Penicillin non-susceptible pneumococci have been listed as one of the major antimicrobial resistance threats among bacterial pathogens (CDC, 2013; WHO, 2017). Although they represent a global public health threat, occurrence and epidemiology of PNSP vary according to the geographic region. Taken our results into consideration, from the mid 1990s until 2010, serotype 14 played a major role in the dispersion of penicillin nonsusceptibility, especially among IPD isolates. Indeed, it was previously shown that an internationally disseminated clone belonging to this serotype (namely ST156), which was also frequently associated with IPD worldwide, was the main reason for PNSP emergence in Brazil in the pre-vaccination era (Barroso

TABLE 5 | Distribution of penicillin minimum inhibitory concentration (MIC) levels among Streptococcus pneumoniae, according to the period of time and clinical source.

aCarriage isolates included strains recovered from nasopharynx or oropharynx specimens while IPD (invasive pneumococcal disease) isolates included those recovered from blood or cerebrospinal fluid.

FIGURE 4 | Distribution over time of capsular types included in the 10-valent pneumococcal conjugate vaccine (PCV10; in green), of those included only in the 13-valent pneumococcal conjugate vaccine (PCV13; in yellow) and of those not included in any PCV currently available (in black) among penicillin non-susceptible pneumococci (PNSP).

et al., 2012; Pinto et al., 2016). After 2010, however, this scenario has changed and a more diversified panel of serotypes has been associated with penicillin non-susceptibility, regardless of clinical source. Among IPD isolates specifically, serotype 19A PNSP emerged significantly, surpassing the previous number of serotype 14 PNSP isolates.

Major limitations of this study are related to the characteristics of the population included. It is known that age of individuals is an important feature and may have an influence on serotype distribution. However, we were not able to assess this issue in detail since information was not available for a large proportion of the isolates analyzed, although we estimate from available data that most of strains were recovered from children. In addition, although Brazil is a country with continental dimensions and, thus, might present discrepancies between regions, the Southeastern region, represented here by five different cities, is the most populated one. According to the last official demographic survey conducted in Brazil (Instituto Brasileiro de Geografia e Estatística [IBGE], 2010), population living in the Southeastern region accounted for nearly half of the whole Brazilian population. Moreover, this region can be considered as representative of the ethnic, social, and economic diversity of the Brazilian population due to the historic high flow of domestic in-migration.

Penicillin non-susceptible pneumococci evolution can be driven by different interventions such as antibiotic therapy policies and vaccine implementation (Guillemot et al., 1998; McCormick et al., 2003; Kim et al., 2016). These aspects usually differ by country; for example, Brazil is one of the 32 countries that have adopted PCV10 in the national immunization program instead of PCV7/PCV13, adopted by other 98 countries (Brazil Ministry of Health, 2010). Therefore, gathering information on PNSP epidemiology over a long period of time can contribute to a better understanding of their evolution and the impact of different vaccination strategies. Overall, our results show the emergence of non-PCV10 serotypes after 2010 in Brazil and the emergence and spread of PNSP associated with carriage. On the other hand, PCV10 has been effective in decreasing PNSP rates and levels among IPD isolates, but it has not avoided serotype replacement. These results reinforce the need of continuous surveillance of PNSP in the postvaccine introduction era and may contribute to the development

#### REFERENCES

of more effective measures to control the spread of drugresistant pneumococci.

#### AUTHOR CONTRIBUTIONS

JP and LT coordinated the study. TP, LT, and JP contributed to the conception and design of the work. TP, FN, AS, LO, NC, CM-S, and LC performed the experiments and analyzed the data. LO performed the statistical analyses. TP, LO, JP, and LT wrote the manuscript. All authors revised and approved the final version of the manuscript.

## FUNDING

This study was supported in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)- Finance Code 001, Instituto Nacional de Pesquisa em Resistência Antimicrobiana (INPRA), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

#### ACKNOWLEDGMENTS

We thank Filomena Soares Pereira da Rocha and Jaqueline Martins Morais for technical support.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.00486/full#supplementary-material

http://www.who.int/medicines/publications/WHO-PPL-Short\_Summary\_ 25Feb-ET\_NM\_WHO.pdf

Zhou, J. Y., Isaacson-Schmid, M., Utterson, E. C., Todd, E. M., McFarland, M., Sivapalan, J., et al. (2015). Prevalence of nasopharyngeal pneumococcal colonization in children and antimicrobial susceptibility profiles of carriage isolates. Int. J. Infect. Dis. 39, 50–52. doi: 10.1016/j.ijid.2015.08.010

**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 Pinto, Neves, Souza, Oliveira, Costa, Castro, Mendonça-Souza, Peralta and Teixeira. 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.

# IncX4 Plasmid Carrying the New mcr-1.9 Gene Variant in a CTX-M-8-Producing Escherichia coli Isolate Recovered From Swine

Vera Manageiro1,2, Lurdes Clemente3 , Raquel Romão1 , Catarina Silva4 , Luís Vieira4 , Eugénia Ferreira1,2 and Manuela Caniça1,2 \*

1 National Reference Laboratory of Antibiotic Resistances and Healthcare Associated Infections, Department of Infectious Diseases, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal, 2 Centre for the Studies of Animal Science, Institute of Agrarian and Agri-Food Sciences and Technologies, University of Porto, Porto, Portugal, 3 Bacteriology and Mycology Laboratory, INIAV – National Institute of Agrarian and Veterinary Research, Oeiras, Portugal, 4 Innovation and Technology Unit, Department of Human Genetics, National Institute of Health Dr. Ricardo Jorge, Lisbon, Portugal

#### Edited by:

Carlos Lodeiro, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Portugal

#### Reviewed by:

María de Toro, Centro de Investigación Biomédica de La Rioja, Spain Zhi Ruan, Zhejiang University, China

\*Correspondence: Manuela Caniça<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 21 September 2018 Accepted: 12 February 2019 Published: 14 March 2019

#### Citation:

Manageiro V, Clemente L, Romão R, Silva C, Vieira L, Ferreira E and Caniça M (2019) IncX4 Plasmid Carrying the New mcr-1.9 Gene Variant in a CTX-M-8-Producing Escherichia coli Isolate Recovered From Swine. Front. Microbiol. 10:367. doi: 10.3389/fmicb.2019.00367 We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus blaCTX-M-8, a blaESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCRencoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.

Keywords: MCR-1.9, plasmid-mediated colistin resistance, IncX4, CTX-M-8, Portugal

## INTRODUCTION

Since the report of a plasmid-mediated colistin resistance (PMCR) mechanism, designated MCR-1, in Escherichia coli and Klebsiella pneumoniae isolated from animals, food and humans in China, further reports exposed the global dissemination of mcr-type gene in various bacterial species isolated from a wide range of different sources (Caniaux et al., 2017). In Portugal, PMCR has also

been detected in a wide range of different sources and species, including humans, food-producing animals and meat, and in the environment (Campos et al., 2016; Figueiredo et al., 2016; Jones-Dias et al., 2016; Kieffer et al., 2017; Manageiro et al., 2017; Tacão et al., 2017; Mendes et al., 2018). Noteworthy, are the recent report of two cases presumably associated with the travel of patients from Portugal, one involving animals: a patient repatriated to France after hospitalization for 2 months in Portugal, in 2015 (Beyrouthy et al., 2017), and a New York state patient returning from Portugal in 2016 after staying on a farm with chickens and pigs (Gilrane et al., 2017).

More worrisome is the presence of mcr genes in Enterobacteriaceae carrying other resistance determinants namely, extended-spectrum β-lactamases (ESBL)- and/or carbapenemase-encoding genes. Since the first report of colocalization of mcr-1 and ESBL- in 2016 in bovines in France, an increase encoding genes in the proportion of mcr-1 genes among ESBL-producing E. coli in animals has been noticed, suggesting that the use of extended-spectrum cephalosporins may have simultaneously favored the spread of mcr-1 (Haenni et al., 2016). Here we describe the first detection of a novel mcr variant, hereafter-named mcr-1.9, identified in a commensal E. coli LV23529 isolated from a swine cecum sample collected at a slaughter, in Portugal.

## MATERIALS AND METHODS

#### Bacterial Isolate

Escherichia coli LV23529 was isolated in 2015 from a swine cecum sample collected at a Portuguese slaughter, during an evaluation study of commensal E. coli recovered from swine samples for antimicrobial susceptibility testing.

#### Antimicrobial Susceptibility Testing

Minimum inhibitory concentrations (MICs) were determined by microdilution method as previously described (Manageiro et al., 2017). In order to assess decreased susceptibility of the strain, interpretation of the results was done according to the epidemiological cut-off values recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST1 ).

## Screening and Characterization of PMCR- and ESBL-Resistance Mechanisms

#### Molecular Detection of mcr-1 and blaESBL-Encoding Genes

Following phenotypic characteristics, PMCR- and ESBLresistance mechanisms were searched and identified by molecular methods, as previously described (Manageiro et al., 2017).

#### Transfer Experiments

Conjugation experiments were performed using sodium azideresistant E. coli J53 as a recipient strain. Transconjugants were selected on McConkey agar supplemented with sodium azide (150 mg/L) and either cefotaxime (2 mg/L) or colistin (2 mg/L). Plasmid DNA was extracted from E. coli LV23529 using a NucleoBond Xtra Plus kit (Macherey-Nagel), and transformed into E. coli TOP10 OneShot chemically competent cells (Invitrogen), accordingly to manufacture's protocol. E. coli transformants were selected on MacConkey agar supplemented with 2 mg/L of colistin. PCR for blaCTX-M-8 or mcr-1-type and MICs of recipients and transformants were determined as mentioned above.

#### Genetic Context of mcr-1.9 Gene

Colistin-resistant E. coli LV23529 was genotypically characterized by whole-genome sequencing (WGS), as previously described (Manageiro et al., 2017). Sequence reads were trimmed and filtered according to quality criteria, and de novo assembled into contigs by means of CLC Genomics Workbench 10.0 (Qiagen). The assembled contigs were analyzed and studied for the presence of antibiotic resistance, virulence genes and plasmid replicon types, serotype, multi-locus sequence type (ST) and fim-type, using bioinformatics tools2 . The NCBI prokaryotic genome automatic annotation pipeline (PGAAP) was used for annotation.

Plasmid sequencing was also performed on a MiSeq Illumina platform using 150 bp paired-end reads, after plasmid DNA extraction from TLV23529 (mcr-1.9) using a NucleoBond Xtra Plus kit (Macherey-Nagel), and quantification using Qubit 1.0 Fluorometer (Invitrogen), as previously described (Manageiro et al., 2017). Sequence reads were trimmed and filtered according to quality criteria, and mapped against E. coli ATCC 25922 genome (NZ\_CP009073). Unmapped reads (80.2%/total reads) were then used for plasmids structure construction by mapping assembly based on the genetic organization of the closest plasmid sequences obtained by BLASTn; this was followed by contig neighbor's prediction from assembly information using CLC Genomics Workbench 10.0 (Qiagen). NCBI Microbial genomes BLAST analysis tool3 was used to search for plasmid sequences. Plasmid alignments and ORF representations were also done using EasyFig v. 2.2.3 (Sullivan et al., 2011).

#### Genomic Epidemiological Analysis

BacWGSTdb database was used for genotyping and source tracking bacterial pathogen (Ruan and Feng, 2016).

#### Nucleotide Sequence Accession Number

The pLV23529-MCR-1.9 and pLV23529-CTX-M-8 nucleotide sequences from this study were submitted to the NCBI GenBank Database with accession numbers KY964067 and KY964068, respectively. The new mcr-1.9 nucleotide sequence was submitted with accession number KY780959.

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession SBIH00000000. The version described in this paper is version SBIH01000000.

1http://mic.eucast.org/Eucast2/

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

3https://www.ncbi.nlm.nih.gov/genome/microbes/

## RESULTS AND DISCUSSION

fmicb-10-00367 March 12, 2019 Time: 19:12 # 3

MIC results showed that LV23529 was non-wild-type to third- and fourth-generation cephalosporins (ceftazidime 2 mg/L, cefotaxime 32 mg/L, cefepime 8 mg/L) with synergy with clavulanic acid; this isolate was also non-wild-type to chloramphenicol (>128 mg/L), sulphamethoxazole (>1024 mg/L), trimethoprim (>32 mg/L), tetracycline (>64 mg/L), and colistin (4 mg/L). LV23529 remained wildtype to carbapenems, fluoroquinolones, aminoglycosides and tigecycline (**Table 1**).

Molecular characterization of the E. coli LV23529 isolate allowed the detection of blaCTX-M-8 and mcr-1-type genes.

Only the transferability of the blaCTX-M-8 gene was achieved by conjugation, with TcLV23529 (blaCTX-M-8) exhibiting the ESBL phenotype from LV23529 isolate (cefotaxime 2 mg/L, cefepime 2 mg/L) with synergy with clavulanic acid, and wildtype to colistin (≤1 mg/L) (**Table 1**). Although conjugation assays for mcr-1-type were negative, the colistin resistance determinant could be transferred to E. coli TOP10 competent cells; transformant TLV23529 (mcr-1-type) showed the respective resistance to colistin (4 mg/L) (**Table 1**).

The WGS assembly of E. coli LV23529 yielded 193 contigs (average 143.7-fold coverage), which together comprised 5,122,415bp, showing a GC content of 50.7%. The largest contig was 320,931 bp long; the N50 statistic, which stands for the minimum contig length of at least 50% of the contigs, was 113,197 bp. The average length of the obtained contigs was 26,541 bp. Overall, the genome sequence comprised 5,124 putative genes, among which 5,037 consisted of protein encoding sequences.

The WGS analysis showed that E. coli LV23529 belongs to serotype O8:H19, usually associated with porcine stx2eproducing E. coli (Zweifel et al., 2006; Bai et al., 2015), and to MLST (Achtman scheme) ST201 [clonal complex 469 (CC469)] and to the FimH-type determinant fimH32. This ST201 was encountered worldwide mainly in isolates collected from livestock samples (Escherichia/Shigella Enterobase database, Alikhan et al., 2018). Three virulence factors were detected: astA (heat-stable enterotoxin 1), lpfA (long polar fimbriae), and gad-type (glutamate decarboxylase).

Further bioinformatics analysis of E. coli LV23529 isolate revealed acquired-genes conferring resistance to β-lactams (blaCTX-M-8 and blaTEM-1), aminoglycosides (aadA1 and aadA2),

TABLE 1 | Phenotypic and genotypic context of CTX-M-8 and MCR-1.9 producing E. coli clinical isolate, transformant, transconjugant, and the respective recipient strains.

