniño et al., 2018).

## Serotype Identification

In most studies, there is lack of information on E. coli serotypes since serotyping is performed by very few laboratories worldwide, hindering epidemiological comparisons. Here, we not only proved that there is a very good correlation between serotyping and SerotypeFinder predictions, but also the advantage of in silico H-antigen identification for those non-mobile (HNM) isolates. It is of note that 15 of the 21 HNM isolates were predicted

1https://castillo.dicom.unican.es/upload/

2https://cge.cbs.dtu.dk/services/

as H39 (**Table 1**), namely O108:H39, O157:H39 and O45:H39 (five genomes, each). Given that the H39 is associated with the most prevalent O antigens within swine colibacillosis, such as O108 and O157, as well as ONT (García-Meniño et al., 2018), it would be probably playing a role in porcine colibacillosis to be considered as a valuable subunit antigen in the formulation of a broadly protective ETEC vaccine (Roy et al., 2009). The remaining six HNM isolates showed different O:H combinations: O138:H14, ONT:H5, O8:H20, O50/O2:H32, and O182:H19. SerotypeFinder also allowed the O45-antigen determination of two non-typeable (ONT) isolates (LREC-141 and LREC-146) and O182 of LREC-172; while LREC-147, belonging to O157 serogroup (**Table 1**), was predicted as ONT, probably due to the limitation of the assembly based on Illumina short reads (100–150 bp paired-end reads here) (Wick et al., 2017).

## Phylogroups, Sequence Types and Clonotypes

The phylogroups established for the 35 genomes were the common ones reported for porcine E. coli isolates (A, B1, D-E) (Shepard et al., 2012; Bosak et al., 2019). However, we found discrepancies in the assignation obtained with the quadruplex PCR of Clermont et al. (2013) in comparison with that predicted by ClermonTyping for seven isolates: phylogroup E by PCR, while phylogroup D in silico (**Table 1**).

MLST and CHTyper tools determined 12 different STs, but mostly belonging to CC10 (21 genomes) and clonotype CH11- 24 (18 genomes) (**Table 1**). The predominance of CC10, and specifically ST10, is in accordance with published data on E. coli isolates of swine origin, independently of the pathogenicity or antibiotic-resistance/susceptibility status (Shepard et al., 2012; Kidsley et al., 2018; Magistrali et al., 2018).

## Resistome, Plasmidome and Phenotypic Expression of Resistances

The resistome analysis revealed that 34 of the 35 genomes encoded mechanisms of antibiotic resistance for ≥three different antimicrobial categories (**Table 1**). Seven E. coli were carriers of blaESBL, namely blaCTX-M-14 (four genomes), blaCTX-M-32 (one) and blaSHV-12 (two). Besides, six different mcr variants were identified within the 35 E. coli: mcr-1.1 (in 18 genomes, including two mcr-4.2 carriers); mcr-1.10 (one); mcr-4.1 (one); mcr-4.2 (13 genomes, including the two mcr-1.1 carriers); mcr-4.5 (two) and mcr-5.1 (two).

PlasmidFinder revealed a high plasmid diversity based on the identified replicons, with two to seven different plasmid types per genome (**Table 1**). Within this heterogeneity, mcr-1.1 genes were found mainly on plasmids of the IncHI2 and IncX4 types (six and four of the 12 mcr-1.1 plasmidlocated genes, respectively); however, mcr-1.1 was also found integrated in the chromosome of LREC-145, LREC-148, LREC-149 and LREC-164 genomes. The mcr-1.10 gene of LREC-151 was located on the chromosome, while mcr-4 and mcr-5 variants were on Col8282-like (mcr-4.1), ColE10 like (for all 13 mcr-4.2 and two mcr-4.5 carriers) and pKP13a-like (mcr-5.1) plasmids. Furthermore, we found that

3http://clermontyping.iame-research.center/

fmicb-10-02469 October 31, 2019 Time: 18:39 # 4

TABLE 1 | Features of the 35 colistin-resistantE. coli genomes of swine origin based onin silicocharacterization (light columns) and on conventional typing (gray columns).

(Continued)

WGS of

mcr-1, mcr-4,

and

mcr-5 E. coli

WGS of

mcr-1, mcr-4,

and

mcr-5 E. coli

fmicb-10-02469 October 31, 2019 Time: 18:39 # 5

TABLE 1 | Continued

TABLE 1 | Continued

fmicb-10-02469 October 31, 2019 Time: 18:39 # 6

WGS of

November 2019 | Volume 10 | Article 2469

(Continued)

fmicb-10-02469 October 31, 2019 Time: 18:39 # 7

#### TABLE 1 | Continued

(Continued)