MICs in mg/L.

aClavulanate 4 mg/L.

bE. coli LV23529 was the clinical isolate harboring the acquired antibiotic resistance genes blaCTX-M-8, mcr-1.9, cmlA1, sul3, tetA, tetM, dfrA12, aadA1, and aadA2. cE. coli TOP10 was the recipient strain in the transformation experiment.

dTLV23529 is a transformant of LV23529 (harboring mcr-1.9).

eE. coli J53AZNa was the recipient strain in the conjugation experiment.

fTcLV23529 is a transconjugant of LV23529 (harboring blaCTX-M-8).

FIGURE 1 | Linear comparison of IncX4-pLV23529-MCR-1.9 with the top six mcr-1-harboring plasmids showing the highest identities (>99.9%, E-value 0.0), in different E. coli isolates. Boxed arrows represent the position and transcriptional direction of ORFs. Gray vertical blocks indicate the shared similarity regions according to TBLASTX identity. Genes associated with pilus and plasmid transfer are colored yellow, antibiotic resistance genes are colored red, mobile genetic elements are colored pink, and other genes are colored gray (hypothetical proteins) or blue (other).

FIGURE 2 | Schematic representation of the genetic environment of mcr-1.9 in comparison with other mcr-1-type representative environments. Boxed arrows represent the position and transcriptional direction of ORFs. Genes are not drawn to scale. Genes associated with pilus and plasmid transfer are colored yellow, antibiotic resistance genes are in red, mobile genetic elements in pink, plasmid maintenance and stability genes in violet, plasmid replication associated genes are in light blue, and other genes are colored gray (hypothetical proteins) or blue (other).

classification.

TABLE 2 | Comparison of IncX4-pLV23529-MCR-1.9 with the top six mcr-1-harboring plasmids showing the highest identities (>99.9%, E-value 0.0), in different E. coli isolates.

aMLST accordingly with Warwick scheme (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli).

phenicol (cmlA1-type and floR-type), sulphamethoxazole (sul3), tetracycline [tet(A)-type and tet(M)-type], trimethoprim (dfrA12), and colistin (mcr-1-type), justifying the phenotype noticed. Additionally, several unknown mutations in the ampC (promoter region), parC, 16S rrsB, 16S rrsC, 23S and pmrB chromosomal genes were detected, the last gene being described as the primary mechanism for the development of chromosomally encoded resistance to polymyxins (Phan et al., 2017).

The named mcr-1.9, differed from mcr-1 by one-point mutation (T1238C), leading to Val413Ala substitution. The MCR-1 protein contains a transmembrane domain and a phosphoethanolamine (PEA) transferase domain with 8α, 12β, and 12η units (Gao et al., 2016). The amino acid substitution of MCR-1.9 occurred in the region between η7 e η8 of the PEA transferase domain, which have been found not to influence the function of MCR-1 (Gao et al., 2016).

The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1 (**Figure 1**), which is in accordance with other studies about mcr-1 gene (Veldman et al., 2016; Sun et al., 2017). The mcr-1.9 gene can be mobilized within an ISApl1-flanked

composite transposon (Tn6330), although many sequences have been identified without ISApl1 or with just a single copy (Snesrud et al., 2018). Indeed, it has been described that initially ISApl1 was presumably involved in the transposition of the mcr-1 cassette and then was lost, contributing for the stability of mcr gene on IncX4 plasmids (Sun et al., 2017; Snesrud et al., 2018).

The PMCR-encoding gene was found in an IncX4 plasmid (pLV23529-MCR-1.9), showing highest identities (>99.9%) with six IncX4-type mcr-1-harboring plasmids identified worldwide, in unrelated E. coli isolates, mainly collected from human patients (**Figure 2** and **Table 2**). Indeed, all belonged to different MLST, which might suggest a resistance plasmid dissemination across strains (plasmid outbreak) rather than clonal transmission of MCR-1-type-producing strains. Furthermore, no E. coli LV23529 closely related isolates were detected among those currently deposited in the public database BacWGSTdb (Ruan and Feng, 2016), which reinforce the importance of the horizontal gene transfer in this study.

Like pLV23529-MCR-1.9, the six plasmids (**Table 2**) doesn't have the ISApl1 element. Hence, similarities may suggest that the one-point mutation (T1238C) in mcr-1.9 occurred on the X4 plasmid, since mobilization of mcr-1 occurs as part of a composite transposon (Tn6330) and that structures lacking the downstream ISApl1 are not capable of mobilization (Snesrud et al., 2018). The IS26 upstream of the mcr-pap2 element is flanked by an 8bp direct repeat (**Figure 3A**), indicating that its insertion wouldn't seems to be related to the mcr-1.9 context, justifying the differences found with other IncX4 mcr-1-harboring plasmids. IncX4 plasmid has been widely implicated in the spread of MCR-1 gene in Europe (Caniaux et al., 2017). In Portugal, this plasmid type is circulating among diverse hosts (humans, pigs, poultry), being responsible for hospital-based outbreak caused by MCR-1 plus KPC-3-producing K. pneumoniae (Mendes et al., 2018), as well as for the diffusion of this PMCR at the farm level (Kieffer et al., 2017). Indeed, IncX4 plasmids seem to be efficiently transferred at different temperatures and different lackof-fitness burdens among bacterial hosts, which may facilitate the transfer of mcr-type among Enterobacteriaceae (Lo et al., 2014; Wu R. et al., 2018). The pLV23529-MCR-1.9 plasmid backbone contains all the core genes common to IncX plasmids involved in segregation, stability, replication, and conjugative transfer of the plasmid (**Figure 3A**), namely the IncX-type pilus synthesis operon (pilX1-pilX11). However, pLV23529-MCR-1.9 was mobilizable, but not self-transmissible. Of note, we found a

#### REFERENCES

one-point mutation (G64T), leading to Asp22Tyr substitution, in the PilX1, a peptidoglycan hydrolase involved in T-DNA plasmid transfer. This mutation might explain why the attempts to conjugate mcr-1.9 from E. coli LV23529 were unsuccessful (Chen et al., 2009).

Further plasmid analysis revealed the presence of two other plasmids: IncF [F2:A-:B-], IncR and the colicinogenic IncI1-ST113-carrying the blaCTX-M-8 (pLV23529-CTX-M-8, **Figure 3B**). Of note, the mcr-1.9-positive isolate, co-harboring blaCTX-M-8 and blaTEM-1 genes, is here reported for the first time in an E. coli isolate of animal origin. In fact, blaCTX-M-8 gene is rarely detected in Europe in isolates of animal origin (Börjesson et al., 2016), but in humans seems to be emerging (Eller et al., 2014). Indeed, a recent phylogenetic study suggested an increasing trend of co-existence and transmission of blaCTX-M and mcr-1 in both clinical medicine and veterinary medicine (Wu C. et al., 2018).

In conclusion, the presence of PMCR-encoding genes, such as MCR-1.9, in food-producing animals represents a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally. As mentioned, in Portugal, PMCR is an emerging problem and its international spread is a worrying reality (Beyrouthy et al., 2017; Gilrane et al., 2017).

## AUTHOR CONTRIBUTIONS

VM designed the study, performed the molecular experiments and bioinformatics analysis, interpreted the data, and wrote the manuscript. LC, RR, and EF performed the microbiological and molecular experiments. CS and LV performed the Illumina genome sequencing experiments. MC designed the study, wrote, reviewed and edited the manuscript. All authors read and approved the final manuscript.

#### FUNDING

VM was supported by Fundação para a Ciência e a Tecnologia (FCT) fellowship (Grant No. SFRH/BPD/77486/2011), financed by the European Social Funds (COMPETE-FEDER), and National Funds of the Portuguese Ministry of Education and Science (POPH-QREN). The authors thank to FCT for the project grant UID/MULTI/00211/2013.

in Salmonella enterica serotype Enteritidis phage type 34. FEMS Immunol. Med. Microbiol. 5, 274–283. doi: 10.1111/j.1574-695X.2009.00612.x

uropathogenic Escherichia coli. J. Antimicrob. Chemother. 72, 2729–2736. doi: 10.1093/jac/dkx204

**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 Manageiro, Clemente, Romão, Silva, Vieira, Ferreira and Caniça. 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.

Institute of Biology,

# Biofilm Forming Antibiotic Resistant Gram-Positive Pathogens Isolated From Surfaces on the International Space Station

Lydia-Yasmin Sobisch1 , Katja Marie Rogowski 1 , Jonathan Fuchs 2 , Wilhelm Schmieder 2 , Ankita Vaishampayan1 , Patricia Oles 1 , Natalia Novikova3 and Elisabeth Grohmann1,2 \*

Institute of Biomedical Problems (IBMP), RAS, Moscow, Russia

1 Life Sciences and Technology, Microbiology, Beuth University of Applied Sciences, Berlin, Germany, 2

University Freiburg, Freiburg, Germany, 3

#### Edited by:

José Luis Capelo, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, Portugal

#### Reviewed by:

Atte Von Wright, University of Eastern Finland, Finland Jose Ruben Morones-Ramirez, Universidad Autónoma de Nuevo León, Mexico

#### \*Correspondence:

Elisabeth Grohmann elisabeth.grohmann@ beuth-hochschule.de

#### Specialty section:

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

Received: 30 November 2018 Accepted: 01 March 2019 Published: 19 March 2019

#### Citation:

Sobisch L-Y, Rogowski KM, Fuchs J, Schmieder W, Vaishampayan A, Oles P, Novikova N and Grohmann E (2019) Biofilm Forming Antibiotic Resistant Gram-Positive Pathogens Isolated From Surfaces on the International Space Station. Front. Microbiol. 10:543. doi: 10.3389/fmicb.2019.00543 The International Space Station (ISS) is a closed habitat in a uniquely extreme and hostile environment. Due to these special conditions, the human microflora can undergo unusual changes and may represent health risks for the crew. To address this problem, we investigated the antimicrobial activity of AGXX®, a novel surface coating consisting of micro-galvanic elements of silver and ruthenium along with examining the activity of a conventional silver coating. The antimicrobial materials were exposed on the ISS for 6, 12, and 19 months each at a place frequently visited by the crew. Bacteria that survived on the antimicrobial coatings [AGXX® and silver (Ag)] or the uncoated stainless steel carrier (V2A, control material) were recovered, phylogenetically affiliated and characterized in terms of antibiotic resistance (phenotype and genotype), plasmid content, biofilm formation capacity and antibiotic resistance transferability. On all three materials, surviving bacteria were dominated by Gram-positive bacteria and among those by Staphylococcus, Bacillus and Enterococcus spp. The novel antimicrobial surface coating proved to be highly effective. The conventional Ag coating showed only little antimicrobial activity. Microbial diversity increased with increasing exposure time on all three materials. The number of recovered bacteria decreased significantly from V2A to V2A-Ag to AGXX®. After 6 months exposure on the ISS no bacteria were recovered from AGXX®, after 12 months nine and after 19 months three isolates were obtained. Most Gram-positive pathogenic isolates were multidrug resistant (resistant to more than three antibiotics). Sulfamethoxazole, erythromycin and ampicillin resistance were most prevalent. An Enterococcus faecalis strain recovered from V2A steel after 12 months exposure exhibited the highest number of resistances (n = 9). The most prevalent resistance genes were ermC (erythromycin resistance) and tetK (tetracycline resistance). Average transfer frequency of erythromycin, tetracycline and gentamicin resistance from selected ISS isolates was 10−5 transconjugants/recipient. Most importantly, no serious human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) were found on any surface. Thus, the infection risk for the crew is low, especially when antimicrobial surfaces such as AGXX® are applied to surfaces prone to microbial contamination.

Keywords: antimicrobial surface, gram-positive human-pathogenic bacteria, antibiotic resistance, biofilm, conjugative transfer, International Space Station, hostile environment

## INTRODUCTION

The International Space Station is an isolated habitat in a hostile environment. Microgravity, solar and cosmic radiation alter the immune-regulatory responses of the crew rendering them more susceptible to bacterial infections (Sonnenfeld, 2005; Crucian et al., 2008; Guéguinou et al., 2009). The microorganisms in the spaceship are human-derived; they originate from the crew and helpers who prepare the mission. The spaceship provides a special environmental niche for microorganisms, which directly or indirectly affect the health, safety or performance of the crew (Taylor, 2015). Microgravity can affect the virulence (Nickerson et al., 2004; Wilson et al., 2007; Rosenzweig et al., 2010; Crabbé et al., 2011), growth kinetics (Klaus et al., 1997; Kacena et al., 1999; Nickerson et al., 2004) and biofilm formation of microorganisms (Mauclaire and Egli, 2010). To assess the risk microorganisms pose to astronauts, the composition and properties of microbial communities in spaceships were analyzed. Two hundred and thirty-four bacterial and fungal species were found on the MIR space station, among those strong biofilm formers. Staphylococcus spp., followed by Bacillus spp. and Corynebacterium spp. were abundant in air as well as in surface samples (Novikova, 2004; Novikova et al., 2006). Schiwon et al. (2013) analyzed ISS samples from air and crewmembers in-flight and post-flight. Bacillus spp., Staphylococcus spp. and Enterococcus spp. were the most prevalent. 75.8% of the isolates exhibited resistance to one or more antibiotics. Corresponding resistance genes were found in 86% of the antibiotic-resistant bacteria. In 86.2% of the isolates horizontal transfer genes were detected. Eighty-three percent of the isolates were able to form biofilms (Schiwon et al., 2013).

Under spaceflight conditions, bacteria were shown to exhibit enhanced secondary metabolite and extracellular polysaccharide production as well as enhanced biofilm formation (Mauclaire and Egli, 2010; Vukanti et al., 2012). In space, the cell wall of S. aureus was significantly thicker than in the same strain grown on Earth (Novikova et al., 2006; Taylor, 2015). Various bacteria exhibited enhanced virulence, increased antibiotic resistance and differential gene expression under space conditions (Horneck et al., 2010; Yamaguchi et al., 2014; Taylor, 2015). Thus, these bacteria could spread their virulence and/or antibiotic resistance genes through horizontal gene transfer (HGT) and turn harmless bacteria into potential pathogens.

HGT is mediated by mobile genetic elements (MGEs), such as conjugative plasmids, conjugative transposons, integron-specific gene cassettes, or phages that are able to facilitate their own transfer. Plasmid-mediated HGT plays a primordial role in the emergence of new pathogens (Frost et al., 2005; Garbisu et al., 2018). Schiwon et al. (2013) found conjugative plasmids in bacterial isolates from the ISS and could demonstrate that some of these strains were able to transfer their antibiotic resistance genes to other bacteria. The HGT rate was shown to be higher in microbial biofilms than in planktonic cultures (Holmes et al., 2015). Biofilms represent a protected mode of microbial growth and confer significant survival advantages in hostile environments (Li et al., 2007; Thallinger et al., 2013). Thus, biofilm forming organisms show increased resistance to antibiotics, either due to decreased penetration of the antibiotic through the biofilm matrix or due to expression of more complex biofilm-specific resistance mechanisms.

Multiple antibiotic resistant and strong biofilm forming Staphylococcus and Enterococcus isolates detected on the ISS could pose an increased health risk on the crew (Schiwon et al., 2013). Several studies report, that bacteria from astronauts inflight were more resistant to antibiotics due to enhanced biofilm formation or changes in cell morphology, e.g., thicker cell walls than isolates obtained from the same individuals either preor post-flight. As medical aid on the ISS is restricted, there is an urgent need for new antimicrobial materials, which can be used there to prevent infections by multi-resistant biofilm forming bacteria.

Heavy metals, e.g., copper and silver, have been known for a long time to possess antimicrobial activity. Silver was officially approved as an antimicrobial agent in the twentieth century (Chopra, 2007; Schäberle and Hack, 2014; Guridi et al., 2015; Vaishampayan et al., 2018). However, after the discovery of antibiotics the use of metals to combat bacterial infections has declined (Chopra, 2007; Grass et al., 2011). Later on, due to the increased occurrence of antibiotic resistant pathogens, silver and copper have again found widespread use, both in medicine and in everyday life (Maillard and Hartemann, 2012; Warnes and Keevil, 2013; Schäberle and Hack, 2014). These metals are easy to use as coatings on a variety of substrates and have a lethal effect on bacteria and fungi via the so-called contact killing (Grass et al., 2011). Silver is one of the best-studied bactericidal agents in water supplies (Russell and Hugo, 1994; Rohr et al., 1999; Vonberg et al., 2008; Vaishampayan et al., 2018). However, as occurred with antibiotics, bacteria have also developed resistance mechanisms against silver (Gupta et al., 1999). Like the excessive use of antibiotics, the extended use of silver is questioned due to its toxicity to the environment as well as to the human body (Landsdown, 2010). Plain ruthenium is not applied as antibacterial agent, but antibacterial activity has been demonstrated for ruthenium(II) polypyridyl complexes (Bolhuis et al., 2011; Li et al., 2011, 2015).

Due to the increasing resistance of bacteria to both antibiotics and commonly used antimicrobial metals, there is an urgent need to develop new approaches to combat bacterial infections. A new antimicrobial surface coating is AGXX R consisting of microgalvanic elements of the two noble metals, silver and ruthenium, surface-conditioned with ascorbic acid (Vaishampayan et al., 2018). Both metals can be galvanically applied to diverse surfaces such as stainless steel, plastics, or cellulose fibers. The coating proved to be active against both Gram-positive and Gramnegative bacteria, but also against filamentous fungi, yeasts and some viruses (Guridi et al., 2015; Landau et al., 2017a,b; Vaishampayan et al., 2018). Recently, we demonstrated that it efficiently inhibits the growth of MRSA (Vaishampayan et al., 2018). The postulated mode of action is based on the formation of reactive oxygen species, particularly superoxide anions (Meyer, C., personal communication), which affect biomolecules, such as nucleic acids, proteins, and lipids. AGXX R has self-regenerating activity based on two coupled redox reactions taking place on the micro-galvanic silver and ruthenium elements on the surface of the material. They result in effective regeneration of the coating (Clauss-Lendzian et al., 2018).

In this study, we investigated the long-term antimicrobial effect of two different antimicrobial coatings. Three sets of V2A steel samples (uncoated, silver-coated, AGXX R -coated) were exposed and analyzed after six, 12, and 19 months on the ISS. Seventy-eight human pathogenic bacteria, which survived on the antimicrobial coatings or on the uncoated steel carrier (control) were phylogenetically affiliated and further characterized. The number of human pathogenic isolates decreased from V2A steel (n = 39) to V2A-Ag (n = 31) to V2A-AGXX R (n = 8). After 6 months of exposure, no bacteria survived on AGXX R , whereas six human pathogens were obtained after 12 and two after 19 months. From all materials, predominantly staphylococci and bacilli were isolated. Multi-antibiotic resistant, plasmid harboring staphylococcal and enterococcal ISS isolates transferred erythromycin, gentamicin and tetracycline resistance with average transfer frequencies of 10−5 transconjugants/recipient.

## MATERIALS AND METHODS

#### Preparation of Antimicrobial Metal Sheets

The material was provided by Largentec GmbH, Berlin, Germany. V2A (DIN ISO 1.4301) stainless steel sheets were used as reference material and as base material for Ag and AGXX R coatings. The coatings were prepared as described in detail in Clauss-Lendzian et al. (2018). Prior to use in the experiments, the metal sheets (coated and uncoated) were autoclaved at 121◦C for 20 min. The metal sheets had a size of 4 cm2 each and were placed on the door to the bathroom of the ISS. Three sets of test sheets, one for each time point, - time points 12 and 19 months thus representing a cumulative bacterial load—were exposed on the ISS.

#### Reference Strains

Bacterial strains used as reference in biofilm formation assays and PCRs or as recipients in mating experiments are listed in **Table 1**. Staphylococcus and Enterococcus strains were grown in Tryptic Soy Broth (TSB, Sigma-Aldrich Chemie GmbH, Munich, Germany) or Brain Heart Infusion broth (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) at 37◦C with shaking. Bacillus strains were grown in Lysogeny Broth (LB, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) at 30◦C with shaking.

#### Bacteria Isolation and Phylogenetic Affiliation

Bacteria were isolated from V2A steel surfaces (uncoated, Ag-coated, AGXX R -coated) exposed on the ISS for 6, 12 and 19 months, respectively. The bacteria were detached from the surfaces by rinsing with Phosphate Buffered Saline (PBS) followed by cultivation in Reasoner's 2A broth (R2A, Lab M Limited, Heywood, England) at 25◦ and 37◦C under shaking. Appropriate dilutions of the cultures were passaged several times onto R2A agar until pure isolates were obtained. Isolates were phylogenetically affiliated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics MALDI Biotyper system) according to the manufacturer's instructions (Bruker Daltonics). Mass spectra were compared with the MALDI-BDAL Database (Version 3.1, 7311rntries). If identification with MALDI-TOF MS failed, the isolate was sent for 16S rRNA gene sequencing (SMB Ruedersdorf, Germany). Analysis of the 16S rDNA sequences was performed with BLAST (http://blast.ncbi.nlm.nih.gov/Blast. cgi?PROGRAM=blastn&PAGE\_TYPE=BlastSearch&LINK\_

LOC=blasthome) and ChromasPro (Version 2.1.8). The isolates are denominated according to following scheme (i) the material they were isolated from, (ii) the exposure time on the ISS in months, and (iii) the order of isolation, e.g., E. faecalis V2A-12-03 was isolated from uncoated V2A steel after 12 months exposure, and it is the third isolate obtained from this material at this time-point.

#### Biofilm Screening Assay

Biofilm formation test was carried out according to Vaishampayan et al. (2018). E. faecalis T9 and S. aureus 04- 02981, both strong biofilm formers, were used as positive controls (Schiwon et al., 2013; Vaishampayan et al., 2018). For Staphylococcus spp., TSB, for E. faecalis, BHI medium was used as negative control (Schiwon et al., 2013). Biofilm formation was measured in EnSpire Multimode Plate Reader 2300-0000 (Perkin Elmer, Turku, Finland) at 570 nm (OD570). The assays were performed in triplicates. Normalized biofilm formation was calculated by dividing the biofilm measure at OD570 by the bacterial growth at OD600. Biofilm classification criteria were applied according to Nyenje et al. (2013).

## Antibiotic Disc Diffusion Method

Antibiotic resistance of the isolates toward 15 different antibiotics was analyzed with the disc diffusion method (discs from Oxoid, Wesel, Germany) on Mueller Hinton agar (Sifin diagnostic GmbH, Berlin, Germany) according to the guidelines of the Clinical and Laboratory Standards Institute, (CLSI, 2013). Details are given in **Table 2**. Each test was performed in triplicates. For sulfamethoxazole (RL25), no comparable data were found for Staphylococci, Enterococci and Bacilli. Thus, isolates lacking an inhibition zone were classified as resistant, those without inhibition zone were classified as susceptible.

TABLE 1 | Bacterial species used as references for AB-R-screening, biofilm formation, plasmid isolation, and in biparental mating.

DSMZ, German collection of microorganisms and cell cultures, Braunschweig. AB-R, antibiotic resistance; StrepR, streptomycin resistance; TetR, tetracycline resistance; GentR, gentamicin resistance; AB-R genes for resistance against ampicillin, ampC (E. cloacae DSM46348), ciprofloxacin, qnrA (E. coli Hm06-20), qnrB (K. pneumoniae K2-78), qnrS (E. coli PS84); erythromycin, ermA (S. aureus 04-02981), ermB (E. faecium SF11770), ermC (S. haemolyticus VPS617), ermD (B. subtilis BD662), ermG (B. subtilis BD1156); gentamicin, aac(6′ )- Ie-aph(2′ )-Ia (E. faecium SF11770), aph(2′ )-ib (E. faecium SF11770), aph(2′ )-ic (E. gallinarum SF9117), aph(2′ )-id (E. casseliflavus UC73); kanamycin, aadD [S. aureus SK5428 (pSK41)], aph(3′ )-III (E. faecalis RE25) against oxacillin, mecA (S. aureus DSM13661); ß-lactams, blaSHV-5 (K. pneumoniae DSM13661), blaZ (S. haemolyticus VPS617); sulfamethoxazole, sul1 (E. coli Hm06-20), sul2 (E. coli PS84), and tetracycline, tetK (S. haemolyticus VPS617), tetL (E. faecium SF11770), tetM (E. faecium SF11770), tetO (E. faecalis TU-79), tetS (L. lactis K214).

## PCR Assays

For the PCR assays, cell lysates prepared from 100 µL overnight cultures were used. Cell pellets were re-suspended in 20 µL lysis buffer (50 mM NaOH, 0.25% sodium dodecyl sulfate) and incubated at 95◦C for 20 min. Prior to use in PCR, they were diluted 1:10 with distilled water. Twenty-five microliter PCR reactions contained 0.125 µL Taq-Polymerase (5 U/µL), 2.5 µL 1x PCR buffer, 0.2µM of each primer (**Table 3**), 0.5 µL of deoxynucleoside triphosphates (200µM) and 1 µL template DNA (lysate). DNA amplifications were carried out in a Biometra T3 Thermocycler (Analytik Jena AG, Jena, Germany). The temperature profiles are given in **Supplementary Table 1**.

#### Plasmid DNA Isolation

Plasmid DNA from Staphylococci was extracted as described in Schiwon et al. (2013) with some minor modifications. After washing the plasmid DNA with 70% ethanol, 1 µL of RNase A (10µg/mL; Merck KGaA, Darmstadt) and 3 µL of Proteinase K (20 mg/mL; Merck KGaA, Darmstadt) were added, followed by 1 h incubation at room temperature. Plasmid DNA extraction from Enterococci was performed as described in (Schiwon et al., 2013).

#### Mating Assays

On basis of multiple antibiotic resistance and occurrence of plasmids >20 kbp, ISS isolates were selected as donors for biparental matings. As recipients, the methicillin resistant clinical isolate, S. aureus 04-02981 and the E. faecalis lab strain OG1X were selected. Details on all of the matings are given in **Table 4**. Overnight cultures of Staphylococci were diluted 1:5 in TSB medium, overnight cultures of Enterococci 1:5 in BHI medium containing the appropriate antibiotics (**Table 4**) and grown until OD600 = 0.5. Donors and recipients were washed with PBS prior to mixing in 1:10 ratio, spotted onto a TSA plate

#### TABLE 2 | Antibiotic disc diffusion method.

For Enterococcus spp., inhibition zones from DA10, E5, K5, KF30, OX5 and for Bacillus spp., inhibition zones from DA10, DO30, K5, KF30, MEM10, OX5, TCG15, TE10 were evaluated as for Staphylococcus spp.

a http://www.liofilchem.net/antibioticdisc/.

for Staphylococcus recipients, on a BHI plate for Enterococcus recipients and incubated for 16 h at 37◦C. Cells were recovered in 1 mL PBS, serial dilutions were incubated at 37◦C on TSA/BHI plates for 16 h to enumerate transconjugants. The number of recipients was also determined after 16 h at 37◦C. Transfer frequencies are given as number of transconjugants/recipient.

#### RESULTS

#### Bacterial Isolates From V2A, V2A-Ag and V2A-AGXX® Surfaces

A total number of 112 bacterial isolates were recovered from the different materials after the three time intervals (6, 12, and 19 months). 73.6% of the isolates are human pathogens. All isolates were identified to species level by MALDI-TOF biotyping or 16S rRNA gene sequencing. In total, 49 isolates were obtained after 6 months, 51 after 12 months and 22 after 19 months exposure of the antimicrobial materials on the ISS. The non-human pathogenic bacteria include Bacillus spp. (n = 20; B. astrophaeus, B. infantis, B. korlensis, B. licheniformis, B. megaterium, B. niacini, B. pumilus, B. tequilensis, and B. thuringiensis), Enhydrobacter aerosaccus (n = 2), Micrococcus yunannensis (n = 1), Paenibacillus polymyxa (n = 1), Pseudomonas psychrotolerans (n = 1), and Staphylococcus capitis (n = 9). To assess the infection risk for the crew, only the human-pathogenic bacteria (n = 78) were characterized in terms of biofilm formation and antibiotic resistance profile. Three Moraxella osloensis strains obtained from V2A (n = 1) and V2A-Ag (n = 2) after 19 months were the only Gramnegative human-pathogenic bacteria. Seventy-five Gram-positive human pathogenic bacteria were selected for the study: 32 from 6 months, 21 from 12 months, and 22 isolates from 19 months exposure.

The longer the exposure time of the three materials, the higher was the bacterial diversity on the materials (**Figure 1** and **Table 5**). All pathogenic isolates recovered from V2A and V2A-Ag after six months belonged to the genus Staphylococcus. No bacteria were recovered from AGXX R after 6 months. In total, 17 Staphylococci and three E. faecalis were detected after 12 months: Seven Staphylococci and one E. faecalis strain from V2A, six Staphylococci from V2A-Ag and four Staphylococci and two E. faecalis strains from AGXX R . After 19 months, seven Staphylococci and seven B. cereus strains were recovered from V2A and three Staphylococci and three B. cereus strains from V2A-Ag. Only one B. cereus and one S. epidermidis strain were isolated from AGXX R after 19 months exposure. In summary, a considerably lower bacterial number survived on AGXX R than on the other two surfaces. Nevertheless, the silver coating also showed a slight antimicrobial effect.