WGS of

mcr-1, mcr-4,

and

mcr-5 E. coli

fmicb-10-02469 October 31, 2019 Time: 18:39 # 8

#### TABLE 1 | Continued

1Year of isolation of the WGS isolates recovered from pig colibacillosis. 2Serotypes, 4clonotypes, 5sequence types, 6replicon/plasmid STs, 7acquired antimicrobial resistance genes and/or chromosomal mutations, 9virulence genes were determined using SerotypeFinder 2.0, CHtyper 1.0, MLST 2.0, PlasmidFinder 2.0, pMLST 2.0, ResFinder 3.1 and VirulenceFinder 2.0 online tools at the Center of Genomic Epidemiology (https://cge.cbs.dtu.dk/services/), respectively; while 3phylogroups were predicted using the ClermonTyping tool at the Iame-research Center web (http://clermontyping.iame-research.center/). 2Serotypes: underlined those antigens that were non-typeable (ONT or HNM) by conventional serotyping but determined by SerotypeFinder. ∗LREC-147 was solved as O157 by conventional typing. 3Phylogroups: " ∗D" indicates that LREC-133, LREC-134, LREC-143, LREC-151, LREC-170, LREC-171, LREC-174 revealed discrepancies between the assignation obtained with the quadruplex PCR of Clermont et al. (2013) and the in silico assignation using ClermonTyping tool, showing phylogroup E by PCR, but phylogroup D in silico. 6Plasmid STs: " ∗" indicates alleles with less than 100% but >95% identity and 100% coverage; "/" indicates alleles with multiple perfect hits found. 7Resistome: chromosomic and plasmid mechanisms associated to colistin resistance are highlighted in bold. Genes coding for ESBLs appear underlined. Acquired resistance genes: beta-lactam: blaTEM-1B, blaTEM-1A, blaOXA-1, blaCTX-M-14, blaCTX-M-32, blaSHV-12, aminoglycosides: aac(3)-II/IV, acc(6 0)-Ib3, aadA, aph(3 0)-I/IIa, aph(300)-Ib, aph(4)-Ia, aph(6)-Id; phenicols: catA1, catB3, cmlA1, floR; fluoroquinolones: aac(6 0)-Ib-cr, qnrS1; macrolides: erm(B), inu(F), inu(G), mdf(A), mph(B); sulfonamides: sul1, sul2, sul3; tetracycline: tet(A), tet(B), tet(M); trimethoprim: dfrA1, dfrA12. Point mutations: quinolones and fluoroquinolones: gyrA S83L: TCG-TTG, gyrA D87G: GAC-GGC, gyrA D87N: GAC-AAT, gyrA D87Y: GAC-TAT, parC S80I: AGC-ATC, parC S80R: AGC-AGG, parC E84G: GAA-GGA, parE L416F: CTT-TTT; colistin: pmrB V161G: GGG-GTG, pmrA S39I: AGC-ATC. 8The mcr gene location was determined using PlasmidFinder/ResFinder predictions, together with PLACNETw building and references (https://castillo.dicom.unican.es/upload/), and Prokka annotations. 9Highlighted in bold, those features defining E. coli pathotypes (STEC, ETEC, EPEC). Virulence genes: itcA, coding for heat labile enterotoxin A subunit; sta1, heat stabile enterotoxin ST-Ia; stb, heat stabile enterotoxin II; fedF, fimbrial adhesin AC precursor; K88, K88/F4 protein subunit; air, enteroaggregative inmunoglobulin repeat protein; astA, EAST-1; capU, hexosyltransferase homolog; cba, colicin B; celB, endonuclease colicin E2; cif, type III secreted effector; cma, colicin M; eae, intimin; efa1, EHEC factor for adherence; eilA, Salmonella HilA homolog; espA, type III secretions system; espB, secreted protein B; espF, type III secretions system; espJ, prophage -encoded type III secretion system effector; etpD, type II secretion protein; fedA, F107; gad, glutamate descarboxylase; iha, adherence protein; iroN, enterobactin siderophore receptor protein; iss, increased serum survival; lpfA, long polar fimbriae; nleA, non LEE encoded effector A; nleB, non-LEE encoded effector B; sepA, Shigella extracellular protein A; tccP, Tir cytoskeleton coupling protein; tir, translocated intimin receptor protein. bp, base pairs; CHType, clonotype (fumC-fimH); ST (CC), sequence type and clonal complex according to Achtman scheme; pMLST, plasmid sequence type. 10Phenotypic resistances interpreted according to the CLSI (intermediate resistance is indicated with an asterisk ∗). AMC, amoxicillin/clavulanate; AMP, ampicillin; AMP/SAM, ampicillin-sulbactam; ATM, aztreonam; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CST, colistin; CTX, cefotaxime; CXM, cefuroxime; CFZ, cefazolin; DOX, doxycycline; FEP, cefepime; FOF, fosfomycin; GEN, gentamicin; LVX, levofloxacin; MI, minocycline; NAL, nalidixic acid; NIT, nitrofurantoin; SXT, trimethoprim/sulfamethoxazole; TIC, ticarcillin; TOB, tobramycin. 11Pathotype of the isolates, established by conventional PCR, based on specific genes encoding toxins (LT, STa, STb, Stx1, Stx2, Stx2e), fimbriae (F4, F5, F6, F18, F41) and intimin (Eae).

fmicb-10-02469 October 31, 2019 Time: 18:39 # 9

there was no mcr plasmid co-occurrence in LREC-141 and LREC-163, but rather the mcr-1.1 and mcr-4.2 genes were located in independent plasmids (IncHI2 and ColE10-like types, respectively). The mcr location remained undetermined for four isolates.