#### Biofilm Formation of Pathogenic ISS-Isolates

Biofilm formation of pathogenic isolates was determined by crystal violet staining, biofilms were classified according to Nyenje et al. (2013). The data are summarized in **Table 5**. Twenty-six V2A-isolates showed strong (66.7% of all pathogenic isolates from V2A-steel), ten moderate (25.5%) and three weak (7.8%) biofilm formation. Twenty-one isolates from V2A-Ag were strong biofilm formers (91.3% of all pathogenic isolates from V2-Ag), one isolate showed moderate (4.3%), one isolate weak (4.3%) biofilm formation. Of the eight AGXX R -isolates, six had strong (75.0% of all pathogenic isolates from V2A-AGXX R ) and two (25%) weak biofilm formation ability. Interestingly, 43 Staphylococci (52 pathogenic Staphylococci in total) formed strong biofilms (82.7%), eight Staphylococci (15.4%) were moderate biofilm formers and one Staphylococcus isolate (1.9%)

 genes.

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formed only weak biofilms. Of the B. cereus isolates (11 in total), one showed strong (9.1%), three moderate (27.3%), and seven (63.6%) showed only weak biofilm formation capacity. In contrast, all three E. faecalis isolates were classified as strong biofilm formers.

#### Prevalence of Antibiotic Resistances in the Pathogenic Isolates

Antibiotic sensitivity testing of the isolates showed that 32.0% of the pathogenic isolates were resistant to <3 of the tested antibiotics (15 antibiotics in total were tested), 68.0% were resistant to three or more antibiotics. Eighteen isolates had three antibiotic resistances (24.0% of the isolates), 23 isolates were resistant to four antibiotics (30.7% of the isolates), six isolates were resistant to five antibiotics (8.0%) and three isolates had six different antibiotic resistances (4.0%). E. faecalis V2A-12-03 (from V2A steel after 12 months) had the highest number of resistances. It was resistant to nine different antibiotics, chloramphenicol, gentamicin, clindamycin, doxycycline, erythromycin, kanamycin, meropenem, sulfamethoxazole, and tetracycline.

In total, 97.3% of the pathogenic Gram-positive isolates were resistant to 25 µg sulfamethoxazole, 74.7% were resistant to 15 µg erythromycin and 61.3% were resistant to 10 µg ampicillin. Interestingly, these resistances were found with similar prevalence on all three surfaces, irrespective of the exposure time. No oxacillin resistant Staphylococcus was detected, whereas all B. cereus isolates (all of the 11 isolates after 19 months) were resistant to oxacillin. One B. cereus (V2A-AG-19-10) isolate showed resistances against six different antibiotics (AMP10, C30, E15, K5, OX5, RL25).

None of the isolates was resistant to vancomycin or cephalothin. Two E. faecalis (V2A-AGXX-12-02,-03) isolates were resistant to six antibiotics (CN10, DA10, E15, K5, RL25, TE10) and one E. faecalis isolate (V2A-12-03) was resistant to nine antibiotics (C30, CN10, DA10, DO30, E15, K5, MEM10, RL25, TE10). Meropenem resistance was detected in three strains, E. faecalis V2A-12-03, S. hominis V2A-12-04, and S. hominis V2A-AG-12-05.

To identify the resistance genes in the isolates resistant to three or more antibiotics (68.0% of the pathogenic isolates), gene-specific PCRs were performed. Gentamicin [aac6-aph2a (n = 1), aph(2)-ic (n = 2)], kanamycin [aadD (n = 4), aph3- III (n = 5)], erythromycin [ermC (n = 19), ermB (n = 1)], and tetracycline [tetK (n = 9), tetL (n = 1), tetM (n = 1)] resistance genes were detected in the number of isolates indicated in parentheses (**Table 5**). ermC and tetK were the most prevalent resistance genes. No sulfamethoxazole (sul1, sul2) resistance gene was found in any of the isolates.

#### Plasmid Profiles of ISS-Isolates

Exemplarily, plasmid DNA profiles of 20 out of total 45 staphylococcal isolates resistant to three or more antibiotics forming moderate or strong biofilms were obtained (**Table 5**). All isolates contained plasmids <20 kbp, the number of plasmid bands varied from one to seven. Interestingly, 17 isolates harbored plasmids >20 kbp likely able to self-transfer. Plasmid

CIP, Ciprofloxacin; CN, gentamicin; E, erythromycin; SM, streptomycin; TE, tetracycline. (-) no transconjugants were obtained. The concentrations of the antibiotics are given in µg/mL.

FIGURE 1 | Number of Gram-positive pathogenic bacteria recovered from the different materials (V2A, V2A-Ag, V2A-AGXX®), after 6 months (A), 12 months (B), and 19 months (C) exposure on the ISS. In black, Staphylococcus spp.; gray, E. faecalis; white, B. cereus.

#### TABLE 5 | Characteristics of all isolates from V2A, V2A-Ag, V2A-AGXX® after 6, 12, and 19 months.

AB-R, antibiotic resistance; + + +, strong biofilm formation; ++, moderate biofilm formation; +, weak biofilm formation; AMP, ampicillin; C, chloramphenicol; CN, gentamicin; DA, clindamycin; DO, doxycycline; E, erythromycin; K, kanamycin; MEM, meropenem; OX, oxacillin; RL, sulfamethoxazole; TCG, tigecycline; TE, tetracycline. The concentration of the antibiotics is given in µg/mL. ermC, ermB, erythromycin resistance genes; aac6-aph2a, aph(2)-ic, gentamicin resistance genes; blaSHV-5, ß-lactam antibiotic resistance gene; aadD, aph3-III, kanamycin resistance genes; tetK, tetM, tetL, tetO, tetracycline resistance genes. n.d., not determined.

DNA profiles were also obtained from the three E. faecalis isolates; all of them were multi-drug resistant and strong biofilm formers. All, E. faecalis V2A 12-03, E. faecalis V2A-AGXX-12-02 and E. faecalis V2A-AGXX-12-03 harbored putative conjugative plasmids >20 kbp. Interestingly, E. faecalis V2A 12-03 showed additionally three small plasmid bands in the size range between 3 and 1.5 kbp.

#### Mating Experiments

Antibiotic resistance transfer of selected ISS-isolates was studied in biparental matings (Laverde et al., 2017). Isolates resistant to tetracycline, gentamicin or erythromycin and harboring a plasmid >20 kbp were selected as donors, plasmid-free S. aureus 04-02981 and E. faecalis OG1X were used as recipients. The results of all of the matings are summarized in **Table 4**. Gentamicin resistance transfer to S. aureus 04-02981 was successful from E. faecalis V2A-12-03 (aac6-aph2a-encoded gentamicin resistance) with a transfer frequency of 8.3 × 10−4 transconjugants/recipient and from E. faecalis V2A-AGXX-12- 03 (aph(2)-ic-encoded gentamicin resistance) with a transfer frequency of 9.2 × 10−7 transconjugants/recipient.

Erythromycin resistance transfer of six Staphylococcus donors harboring the ermC resistance gene and of three Staphylococcus donors harboring an unknown erythromycin resistance gene to E. faecalis OG1X was successful with transfer frequencies in the range of 1.1 × 10−6 to 4.2 × 10−4 transconjugants/recipient. Tetracycline resistance transfer from four S. hominis strains and two S. haemolyticus strains to S. aureus 04-02981 was successful. Three of the staphylococci harbored only the tetK resistance gene, one only tetO. One S. hominis strain harbored tetK and tetO, while another harbored the resistance genes tetK and tetL. Tetracycline resistance transfer frequencies varied considerably ranging from 3.3 × 10−8 to 6.8 × 10−4 transconjugants/recipient.

Ten out of the 17 successful matings were randomly chosen for plasmid DNA isolation of the transconjugants. In nine of the ten matings large plasmid bands comparable in size to those of the donors were detected in the transconjugants (data not shown).

## DISCUSSION

We proved that the novel antimicrobial coating AGXX R strongly reduced the bacterial load on surfaces on the ISS particularly prone to microbial contamination. However, over time with exposure times >6 months—some nosocomial pathogens survived even on the novel antimicrobial coating. Moreover, an interesting shift in the composition of the microbial communities was observed over time.

#### Bacterial Survivors Isolated From V2A, V2A-Ag and V2A-AGXX® Surfaces

The bacterial community isolated from the surfaces was always dominated by Staphylococcus spp. (63.4% of 112 isolates) and Bacillus spp. (24.1%) irrespective of the exposure time. 46.4% of the Staphylococci are affiliated to the coagulase-negative Staphylococci, including pathogens such as S. epidermidis, S. lugdunensis, S. haemolyticus, S. hominis, and S. caprae. Coagulase-positive Staphylococci such as S. aureus (8.9% of all isolates) were only found on V2A and V2A-Ag surfaces after 6 months exposure. B. cereus (9.8% of all isolates) was the only pathogenic Bacillus. Only three E. faecalis (2.7% of all isolates) were recovered from V2A and V2A-AGXX R surfaces after 12 months. Schiwon et al. reported that predominantly S. hominis, S. aureus, and S. epidermidis were detected on crewmembers and in air-filters on the ISS (Schiwon et al., 2013). S. hominis and S. epidermidis were the most prevalent Staphylococci associated with debris collected from the crew's quarters on the ISS (Venkateswaran et al. (2014). In addition, 13 E. faecalis and eight B. cereus strains were isolated from the crew and air-filters on the ISS (Schiwon et al., 2013). Taking the data of this study and others together (Van Houdt et al., 2012; Schiwon et al., 2013; Venkateswaran et al., 2014; Mayer et al., 2016) it can be concluded that the bacteria that survived on the different surfaces were predominantly human-associated.

Microbial diversity on the test materials increased over time. After 6 months only Staphylococci and Bacilli were found, after 12 months Staphylococci, Bacilli, E. faecalis and one P. polymyxa strain were isolated while after 19 months, Staphylococci, Bacilli, E. aerosaccus, M. osloensis, M. yunnanensis, and P. psychrotolerans were recovered. Novikova (2004) reported a similar diversity on surfaces on the MIR station including Staphylococci, Bacilli, Micrococcus, Moraxella, and Pseudomonas.

A decline of the number of Gram-positive human-pathogens recovered from V2A (n = 39) to V2A-Ag (n = 28) to V2A-AGXX R (n = 8) was observed. In total, only 12 bacteria were recovered from AGXX R -coated surfaces after 12 and 19 months exposure. AGXX R showed a pronounced antimicrobial effect, it reduced the microbial load by 79.5%. Silver also had a slight antimicrobial effect, it reduced the microbial load by 28.2%.

The antimicrobial test-materials are static surfaces, where dead cells, dust particles and cell debris can deposit. These deposits might interfere with the direct contact between the antimicrobial surface and the bacteria, which is required for effective antimicrobial activity of contact catalysts, such as Ag and AGXX R . Over time the deposits might have grown in size and thickness resulting in increasing interference with the antimicrobial activity. Possibly, this effect could explain that after 6 months no bacteria were recovered from AGXX R , whereas with prolonged exposure time a few bacteria escaped the antimicrobial action.

#### Strong Biofilm Forming ISS Isolates

Biofilms provide microbes shelter from antimicrobials and the host immune system (Foulquié Moreno et al., 2006; Chen and Wen, 2011; Rafii, 2015; Qi et al., 2016; Hall and Mah, 2017). Bacterial biofilms have been associated with diseases such as cystic fibrosis, periodontitis, and nosocomial infections on catheters and prosthetic heart valves (Storti et al., 2005; Delle Bovi et al., 2011). Eradication of biofilms is difficult due to impaired penetration of antibiotics and the decreased host immune response. Thus, they can pose a health risk to immunosuppressed people, such as the crew on the ISS.

Most Staphylococcus and all Enterococcus isolates from this study formed strong biofilms. B. cereus isolates were more diverse in terms of biofilm formation: Seven isolates produced a weak, three a moderate and only one produced a strong biofilm. The fact that all bacterial isolates were able to form biofilms could be due to the long exposure to adverse space conditions.

### Prevalence of Antibiotic Resistances in Human Pathogenic Isolates

Astronauts have a suppressed immune response in-flight and as a consequence they are more susceptible to bacterial infections (Van Houdt et al., 2012; Taylor, 2015). The potential infection by pathogenic Staphylococci and Enterococci increases with duration of the mission (Schiwon et al., 2013). Therefore, treatability of bacterial infections on the ISS and on even longer space missions with limited amounts of antimicrobial drugs available is a health concern which has to be tackled.

In this study, all Gram-positive pathogenic isolates were resistant to at least one antibiotic. 68.0%, mostly Staphylococci, were multidrug resistant (resistant to more than three antibiotics). After 12 months exposure, also multi-resistant Enterococci occurred, one E. faecalis strain from V2A steel and two E. faecalis strains from V2A-AGXX R . E. faecalis V2A-12-03 had with nine resistances the largest number of resistances.

In total, the isolates were tested against 15 different antibiotics. Seven different antibiotic resistances were found after 6 months, 13 after 12 months and after 19 months, the number of resistances equalled the number after 6 months. This could be partly due to the fact, that the number of resistances in the Staphylococci declined after 19 months (most isolates had only one or two resistances), while Bacillus strains with more than three resistances came up.

All Staphylococci had similar antibiotic resistance profiles. The B. cereus isolates after 19 months exposure also showed similar resistance profiles. Most Bacilli and Staphylococci were resistant to ampicillin and erythromycin. Gentamicin resistance only occurred in E. faecalis isolates. Interestingly, all of them were also resistant to kanamycin. E. faecalis strains are known to be intrinsically resistant to low-level aminoglycosides (gentamicin, kanamycin) or have acquired high-level aminoglycoside resistance e.g., by uptake of aac6 aph2a or aph(2)-ic(Chow, 2000; Wendelbo et al., 2003; Dadfarma et al., 2013). As E. faecalis V2A-12-03 encodes aac6-aph2a and E. faecalis V2A-AGXX-12-02 and−03 encode aph(2)-ic, they are likely high level gentamicin resistant. aac6-aph2a was found on plasmids pSK41, pGO1, pLW1043, pSK1, pTEF1, on Tn4001-like transposons and on the chromosome (Schiwon, 2011). aph(2) ic was found on conjugative plasmid pYN134 (Hollenbeck and Rice, 2012) in E. gallinarum but was shown to readily transfer to E. faecalis (Chow et al., 1997). Therefore, it is likely that gentamicin resistance spreads via these conjugative plasmids (Chow et al., 1997).

Most ISS-isolates were resistant to sulfamethoxazole, which interferes with bacterial synthesis of folic acid. It could be speculated that changes in the thickness of the cell wall due to exposure to space conditions might be involved in resistance to sulfamethoxazole by inhibiting the uptake of the antibiotic.

Most abundant resistance genes in the ISS-isolates were ermC and tetK coding for erythromycin and tetracycline resistance, respectively. Both genes are plasmid-borne and have been detected in Staphylococci of human origin (Schiwon et al., 2013). ermC was found on pSK41-like conjugative plasmid pUSA03 isolated from the community-acquired MRSA strain USA300 (Grohmann et al., 2003; Smillie et al., 2010; Schiwon et al., 2013). A pSK41-like plasmid could have spread ermC among the S. aureus strains V2A-6-13 and V2A-6-16, and between V2A-AG-6-03 and V2A-AG-6-04 isolated from the same material. Indeed, from S. aureus V2A-6-16 a plasmid >20 kbp was isolated. ermB is another plasmid-encoded erythromycin resistance gene. It is one of the 33 erythromycin resistance genes found in Staphylococci (Schiwon et al., 2013). However, ermB is not abundant in Staphylococci. No ISS-isolate from crew and airfilters harbored ermB (Zmantar et al., 2011; Schiwon et al., 2013). Also in this study, only E. faecalis V2A-AGXX-12-03 encoded ermB. De Leener et al. (2005) reported that ermB is present on Tn1545-like elements and that is likely associated with the occurrence of the tetracycline-resistance gene tetM. Interestingly, E. faecalis V2A-AGXX-12-03 harbored tetM along with ermB.

tetK is found on small mobilizable plasmids, which can be integrated into the Staphylococcus chromosome or into larger staphylococcal plasmids (Gillespie et al., 1987; Needham et al., 1994; Roberts, 2005). tetO and tetK can be found on pT181 like small mobilizable plasmids (Khan and Novick, 1983; Chopra and Roberts, 2001). S. hominis V2A-AGXX-12-01 (tetK, tetO) and S. haemolyticus V2A-AGXX-12-05 (tetK) likely carry pT181 like plasmids as small plasmid bands in the range of 2000–6000 bp were observed on the gel (data not shown). Both strains were isolated from the same material after the same time-period. Thus, the resistance genes might have spread via HGT among them. Along with tetK, pT181-like plasmids can carry tetL as well (Chopra and Roberts, 2001). Both genes were found in S. hominis V2A-AGXX-12-01.

Kanamycin resistance occurred both in Staphylococci and Enterococci. The kanamycin-resistance gene aph3-III was found in S. hominis V2A-12-04, S. hominis V2A-AG-12- 05 and E. faecalis V2A-12-03, E. faecalis V2A-AGXX-12-02, and E. faecalis V2A-AGXX-12-03, all isolated from V2A and the two antimicrobial surfaces after 12 months. aph3-III is located on transposons of the Tn916-Tn1545 type encoding a broad spectrum of resistances, toward tetracycline, macrolides, lincosamides, streptogramins, and kanamycin (Fons et al., 1997; Soge et al., 2008; Roberts and Mullany, 2011). The kanamycinresistance gene aadD was detected in S. hominis V2A-AG-12-03, V2A-AG-12-05 and in E. faecalis V2A-AGXX-12-02 and V2A-AGXX-12-03. aadD is encoded on S. aureus plasmid pUB110 (4548 bp) (McKenzie et al., 1986; Allignet et al., 1998). As the two S. hominis and two E. faecalis strains were isolated from the same materials, V2A-Ag and V2A-AGXX R , respectively, transfer of the aadD gene might have taken place. S. hominis V2A-AG-12-05 showed plasmid-bands in the range of 2000-3000 bp and around 7000 bp likely indicating the presence of pUB110-like plasmids (data not shown). Occurrence of aph3-III and aadD genes in Staphylococcus and Enterococcus isolates from the ISS has already been reported (Schiwon et al., 2013).

### Antibiotic Resistance Transfer of the ISS-Isolates

Plasmids are the key players in HGT of antibiotic resistances (Kohler et al., 2018). Twenty multidrug-resistant, biofilm forming human-pathogenic staphylococcal isolates obtained from the three different materials after 6, 12, and 19 months were applied to plasmid DNA isolation. All isolates harbored plasmids <20 kbp and 17 of them also harbored plasmids >20 kbp. Commonly, S. aureus strains contain one or more plasmids ranging in size from <2000 bp to >60 kbp (Kwong et al., 2008).

Fourteen of the 17 Staphylococcus isolates with large plasmids were applied as donors to biparental matings to test the transferability of tetracycline and erythromycin resistance. In total, six out of seven tetracycline resistance transfer experiments (S. hominis V2A-6-05, S. hominis V2A-6-06, S. haemolyticus V2A-12-08, S. hominis V2A-AG-12-06, S. hominis V2A-AGXX-12-01, and S. haemolyticus V2A-AGXX-12-05) were successful whereas nine out of 18 erythromycin resistance transfer experiments (S. hominis V2A-6-03, S. hominis V2A-6-06, S. hominis V2A-6-11, S. aureus V2A-6-13, S. aureus V2A-6-14, S. hominis V2A-12-04, S. haemolyticus V2A-12-08, S. hominis V2A-AG-12-06, and S. hominis V2A-AGXX-12-06) were successful. Thus, these nine isolates likely harbor conjugative elements encoding erythromycin resistance. Indeed, in E. faecalis OG1X transconjugants of four of these matings large plasmids similar in size to those of the donors were found. pSK41 (46.4 kbp) and pUSA03 (37 kbp) are well known staphylococcal conjugative plasmids. Both carry ermC (Berg et al., 1998; Kennedy et al., 2010; Smillie et al., 2010) which was also detected in five of the successful donors.

Tetracycline resistance transfer frequencies from S. hominis V2A-6-05 (tetK), S. hominis V2A-6-06 (tetK, tetO), S. haemolyticus V2A-12-08 (tetK), S. hominis V2A-AG-12-06 (tetK), S. hominis V2A-AGXX-12-01 (tetK, tetL), S. haemolyticus V2A-AGXX-12-05 (tetO) to S. aureus 04-02891 ranged from 1.2 × 10−7 to 6.8 × 10−4 transconjugants/recipient. tetK is only rarely found on large staphylococcal plasmids. It is rather encoded on small mobilizable staphylococcal plasmids in the size range of 4.4 to 4.7 kbp, such as pT181 (Chopra and Roberts, 2001). Thus, in the successful matings with donors harboring tetO or tetK mobilizable pT181-like plasmids might have played a role in the transmission of the resistance to S. aureus 04-02981. As pT181 is non self-transmissible another conjugative element has participated in the transfer of the tetracycline resistance. All donors that were successful in the tetracycline resistance matings contained in addition to plasmid-bands <20 kbp at least one plasmid-band >20 kbp, which could represent the conjugative plasmid. Thus, it is likely that the successful donors harbor a pT181-like plasmid which was transferred by the help of a conjugative plasmid. Indeed, in S. aureus 04-02981 transconjugants from all of those matings large plasmids similar in size to those of the donors were detected. In addition, small plasmids in the size range of pT181-like plasmids were found in transconjugants of three of these matings.

Transfer frequency of gentamicin resistance (8.3 × 10−4 transconjugants/recipient) from E. faecalis V2A-12-03 (aac6 aph2a) to S. aureus 04-02891 was higher than from E. faecalis V2A-AGXX-12-03 (aph(2)-ic) to the same recipient (9.2 × 10−7 transconjugants/recipient). aac6-aph2a can be found on conjugative plasmids, such as pSK41, pGO1, pLW1043, pSK1, pTEF1, Tn4001-like transposons but also on the chromosome (Schiwon, 2011). The uptake of aac6-aph2a by S. aureus 04-02891 indicates that E. faecalis V2A-12-03 likely harbors one of these conjugative elements. Indeed, this observation was corroborated by isolation of a plasmid >20 kbp from E. faecalis V2A-12- 03. aph(2)-ic was found on the 34-kbp conjugative plasmid pYN134 (Chow et al., 1997; Hollenbeck and Rice, 2012). The uptake of aph(2)-ic by S. aureus 04-02891 suggests that E. faecalis V2A-AGXX-12-03 likely harbors a pYN134-like plasmid. This argument was corroborated by the observation of a plasmid band >20 kbp for E. faecalis V2A-AGXX-12-03.

The data of this study confirm erythromycin and tetracycline resistance transfer in ISS-isolates from air-filters and the crew as reported by Schiwon et al. (2013). Further transfer studies between ISS-isolates could deepen our knowledge in the transmissibility of antibiotic resistances. However, no methicillin resistant Staphylococci and no vancomycin resistant enterococci

#### REFERENCES

Allignet, J., Liassine, N., and El Solh, N. (1998). Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and were found. Thus, the generation of serious multi-resistant pathogens by horizontal transfer is unlikely.

#### Further Applications of the Antimicrobial Surface

AGXX R proved to be a long-term efficient antimicrobial, even under the harsh conditions on the ISS. The antimicrobial coating has been also successfully applied against other Gram-positive and Gram-negative pathogens. It also strongly reduced the bacterial load of Legionella and the highly pathogenic Shiga toxin-producing E. coli O104:H4 strain (Guridi et al., 2015). It is available in diverse application forms, such as powders, thin sheets, as coating on diverse plastic materials and on cellulose fleece and will be recently tested in the 4 months SIRIUS isolation study for future lunar flights.

#### DATA AVAILABILITY

All datasets generated for this study are included in the manuscript and/or the supplementary files.

#### AUTHOR CONTRIBUTIONS

EG designed the project and supervised all the experiments. L-YS, KR, JF, WS, PO, and AV performed the experiments. L-YS, KR, and EG wrote the manuscript and designed the figures and tables. NN provided us access to the BIORISK experiment on the ISS and contributed with insightful discussions on the experimental design. All authors read and revised the manuscript.

### FUNDING

This work was partially supported by the Russian Academy of Science (Topic # 65.5).

#### ACKNOWLEDGMENTS

We thank U. Landau and C. Meyer from Largentec GmbH, Berlin, for providing us with the antimicrobial materials and for helpful discussions. We thank Zeliha Kaban, Ghazoua Cheibi and Maxim Gabor Bogisch for their help with the strain collection and Ali Younes for helping with plasmid DNA isolations. Funding by DLR, German Aerospace Center (grants 50WB1166 and 50WB1466 to EG) is acknowledged.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.00543/full#supplementary-material

B and similar antibiotics. J. Antimicrob. Chemother. 42, 1794–1798. doi: 10.1128/AAC.42.7.1794

Andrews, J. M. (2007). BSAC standardized disc susceptibility testing method (version 6). J. Antimicrob. Chemother. 60, 20–41. doi: 10.1093/jac/ dkm110

and future perspectives. Microb. Environ. 29, 250–260. doi: 10.1264/jsme2.ME 14031

Zmantar, T., Kouidhi, B., Miladi, H., and Bakhrouf, A. (2011). Detection of macrolide and disinfectant resistance genes in clinical Staphylococcus aureus and coagulase-negative staphylococci. BMC Res. Notes. 4:453. doi: 10.1186/1756-0500-4-453

**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 Sobisch, Rogowski, Fuchs, Schmieder, Vaishampayan, Oles, Novikova and Grohmann. 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.

# Clonally Diverse Methicillin and Multidrug Resistant Coagulase Negative Staphylococci Are Ubiquitous and Pose Transfer Ability Between Pets and Their Owners

Elena Gómez-Sanz1,2 \*, Sara Ceballos3 , Laura Ruiz-Ripa3 , Myriam Zarazaga3 and Carmen Torres3

1 Institute of Food, Nutrition and Health, ETH Zürich, Zurich, Switzerland, 2 Área de Microbiología Molecular, Centro de Investigación Biomédica de La Rioja (CIBIR), Logroño, Spain, 3 Área Bioquímica y Biología Molecular, Universidad de La Rioja, Logroño, Spain

#### Edited by:

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

#### Reviewed by:

Magaly Toro, Universidad de Chile, Chile Ana P. Tedim, Neiker Tecnalia, Spain

\*Correspondence: Elena Gómez-Sanz<EMAIL_ADDRESS>
#### Specialty section:

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

Received: 29 November 2018 Accepted: 25 February 2019 Published: 26 March 2019

#### Citation:

Gómez-Sanz E, Ceballos S, Ruiz-Ripa L, Zarazaga M and Torres C (2019) Clonally Diverse Methicillin and Multidrug Resistant Coagulase Negative Staphylococci Are Ubiquitous and Pose Transfer Ability Between Pets and Their Owners. Front. Microbiol. 10:485. doi: 10.3389/fmicb.2019.00485 Sixty-eight owners and 66 pets, from 43 unrelated pet-owning households were screened for methicillin-resistant coagulase negative staphylococci (MRCoNS), potential cases of MRCoNS interspecies transmission (IT), and persistence. MRCoNS isolates were identified by microbiological and molecular tests. MLST-based phylogenetic analysis was performed in Staphylococcus epidermidis isolates. Antimicrobial susceptibility was evaluated using phenotypic and molecular methods. SCCmec type and the presence of biofilm-related ica locus was PCR-tested. Isolates suspected for MRCoNS IT cases were subjected to SmaI-PFGE analysis and individuals from positive households were followed-up for 1 year for carriage dynamics (every 3 months, T0– T4). Nineteen MRCoNS isolates from owners (27.9%) and 12 from pets (16.7%) were detected, coming from 20 households (46.5%). S. epidermidis was predominant (90 and 67% of human and animal strains, respectively), showing high phylogenetic diversity (16 STs among 24 strains). Methicillin-resistant S. epidermidis (MRSE) strains belonged to CC5 (75%), CC11 (12.5%), singleton S556 (8.3%), and S560 (4.17%). Significant host-associated differences were observed for resistance to aminoglycosides, cotrimoxazole, chloramphenicol (higher in animal isolates) and tetracycline (higher among human strains). Multidrug resistance (MDR) was common (68.4%) and associated with human strains. Great diversity of ccr and mec complexes were detected, most strains being non-typeable, followed by SCCmecIV and V. Over one third of isolates (most from owners), carried the ica locus, all MRSE CC5. Two sporadic IT cases (T0) were identified in owners and dogs from two households (4.7%), with diverse interspecies-exchanged clones detected along the sampling year, especially in dogs. A comparative analysis of all MRCoNS, with all nasal coagulase positive staphylococci (CoPS) recovered from the same individuals at T0, revealed that CoPS alone was predominant in owners and pets, followed by co-carriage of CoPS and MRCoNS in owners but single MRCoNS in pets. Statistical analyses revealed that owners are more prone to co-carriage and that co-existence of IT cases and co-carriage are positively interrelated. MRCoNS from

healthy owners and their pets are genetically heterogeneous MDR strains that are spread in the community. Therefore, pets also contribute to the dissemination of successful human clones. Owner-pet inhabitancy increases the risk for staphylococcal temporal concomitance with its subsequent risk for bacterial infection and genetic exchange.