Since the mcr-1 plasmid gene was first described (Liu et al., 2016), it has been identified in members of the Enterobacteriaceae family encoded in different plasmid types, including IncI2, IncX4, IncHI1, IncHI2, IncFI, IncFII, IncP, IncK (Sun et al., 2018). Different authors corroborate that large conjugative plasmids of types IncHI2, IncX4 and IncI2 would be the maximum responsible for the dissemination of the mcr-1 gene among E. coli isolates from different sources and geographical locations (Doumith et al., 2016; Li et al., 2017; Manageiro et al., 2019). To date, other mcr genes (2–9) have been described (Carroll et al., 2019); among them, the mcr-4 and mcr-5 genes appear mostly encoded in small and non-conjugative ColE-like type plasmids (Sun et al., 2018). Here we found similar results, since mcr-1.1 genes were located mainly on IncHI2 and IncX4 types, and mcr-4 on ColE10-like plasmids. It is of note that the mcr-5.1 gene, predicted in LREC-152 and LREC-177, was linked to a Kp13-like plasmid (CP003996.1), location previously described by Hammerl et al. (2018) for one mcr-5 isolate recovered from a fecal pig sample at farm. Chromosomally-encoded mcr-1 location remains rare, however, it was described soon after the discovery of this plasmid-borne gene (Falgenhauer et al., 2016; Veldman et al., 2016). Here, we determined chromosomal location in five genomes by means of PLACNETw, and according to the predictive annotation of the mcr-contigs, the only common element flanking the mcr-1 was a putative ORF, pap2, which is part of the Tn6360 and encodes a Pap2 superfamily protein. Thus, Pap2 was detected in LREC-145, LREC-148, LREC-149, and LREC-164, while the ISApI1 element typically associated with the initial mobilization of mcr-1, was missing within the five contigs (Snesrud et al., 2018).

Overall, our findings are in accordance with those reported by Magistrali et al. (2018) on 13 mcr-positive E. coli isolated from swine colibacillosis in Belgium, Italy and Spain. Both studies show common features in relation to a prevalent clonal group (CC10), serotypes (O108:H39, O138:H10, O139:H1, O141:H4), and mcr-plasmid types. The confirmation of these similarities are of interest for the global design of alternatives to antibiotics that would curb the dissemination of specific clones in the pig farming.

The in vitro analysis of resistances showed that 30 of the 35 E. coli were multidrug-resistant (MDR) according to Magiorakos et al. (2012) definition (**Table 1**). Phenotypic results corresponded broadly to those predicted by ResFinder (**Supplementary Table S1**) as detailed below.

The quinolones/fluoroquinolones (FQ), together with polymyxins and 3rd–4th-generation cephalosporins, all are included in Category B of restricted antimicrobials in the EMA categorization, considering that the risk to public health resulting from its veterinary use needs to be mitigated by specific restriction (EMA/CVMP/CHMP, 2019). Two major mechanisms are implicated in the resistance to FQ, namely, mutations in the genes for DNA gyrase and topoisomerase IV, and decreased intracellular drug accumulation. In addition, plasmid-mediated quinolone resistances also play a role but usually conferring lowlevel FQ resistance (van Duijkeren et al., 2018). Phenotypically, 17 of the 35 isolates showed resistance to both nalidixic acid and ciprofloxacin, and other eight resistance to nalidixic acid only (**Supplementary Table S1**). In the majority of cases, phenotypic results correlated with those predicted by ResFinder. Particularly, 28 of the 35 genomes carried the gyrA S83L substitution, with 12 of those 28 showing double-serine mutations (gyrA S83L and parC S80I). An additional substitution (gyrA D87N) was detected in two of the 12 gyrA S83L/parC S80I genomes. Thus, nalidixic acid resistance in vitro corresponded to one single substitution (gyrA S83L), and FQ resistance to double or triple substitutions (gyrA S83L/parC S80I/gyrA D87N). Plasmid-mediated quinolone resistant genes acc(60 )-Ib-cr and qnrS1 were also present together with chromosomal mutations in LREC-147 and LREC-169, respectively. Double-serine mutations in specific positions of the gyrA and parC genes have been reported as a dominant feature of MDR lineages within E. coli, S. aureus and K. pneumoniae, with favorable fitness balance linked to high levels of resistance to FQ (Fuzi et al., 2017). This finding, in 12 out of the 28 in silico predicted FQ-resistant could be conferring adaptive advantages to certain widely spread pig pathogenic clonal groups of E. coli, such as the ST10-A (CH11-24) (García et al., 2018). This hypothesis is presently assumed for ST131 and other risk clones linked to high FQ-resistance (Johnson et al., 2015; Fuzi et al., 2017).