Keywords: methicillin-resistant coagulase negative staphylococci, Staphylococcus epidermidis, multidrug resistance, interspecies transmission, carriage dynamics, co-carriage, owner, pet

#### INTRODUCTION

Staphylococci are normal commensal bacteria of the skin and mucous membranes of humans and other animals. They can be differentiated by their ability to produce coagulase. Coagulase positive staphylococci (CoPS), with Staphylococcus aureus as major representative in humans and Staphylococcus pseudintermedius in dogs, pose, in general, higher pathogenic potential than coagulase negative staphylococcal (CoNS) species (Becker et al., 2014). CoNS are less often involved in communityassociated diseases, but represent one of the major nosocomial pathogens, and have a substantial impact on human life and health (Becker et al., 2014; May et al., 2014). In humans, Staphylococcus epidermidis is the most common species among CoNS infections (24–80%), and the most frequent cause of medical device-associated infections (Miragaia et al., 2009; Becker et al., 2014). Regardless of the sparse data available, CoNS have occasionally been confirmed as causative agents for different site infections in dogs (Malik et al., 2006; Kern and Perreten, 2013; LoPinto et al., 2015; Couto et al., 2016). Yet, their zoonotic potential and importance in veterinary medicine is unclear.

Staphylococci, especially CoNS, are notorious for their ability to accrue antimicrobial resistance (AMR) determinants and to produce a biofilm, which makes associated infections particularly difficult to treat (Miragaia et al., 2009; Becker et al., 2014). Further, methicillin resistance is normally associated with additional resistances, which may pose a risk for the AMR gene transfer between staphylococci with higher pathogenic properties, such as S. aureus (Bloemendaal et al., 2010). On top of this, multidrug resistant (MDR) strains drastically limit the therapeutic options available and represent a human and animal health problem.

Nasal S. aureus and S. pseudintermedius can be exchanged between owners and cohabitant pets, and such acquisition can persist over time (Gomez-Sanz et al., 2013a,b). However, no data are available on the incidence and diversity of MRCoNS in healthy owners and their companion animals at the household, on potential cases of interspecies transmission (IT) and on its persistence over time.

The potential association between owner-pet companionship and the concomitant carriage of more than one staphylococcal type (CoPS and MRCoNS), as well as the potential host tropism for these subpopulations is unknown, but is essential to appraise potential owner-pet cohabitation as a risk factor for staphylococcal acquisition, infection and transmission. In addition, simultaneous carriage of CoPS and MRCoNS represents a potential risk for AMR transfer, which is barely considered in AMR surveillance studies.

The **goal** of this study is to determine the nasal occurrence, diversity, clonal distribution, and molecular characterization of MRCoNS in healthy owners and their pets, residing in common households, as well as to address potential IT cases and their carriage dynamics. We subsequently analyzed the MRCoNS and concomitant CoPS nasal patterns to determine whether there was any bacterial species- and/or host-associated tropism.

#### MATERIALS AND METHODS

#### Study Population and Sampling Criteria

Individuals from 43 unrelated pet-owning households were sampled in La Rioja region (Northern Spain) for the nasal carriage of MRCoNS and for IT potential cases. IT was defined as the presence of the same MRCoNS clone in owner and cohabitant pet. Samples were taken from March 2009 to February 2011. Individuals tested were, in parallel, sampled for the nasal occurrence of CoPS (Gomez-Sanz et al., 2013b). Only MRCoNS were further characterized in this study. Inclusion criteria for households tested included healthy humans whose profession did not involve any direct animal contact. None of the individuals tested had received antimicrobial treatment within the 4 months prior sampling. Household recruitment was on a voluntary basis. Sixty-eight humans and 66 animals (54 dogs, 12 cats) were included (Gomez-Sanz et al., 2013a,b). All individuals gave written informed consent to participate in this study, as well as for the sampling of their animals. This study was included in a project approved by the Ethical Committee of Clinical Research of La Rioja (reference: METC 09-399/C). One to five owners and one to five pets were tested from each household, showing 10 different combinations. In most cases (19, 44.2%), only one person and one animal were sampled per household. Nine and 11 of the 43 household units included more than one pet (20.9%) and more than one owner (25.6%), respectively. Four households included both more than one animal and more than one owner (9.3%). In total, 36 of 66 pets lived with other sampled animals (dog/cat) (54.5%), while 40 of 68 owners lived with other sampled humans (58.8%). Of note, all cohabitant pets within a sampled household were included in the study whereas owners were not always all sampled. Swabs were transported to the lab within 5 h after sampling and were either immediately analyzed or stored at -20◦C until further analysis.

#### Isolation and Identification of MRCoNS

Sampled nasal swabs were inoculated into Brain-Heart-Infusion broth (BHI, Difco) supplemented with 6.5% NaCl and incubated at 37◦C for 24 h. One-hundred microliters were inoculated

on Oxacillin-Resistant-Staphylococcal-Agar-Base (ORSAB; OXOID) plates supplemented with 2 mg/L of oxacillin. Plates were incubated at 35◦C for 24–48 h. All blueish to white (potential MRCoNS) colonies with different morphologies were sub-cultured on BHI agar and further studied. Preliminary identification of MRCoNS isolates was based on colony morphology, Gram staining, and catalase and DNase activities. Presence of the mecA gene was investigated by PCR in all isolates (Gomez-Sanz et al., 2013a). Identification of MRCoNS was performed by amplification and sequencing of the sodA gene in all mecA positive CoNS isolates (Poyart et al., 2001). In addition, isolates that were difficult to type by Multi Locus Sequence Typing (MLST) were also identified by amplification and sequencing of the 16S rRNA (Hogg and Lehane, 1999), and by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). When different isolates from the same individual were recovered, which belonged to the same bacterial species and shared the same AMR phenotype, only one isolate was further characterized. Individual nomenclature was as follows: household number (1–43) – isolate host [Human (H); dog (D); cat (C)]. – number of individuals when more than one (1–5).

#### Multi-locus Sequence Typing (MLST) of Methicillin Resistant S. epidermidis (MRSE) Isolates

All 25 MRSE isolates were subjected to MLST as recommended by Thomas et al. (2007). Two novel sets of primers for aroE (aroE-fw2: 50 -TTCATTATCGCATTGATGC-30 , aroE-rv2: 50 - TCAGCACCTTGATGAACGAA-30 ) and tpi (tpi-fw2: 50 -TAGCC GGAAACTGGAAAATG-30 , tpi-rv2: 50 -GCACCTTCTAACAAT TGTACG-30 ) alleles were employed for isolates that could not be amplified with the standard primers. Allele and ST identification was used following the S. epidermidis MLST database1 . The MLST data were analyzed using the goeBURST algorithm2 for ST clustering within clonal complexes (CC) (as of November 2017). For this, Phyloviz2 grouping was generated by Hierarchical Clustering (Hamming Method, UPGMA) using allelic profiles (Nascimento et al., 2017). In addition, a phylogenetic relationship of concatenated sequences was investigated by the construction of a distance tree including metadata on isolates characteristics for each of the different MLST profiles obtained (CLC Genomics Workbench 10.0.1, Qiagen Bioinformatics).

### Staphylococcal Cassette Chromosome mec (SCCmec) Classification

The SCCmec type was determined based on the chromosomal cassette recombinase ccr gene/s and on the type of mec complex as described by Kondo et al. (2007), while confirmation of SCCmec type was tested using SCCmec primers described by Zhang et al. (2005). In addition, allele ccrAB4, present in SCCmec types VI and VIII (Oliveira et al., 2006) was included. Following this approach, cassettes I–IX could be identified.

1https://pubmlst.org/sepidermidis/

2http://goeBURST.phyloviz.net

Typeability of the SCCmec cassettes was defined as follows: (i) Typeable (T) SCCmec cassettes were considered those for which ccr, type of mec complex (Kondo et al., 2007) and/or SCCmec (Zhang et al., 2005) were identified; (ii) Non-Ascribed (NA) SCCmec types were those with a novel combination of ccr, mec complex, and/or SCCmec, and (iii) Non-Typeable (NT) were considered those that did not yield positive results with the primer sets used, per scheme. New SCCmec were defined as those enclosed within NA and NT categories.

#### Characterization of Antimicrobial Resistance Profile

Susceptibility to 17 antimicrobial agents was performed using an agar disk-diffusion method (CLSI, 2013). Antimicrobial agents tested were as follows (class of agent/s): penicillin, oxacillin [+ 2% NaCl], cefoxitin (β-lactams); gentamicin, kanamycin, tobramycin, streptomycin (aminoglycosides); co-trimoxazole (aminopyrimidine/sulfonamide); erythromycin (macrolides); clindamycin (lincosamides); tetracycline (tetracyclines); chloramphenicol (amphenicols); vancomycin (glycopeptides); ciprofloxacin (fluoroquinolones); mupirocin (pseudomonic acid); fusidic acid (steroids); and linezolid (oxazolidinones). Procedures and breakpoints were those proposed for CoNS in CLSI document M100-S23 (CLSI, 2013). For streptomycin and fusidic acid, the methods and breakpoints employed were those recommended by the Société Française de Microbiologie3 . The double-disk diffusion test (D-test) was performed on all isolates to detect inducible clindamycin resistance (CLSI, 2013). Multidrug resistance (MDR) was considered when a resistance to > 3 antimicrobial classes was observed.

The presence of 33 AMR genes, in addition to the mecA gene, was investigated by PCR: blaZ, tet(K), tet(M), tet(L), erm(A), erm(B), erm(C), erm(T), erm(F), mph(C), msr(A)/msr(B), lnu(A), vga(A), vga(C), aacA-aphD, aphA3, aadE, aadD, aadA, str, dfr(A), dfr(D), dfr(G), dfr(K), mupA, fexA, cfr, catpC194, catpC221, catpC223, fusB, and fusC (Gomez-Sanz et al., 2013a,b). Positive controls from the collection of the University of La Rioja were included in each reaction.

Mutations within the quinolone resistance determining region (QRDR) of gyrA and gyrB genes (DNA gyrase subunits), and within parC and parE genes (DNA topoisomerase IV subunits) were investigated in ciprofloxacin resistant isolates (Yamada et al., 2008). The corresponding genes of the quinolone susceptible S. epidermidis strain ATCC 12228 (GenBank ac. no NZ\_CP022247.1) were used as a reference for mutation detection and positioning within the gene.

#### Presence of Virulence Genes Involved in Biofilm Formation

PCR based determination of several genes involved in biofilm formation was implemented. Genes tested were the S. aureus biofilm matrix protein bap (Cucarella et al., 2001); the Staphylococcal intercellular adhesin (icaADBC) operoncontaining genes icaA, icaB, icaC, and icaD, responsible for

3http://www.sfm-microbiologie.org/

the synthesis of the biofilm matrix polysaccharide intercellular adhesion (PIA) (Ziebuhr et al., 1999; Arciola et al., 2006); the transcriptional repressor of the ica locus, the icaR gene (Conlon et al., 2002); as well as the insertion sequence IS256, which has been observed to play a role in phase variation of virulence by ica locus in S. epidermidis (Ziebuhr et al., 1999).

#### Determination of Cases of Interspecies Transmission (IT)

The genetic relatedness of MRCoNS isolates suspected for cases of direct IT – i.e., those isolates of the same species recovered from cohabiting individuals that exhibited identical AMR profile, MLST for S. epidermidis, and SCCmec type – was addressed by Pulsed Field Gel Electrophoresis (PFGE) of the total DNA digested with a SmaI macro-restriction enzyme following the HARMONY protocol (Murchan et al., 2003).

#### Longitudinal Approach: Carriage Status Definition and IT Dynamics

All individuals from households with cases of direct IT were followed-up with for a year. For this, nasal samples from the anterior nares of owners and pets were studied once every 3 months (five sampling times in total, T0–T4) with a total of 24 additional samples analyzed (T0–T4). Studied subjects positive for MRCoNS in at least four of the five samplings (including T0) were considered persistent carriers; those positive in two or three samplings were defined as intermittent carriers; individuals positive in a single sampling were reported sporadic carriers; and those negative throughout the study were defined as noncarriers. Dynamics of the IT cases over time was defined likewise (persistent, intermittent, and sporadic).

## MRCoNS and Coagulase Positive Staphylococci (CoPS) Individual and Household Concomitance

In a former study Gomez-Sanz et al. (2013a,b), all coagulase positive staphylococcal (CoPS) isolates recovered from the same individuals at the same sampling (T0) were characterized (36 S. aureus and 18 S. pseudintermedius). At this stage, we aimed at making a summative and comparative analysis of the MRCoNS and CoPS concomitant carriage of individuals tested in T0 and, subsequently, of respective households. Such concomitance was also analyzed along the longitudinal study with the individuals from households with cases of IT (**Supplementary File S1**). Potential association of concomitant carriage, host, and/or being involved in an IT case was evaluated.

#### Statistical Analysis

The characteristics of the owner and pet isolates were compared for consistent differences. Statistical analysis tests were performed in R (R Development Core Team, 2018). SCCmec, AMR, and ica locus profiles between owners and dogs were compared using the Fisher's Exact test. Potential significant differences in MRCoNS carriage and MRCoNS/CoPS co-carriage between owners and pets at individual and household level were likewise evaluated. Correlations between presence of ica locus and (i) bacterial species, (ii) CC, (iii) host, and (iv) household of origin were analyzed by dependence measure of variables using multivariable Logistic Regression test. Correlations between owner and pet cohabitation and bacterial nasal carriage, as well as between involvement in **IT** cases and bacterial simultaneous carriage (MRCoNS; CoPS), at individual and household level, were likewise evaluated [variables: (i) host, (ii) presence of more than one pet per household, (iii) involvement in IT case, (iv) bacterial concomitance]. Correlation analyses were performed using the Corrplot R package. All analyses were performed at a 95% confidence interval (CIs). The degree of genetic diversity for ST and SCCmec types was assessed by Simpon's Index of Diversity (SID). SID represents the probability (0 = low diversity, 1 = high diversity) that any two randomly selected species from the sample will be different. In this analysis, each ST or SCCmec element (ccr, mec complex combination) was considered a "type" or "species."

## RESULTS

#### Occurrence of MRCoNS in Individuals and Households

Thirty-one MRCoNS isolates, 19 isolates from 19 owners (27.9%) and 12 isolates from 11 pets (16.7%) (14.81% dogs, 25% cats) were detected. MRCoNS species distribution in owners and pets is shown in **Figure 1**. S. epidermidis and Staphylococcus lentus were detected in both owners and pets. Staphylococcus haemolyticus was detected in one owner only and Staphylococcus cohnii and Staphylococcus vitulinus in dogs (the latter in two cohabitant dogs). One dog (1-D1, from household no. 1) carried one S. lentus and one S. epidermidis isolate (**Table 1**). For both, owners and pets, S. epidermidis was the predominant species, accounting for 89.5 and 66.0% of strains, respectively. In total, 25% of owners and 12.1% of pets (9.3% among dogs, 25% in cats) carried MRSE.

In 20 of the 43 (46.5%) tested households there was at least one individual (either owner and/or pet) positive for MRCoNS. In two households (4.6%) (numbered 1–2) there was concurrent MRCoNS carriage of at least one owner-pet pair (**Table 1**). Instead, in 11 residences (25.6%) (no. 3–13) only owners were positive for MRCoNS whereas in seven households (16.3%) (no. 14–20) only pets carried MRCoNS (**Table 1**).

TABLE 1 |

Molecular

characterization

 of the 31

MRCoNS strains recovered from healthy owners and their pets from 20 households.

T, tetracycline;

 G, gentamicin;

ciprofloxacin;

SCCmec classification

 F, fusidic acid.

 schemes.

 To, tobramycin;

eSequence

 type for which aroE and tpi alleles could not be amplified with standard primers.fNT,

 K, kanamycin;

 S, streptomycin;

 M, mupirocin;

 X,

trimethoprim/sulfamethoxazole;

 Xi, intermediate

 non-typeable.

 Cells with gray background

 resistance

trimethoprim/sulfamethoxazole;

 highlight the concordant

 to Ch,

 results between both

chloramphenicol;

 Cp,

Not significant differences were observed in MRCoNS carriage among owners or pets where more than one pet was in the house (p = 0.7946 versus 0.4321, respectively).

#### Clonal Lineages of MRCoNS Isolates

Molecular characterization of the 31 MRCoNS isolates recovered is displayed in **Table 1**. In total, 24 of the 25 MRSE isolates were typed by MLST, with 16 different STs detected. One human MRSE isolate (C3938) could not be typed due to reiterate lack of amplification of several of the MLST-schemed alleles (gtr, pyr, yqil, and mutS), regardless MALDI-TOF confirmed that it was S. epidermidis. In addition, four isolates were not typeable using the standard aroE and tpi primers4,5 , but did amplify with in-house designed primers (ST290 and the novel ST558, ST559, and ST560).

Nine of 24 (37.5%) MRSE strains revealed novel STs (seven different ones), with either novel allele (ST553, ST554, ST555, ST556) or novel allele combination (ST558, ST559, ST560) (**Supplementary Table S1**). Fifteen MRSE strains (62.5%) belonged to already known STs (nine different STs). ST5 (primary ST founder of CC5) was predominant, being present in two MRSE from unrelated owners and two from related dogs. Most MRSE belonged to CC5 (75%), which is the major group within the S. epidermidis MLST scheme, three strains belonged to CC11 (12.5%) and three strains were singletons [S556 (8.3%) and S560 (4.2%)] (**Figure 2A**). All previously known STs (ST2, ST35, ST22, ST60, ST20, ST130, ST83, and ST290) represented subgroup founders (by default settings, i.e., an ST with at least three links to other STs, including the link to its assumed progenitor), with ST2 as the biggest subgroup founder within CC5 (formerly compiling CC2) (**Figure 2A**).

The distance tree of the 16 concatenate ST sequences detected among the 24 MRSE strains revealed high profile diversity. All cases were concordant with the CC and STs subgroup clusters (based on allelic profile) represented by Phyloviz2 clustering using the goeBURST algorithm, except for ST35, which formed an independent branch from the closest variants (ST2, ST22, and ST553). Remarkably, all four STs that could not be amplified using the standard primers (all 3 CC11 and S560) clustered together in a distant branch from the rest of STs (**Figure 2B**).

All canine MRSE strains exhibiting STs were also detected in owners (1 ST155, 2 ST5, 2 ST130, all CC5), while all feline (ST60- CC5, ST560-S560, ST558-CC11) and some human MRSE strains were unique (**Figure 2B**).

The Simpon's Index of Diversity (SID) was remarkably high (0.96), reflecting a 95.6% chance of randomly picking two strains from the sample cohort that are different.

#### ccr and mec Complex Diversity Among MRCoNS Isolates (SCCmec Profile)

Based on a scheme by Kondo et al. (2007), high diversity of ccr types, mec complexes and ccr-mec complex combinations were detected among the 31 studied isolates (**Table 1**). ccrAB2 (n = 21) and mec complex A (n = 13) were predominant within

4https://pubmlst.org/sepidermidis/

5http://sepidermidis.mlst.net

their respective category (see **Supplementary Table S2**). All eight SCCmec cassettes carrying ccrC presented additional ccr genes (ccrAB2, n = 5; ccrAB1, n = 2; or ccrAB1+ccrAB2, n = 1) (**Supplementary Table S2**). More than one ccr type was detected in 11 isolates (35.5%). A total of 21 SCCmec cassettes were either NT or NA (67.7%), nine were SCCmec IV (29%), and one was SCCmec V (3.2%).

According to scheme by Zhang et al. (2005), 20 strains were either SCCmec NT or NA (64.5%), seven were SCCmec V (22.6%), three were SCCmec IV (9.7%), and one was SCCmec III (3.2%). Four strains were positive for more than one SCCmec cassette.

Eight of 31 strains (25.8%) were concordantly typed with both typing schemes (**Table 1** and **Supplementary Table S2**). Among them, SCCmec NT was predominant (n = 4), followed by SCCmec IV (n = 2) and SCCmec V and SCCmec NA (one each), respectively. Both schemes categorized seven additional cassettes in different strains, with a SID of 0.89 by Kondo et al. (2007) and SID 0.71 by Zhang et al. (2005). In total, as a consensus of both schemes, 18 strains of SCCmec were NT (58.1%), 10 NA (32.3%), two SCCmec IV (6.5%), and one SCCmec V (3.2%) (**Supplementary Table S2**).

Comparing owner versus pet MRCoNS isolates by Kondo et al. (2007), SCCmec NT or NA were predominant among both human and animal strains (combined 63.2% for owners versus 91.7% for pets) (p = 0.02203). SCCmec IV was the most commonly known SCCmec cassette among both host isolates (six from humans, 31.6%; three from dogs, 25%), while SCCmec V was only detected in two owners (10.5%).

According to both schemes performed, one MRSH from an owner and one MRSE from her pet (1-H2 and 1-D1, household 1) shared the same SCCmec cassette (**Table 1** and **Supplementary Table S2**).

#### Antimicrobial Resistance (AMR) Pattern

Prevalence of resistance to non β-lactams among human and animal isolates, as well as the detected resistance genes, is shown in **Figure 3**. Erythromycin resistance [erm(A), erm(C)] was the most common pattern (51.6% of isolates), followed by mupirocin (mupA) (29%) and clindamycin [vga(A), lnu(A)] (29%) resistance. Subsequently, MLS was the antimicrobial class to which most strains exhibited resistance. Mupirocin resistance was only present in MRSE strains (36% of MRSE). Inducible clindamycin resistance was only observed in the three isolates carrying the erm(C) gene (see **Table 1**).

Mutations identified in the QRDR of the gyrA, parC and parE genes of the five MRSE ciprofloxacin resistant strains are summarized in **Table 2**. All detected substitutions are displayed in **Supplementary Table S3**. No mutation was observed in any strain within the gyrB gene sequence region. The most common mutation was Ser84Phe (5/5) and Ser84Tyr (3/5) in GyrA and ParC, respectively.

Resistance to aminoglycosides (p = 0.008–0.016), cotrimoxazole (p = 0.016) and chloramphenicol (p = 0.007) was significantly higher in animal isolates (with the latter being exclusively detected in pets), whereas resistance to tetracycline was only present and abundant in owner isolates (p = 2.95E-07).

FIGURE 2 | (A) Clustering analysis of the S. epidermidis STs detected in this study by goeBURST algorithm using Phyloviz 2 software (Nascimento et al., 2017). The most restricted level [level 1 – Single Locus Variant (SLV)] was used, requiring six of seven alleles shared to the linked ST. Cyan STs indicate probable ancestors (group founders) and green STs constitute subgroup founders. Blue STs correspond to STs that share the same background (CC). Circles in red indicate the STs detected in this study. Specific location of ST5 (CC5 ST primary founder) and ST2 (major subgroup founder of the cluster) within CC5 are indicated. (B) Distance tree of the 16 concatenate ST sequences detected among the 24 S. epidermidis isolates constructed using CLC Genomics Workbench 10.0.1 (https://www.qiagenbioinformatics.com/). Sequences were aligned using internal parameters, and the tree was built with a Neighbor Joining method using Jukes-Cantor as Nucleotide Distance measure, with a bootstrap analysis of 500 replicates. The bar length indicates the number of substitutions per site. STs in black color are those with new ST, either by the presence of a new allele or new allele combination.

FIGURE 3 | Percentage of resistance to non β-lactams and antimicrobial resistance genes detected among the 31 MRCoNS isolates investigated in T0. FUS, fusidic acid; CHL, chloramphenicol; TET, tetracycline; CIP, ciprofloxacin; SXT, co-trimoxazole; GEN/TOB/KAN/STR, gentamicin/tobramycin/kanamycin/streptomycin; MUP, mupirocin; ERY/CLI, erythromycin/clindamycin. All isolates were susceptible to vancomycin and linezolid. Individual P-value (Fisher's Exact Test for count data) to account for significant difference at 95% confidence interval is indicated at the bottom of the histogram. Asterisks (blue or red) above the bars represent those agents for which statistical differences were detected, with the asterisk color remaking the host (owner or pet, respectively) of the bacteria involved in the significance.

TABLE 2 | Mutations in the quinolone resistance determining regions (QRDR) of GyrA (DNA Gyrase), ParC, and ParE (DNA topoisomerase IV) of the quinolone resistant strains.

GyrB is not represented given that no mutations were observed. Synonymous substitutions are marked in bold.

Resistance to fusidic acid and streptomycin were only detected in human isolates at low rates, but no significant differences were observed with the Fisher's Exact test.

Remarkably, one methicillin-resistant S. lentus (MRSL) clone (isolates C3030 and C3031, from owner 1-H1 and cohabitant dog 1-D1) showed intermediate resistance to trimethoprim and co-trimoxazole but did not harbor any of the trimethoprim resistance genes so far described in staphylococci. The human MRSE-S556 strain (C5112) also showed hetero-resistance to clindamycin but was negative for the corresponding genes tested. This strain was also resistant to fusidic acid and lacked the acquired fusB and fusC genes.

Significant differences were observed between the rate of owners and pets carrying MDR MRCoNS isolates (68% versus 33%) (p = 1.205E-06). In total, 54.84% of isolates were MDR.

#### Presence of Determinants for Biofilm Formation

A total of 32.3% of isolates were positive for the genes enclosed within the ica locus (icaADBC) as well as for the icaADBC transcriptional regulator icaR (**Table 1**); all of which were MRSE of the CC5 lineage (see **Figure 2B**). If divided by the bacterial host, 47.4% of human isolates and a single MRSE canine strain (C3029) (8.3%) were positive (p = 4.49e-10).

Subsequently, the presence of the ica locus gene cluster in human MRSA-CC5 isolates was strongly positively correlated. Through logistic regression analysis, positive association was observed between presence of the ica locus and owners, only when the variable household of origin was not considered in the equation (association was observed at 0.1 significance code otherwise).

The IS256 was detected in four icaADBC-negative isolates (12.9%). These isolates also contained the bifunctional aminoglycoside resistance aacA-aphD gene, which is normally enclosed within Tn4001 (IS256\_aacA-aphD\_IS256).

#### Owner/Pet MRCoNS IT Cases and Longitudinal Overview

Based on all molecular techniques performed, two cases of IT were identified in the owner and cohabitant dog in two unrelated households (4.7% of tested residences; 10% of households with MRCoNS-carrying individuals): (i) a MDR MRSL clone (1-H1 and 1-D1), resistant to erythromycin/clindamycin and gentamicin/tobramycin/kanamycin; and (ii) a MRSE-ST130- CC5 clone (2-H1 and 2-D1) resistant to tobramycin/kanamycin (**Table 1** and **Figure 4**).

According to the 1 year longitudinal study, in case 1, sporadic carriage by the involved MRSL clone was observed in both individuals (1-H1, 1-D1). Instead, the involved dog (1-D1) also carried a MRSE ST155-CC5-SCCmecV strain (resistant to erythromycin) in T0 which was also present when sampling T3 in the same animal (intermittent carrier of such clone) as well as in the other cohabitant owner (1-H2), representing an additional S. epidermidis sporadic IT case (**Figure 4**). In total, three different MRCoNS species (S. lentus, S. epidermidis, S. haemolyticus) and one clone of each were detected along the sampling year. Dog 1-D1 carried two of these clones while the owners carried one clone each.

In case 2, sporadic carriage by the involved MRSE ST130- CC5 clone was also observed. Notably, the same owner and dog (2-H1, 2-D1) carried an identical non-concurrent MRSE clone (only resistant to β-lactams) in different samplings: T2 for the owner and T3 for the dog (**Figure 4**), indicating transient carriage and suggesting that such a clone might be circulating within the household. Along the sampling year, these two subjects revealed to be intermittent carriers of different S. epidermidis clones with different resistance patterns (**Figure 4**). In total, a single MRCoNS species (S. epidermidis) was detected throughout the sampling year, however, five different MRSE clones were observed, three of them found in dog 2-D1, three in owner 2-H1 and a single clone in owner 2-H2.

None of the individuals, from both cases, were persistent carriers by any of the recovered MRCoNS strains. None of the ITinvolved isolates in T0 exhibited any of the genes of the ica locus. However, the MRSE C3029 clone (from case 1), which carried the ica-locus, was detected again in this animal and one owner in T3 (IT case).

The dynamics of all CoPS staphylococci detected in the same samplings (T0–T4) are described in the **Supplementary File S1** as well as in **Supplementary Figure S1**.

## Individual and Household MRCoNS and/or CoPS Concomitance

Eighty-five staphylococcal strains [MRCoNS (n = 31) and CoPS (n = 54)] (Gomez-Sanz et al., 2013b) from the 68 positive individuals recovered at the same sampling point were compared here (**Supplementary Table S4**). This comprehensive picture revealed a total of nine cases of IT (two MRCoNS, 7 CoPS) at sampling T0 (11.9% of subjects coming from 18.6% of tested households) (Gomez-Sanz et al., 2013a,b). Altogether, 55.9% of owners and 45.5% of pets were positive for MRCoNS and/or CoPS (**Supplementary Table S5**).

Single presence of CoPS was the most common pattern, with owners and pets predominantly carrying only S. aureus (26.5%) or S. pseudintermedius (22.7%), respectively (**Figure 5A**). The carriage rate of MRCoNS as the single species recovered was similar in owners and pets tested (ca. 10.5%) (**Figure 5A**). Alternatively, 17.7% of owners and 6.1% of pets simultaneous carried both bacterial types (p = 0.015) (**Table 3**). Concomitant carriage of MRCoNS and S. aureus was significantly higher among owners than pets (14.7% versus 1.5%), while no significant differences were detected for co-carriage of MRCoNS and S. pseudintermedius (2.9% versus 4.6%) (**Figure 5A** and **Supplementary Table S5**).

Eleven of the 17 MRCoNS (64.7%) strains involved in the simultaneous carriage were MDR and six of 17 (35.3%) contained the ica-locus genes, involved in biofilm formation (**Table 3**). IT cases were more common among individuals with concomitant carriage (6/16, 37.5%) (p = 0.004).

At the household level, based on the strains recovered from individuals tested, 32 households were positive for any of the tested bacterial species (74.4%; 34.9% positive for one bacterial type, 39.5% positive for both MRCoNS and CoPS) (**Figure 5B**). Co-presence of S. aureus and MRCoNS was the most predominant pattern (18.6%), followed by S. aureus alone (16.3%), and co-presence of S. aureus, S. pseudintermedius and MRCoNS (14%). Considering the 32 positive residences, S. aureus was the predominant species among households with a single bacterial type (21.9%), and half (50%) presented both MRCoNS and CoPS bacterial types (**Figure 5B**).

In total, 23.3% of households contained individuals simultaneously harboring both bacterial types (**Table 3**). Half (5/10) of these households enclosed subjects directly involved in IT cases (p = 0.011). Further, all four pets and seven of the 12 owners who tested positive for concomitant MRCoNS and CoPS (11/16, 68.8%) originated from households where IT cases occurred, even if they were not the individuals directly involved in the case (**Table 3**).

#### Association Between MRCoNS and CoPS Concomitance, IT Cases, and Host

Logistic regression analysis confirmed a strong positive correlation between individual staphylococcal concomitance and involvement in IT case (0.001 significance code). A positive association (0.05 significance code) was observed between concomitance and owners, only when the household of origin

FIGURE 4 | (A) Schematic representation of the methicillin resistant coagulase negative staphylococcal carriage dynamics of both households investigated along 1 year. IT, bacterial species responsible for interspecies transmission. T0–T4 indicate the different sampling times along the sampling year. Individuals are named H (for human) or D (dog) followed by the case number (1 or 2) and a lower-case letter to differentiate subjects per household. (B) Pulsed-field Gel Electrophoresis (PFGE) profile of genomic DNA digested with SmaI restriction enzyme of isolates recovered from individuals involved in both cases of possible direct interspecies transmission. Upper lane in PFGE per case corresponds to MidRange PFGE Marker (New England Biolabs). Antimicrobial resistance (AMR) genes detected in each strain are also indicated.

FIGURE 5 | (A) Left panel, bar chart showing the percentage of owners and pets that carried Coagulase Positive Staphylococci (CoPS), i.e., S. aureus (SA) and/or S. pseudintermedius (SP); MRCoNS; or CoPS + MRCoNS in sampling T0 (Gomez-Sanz et al., 2013b). Right panel, graphical view of the distribution of CoPS and/or MRCoNS detected among the individuals positive for such bacterial species. (B) Left panel, bar chart displaying the percentage of households with individuals positive for CoPS (SA, SP), MRCoNS or CoPS + MRCoNS in sampling T0. Right panel, graphical representation of the distribution of CoPS and/or MRCoNS detected among the households with individuals positive for such bacterial species. Colored stars indicate values with significant differences between human and animal strains.

was not considered in the equation (association was observed at 0.1 significance code otherwise).

No significant differences were observed between the presence of more than one animal in the house (animal cohabitation) and (i) staphylococcal carriage (p = 0.3145 for pets, p = 0.1644 for owners), or (ii) MRCoNS and CoPS individual co-carriage (p = 1 for pets, p = 0.7781 for owners).

#### DISCUSSION

The present study provides novel information on frequency, population structure, genetic diversity, AMR and virulence potential among MRCoNS from companion animals and their owners within the household, as well as on staphylococcal human-pet interaction and persistence. The MRCoNS carriage rate detected among healthy owners (28%) is remarkably higher than those detected in former studies among healthy individuals in non-healthcare settings, with rates ranging between 7 and 17% (Barbier et al., 2010; Rolo et al., 2012; Du et al., 2013; Abadi et al., 2015; Xu et al., 2018). Higher nasal MRCoNS rates (30, 47–51%) were detected in Japanese children in daycare centers and kindergartens (Jamaluddin et al., 2008) and among a remote population in French Guiana (Lebeaux et al., 2012). On the other hand, a recent international study on nasal staphylococcal colonization among healthcare workers from 75 different countries revealed a nasal MRCoNS carriage rate of 21.4% (Morgenstern et al., 2016). All these data reflect that nasal distribution of MRCoNS markedly depend on the cohort studied. Remarkably, scarce data are available on the nasal MRCoNS colonization rate among pet owners, and or the animal-owner contact as a possible contributor in increased MRCoNS carriage. Only a couple of recent studies analyzed the risk factors of MRS carriage among individuals in contact with companion animals (Han et al., 2016; Rodrigues et al., 2018). Rodrigues et al. (2018) reported an overall prevalence of MRCoNS of 54.2% among healthy humans in professional daily contact with companion animals in Portugal. In this report, being a veterinary professional was identified as a risk factor for methicillin-resistant staphylococcal carriage (both CoNS and CoPS) colonization (Rodrigues et al., 2018). The relatively high MRCoNS rate detected here might therefore be due, at least partially, to direct pet-human contact, and might be considered as a risk factor for colonization. However, the lack of a "control" population in the current study forces us to interpret these data with caution. Among pets, very sparse data are available on the specific nasal MRCoNS rates. Lower rates (1–15%) than those detected here (17%) have been observed among healthy dogs from several body sites (nasal, rectal, oral, anal, belly) (Vengust et al., 2006; Bagcigil et al., 2007; Aslantas et al., 2013; Gandolfi-Decristophoris et al., 2013; Garbacz et al., 2013; Chah et al., 2014; Davis et al., 2014; Wedley et al., 2014; Siugzdaite and Gabinaitiene, 2017). Interestingly, MRCoNS was isolated from 42% of healthy non-vet visiting and non-antimicrobial treated Labrador retrievers in the United Kingdom (Schmidt et al., 2014). In the latter study, both nasal and perineal samples were collected, suggesting that different sampling methodologies may affect observed prevalence.

In humans, S. epidermidis is the most frequently recovered staphylococcal species, colonizing the body surface (Becker et al., 2014; Schmidt et al., 2014). Moreover, the S. epidermidis group (predominantly S. epidermidis and S. haemolyticus) is the most significant species within CoNS representing one of the major nosocomial pathogens (Becker et al., 2014). As such, MRSE was the MRCoNS predominant species detected (25%, 99% of human strains). S. epidermidis was also the predominant species among tested animals, with an overall prevalence of 12.1% (9.3% among dogs versus 25% in cats), corresponding to 66% of strains. A diverse range of MRCoNS species have been detected among dogs, such as Staphylococcus sciuri, Staphylococcus warneri, S. lentus, S. vitulinus, or Staphylococcus fleurettii (Bagcigil et al., 2007; Becker et al., 2014; Chah et al., 2014; Davis et al., 2014; Schmidt et al., 2014; Siugzdaite and Gabinaitiene, 2017). Regardless S. epidermidis has a more defined role in humans; it may also form part of the normal microbiota of animals and, although at lower rates, has been detected as the predominant CoNS species among healthy dogs (Aslantas et al., 2013; Schmidt et al., 2014; Han et al., 2016). Nevertheless, S. epidermidis is a predominantly human associated bacterium and the observed distribution here may be influenced by the human-pet direct or indirect contact within the household.

S. epidermidis is the most studied species within CoNS and it is characterized by pronounced genomic diversity (Becker et al., 2014). This agrees with the high diversity of MRSE STs detected (SID of 0.96). In spite of the scattered data available on MRSE lineages from healthy individuals, former reports have also reported high rates of novel STs among S. epidermidis isolates (Xu et al., 2018), evidencing high intra-species diversity. MRSE CC5 was predominant, clustering 75% of MRSE isolates from owners. This clonal lineage (with ST5 as primary founder) represents the biggest group within the MLST scheme for this species. MRSE ST2 and ST22, among others, currently enclosed within CC5 but traditionally constituting its own CC (CC2), have been shown to be predominant among hospital environments (Miragaia et al., 2007; Rolo et al., 2012; Cherifi et al., 2013; Becker et al., 2014; Widerstrom et al., 2016; Gordeev et al., 2017). In the community, a high diversity of STs have been identified among healthy individuals (Miragaia et al., 2007; Rolo et al., 2012; Cherifi et al., 2013; Becker et al., 2014; Widerstrom et al., 2016; Gordeev et al., 2017). In contrast, recent studies have revealed a high diversity of lineages among MRSE from both clinical and healthy individuals, with either no increased abundance of CC5 strains among clinical isolates (Jena et al., 2017) or with CC5 predominance in both settings (Rolo et al., 2012; Du et al., 2013). This may be due to the fact that most STs already cluster into CC5 by eBURST/goeBURST analyses, which may hamper attempts to identify lineages that might be associated with different regimes (Thomas et al., 2014). For this reason, a couple of recent studies implemented a Bayesian clustering approach to appraise the real species-wide population structure and ecology of S. epidermidis, detecting six genetic cluster (GCs) based on their adaptation to nosocomial or commensal lifestyles (Thomas et al., 2014; Tolo et al., 2016). Following this classification for the already known

characteristics.

fmicb-10-00485 March 22, 2019 Time: 17:57 # 12

TABLE 3 |

Individuals concomitantly

 carrying at least one MRCoNS and one CoPS isolate ranked by household type (based on carriage and IT) and major strain

**502**

non-typeable.

STs, (i) ST2 and ST22 were more suited to a nosocomial lifestyle (GC5); (ii) ST290 to a more commensal lifestyle (GC4), (iii) ST5, ST83, and ST130 were adapted to a more generalist-to-nonhospital sources (GC1); and (iv) ST20, ST35, and ST60 were better suited for generalist-to-infection-associated lifestyles (GC6).

Very scarce data are available on MRSE lineages among pets. A few studies among clinical samples detected ST5 and/or ST2 (both CC5) as predominant, in line with data from humans (Kern and Perreten, 2013; Weiss et al., 2013; Couto et al., 2016). However, data on the circulating MRSE lineages in the community and whether they reflect the human circulating lineages within a target system, are lacking. Here, clear clustering of human and canine strains was observed, as all STs detected among dogs were also detected among different owners from different households. This lack of host tropism of specific lineages suggests the adaptability potential of MRSE to different hosts within a shared habitat and/or the easiness of host sporadic acquisition of circulating lineages. In contrast, the three STs detected among MRSE from feline isolates were unique. This might indicate that, while dogs tend to share the same clonal lineages as owners, cats might pose felineassociated lineages. Further studies with a bigger sample size are needed on the ecology of MRCoNS and MRSE among different inhabitant species, and how cohabitation may influence host staphylococcal profiles.

High diversity of SCCmec types was detected, most being either NT or NA (90.3%, 28/31). These values are notably higher than those detected among both clinical and community MRCoNS human isolates (Barbier et al., 2010; Lebeaux et al., 2012; Aslantas et al., 2013; Abadi et al., 2015; McManus et al., 2015). This high rate may be partially due to the higher discriminatory power of using two schemes. Remarkably, slightly similar values (83%) were recently detected among MRSE from the nares of neonates at hospital admission (Salgueiro et al., 2017). It is challenging to define whether the NTs cassettes identified here are identical to those previously described as NTs, due to variances in typing methods and the lack of full analysis of the genetic organization and composition of these elements. For this, further in-depth analyses, such as whole genome sequencing (WGS), are definitively needed.

Lack of robust concordance was observed between results obtained by both schemes, with guidelines from Kondo et al. (2007) showing a remarkable high diversity index (SID 0.89 versus 0.71), and reflecting the high intergenic diversity within MRCoNS cassettes. SCCmec IV was the predominant typeable cassette for both owner and pets, and despite, additional cassettes have been sporadically detected, it is also the most prevalent cassette among humans and companion animals (Ruppe et al., 2009; Barbier et al., 2010; Lebeaux et al., 2012; Aslantas et al., 2013; Kern and Perreten, 2013; Park et al., 2013; Weiss et al., 2013; Becker et al., 2014; Abadi et al., 2015; McManus et al., 2015; Couto et al., 2016).

Several ccr genes were detected in 35.5% of strains, showing a high variety of site-specific recombinases among MRCoNS. The possibility that primers are not specific enough for potential new ccr cannot be discarded. Further, ccr2 and ccrC were copresent in all but one detected cases, suggesting that clustering of both ccr genes might imply and adaptive advantage. Further analyses should be performed to unveil the real presence and functionality of redundant ccr genes, and whether this implies an adaptive advantage under specific conditions. The high SCCmec variability, lack of typeability and presence of novel ccr and mec complex combinations reflect an ever-increasing complexity among SCCmec cassettes among CoNS from healthy individuals. Such mobile elements may represent a source for the potential transfer to concurrent staphylococci sharing the same niche. In this study, however, transmission of β-lactams resistance between MRCoNS and CoPS appears negligible among the population tested.

Macrolides-Lincosamides-Streptogramins (MLS), especially erythromycin, was the antimicrobial class for which most strains exhibited resistance among owners and pets (64.5%). MLS are important antibiotics for treatment of staphylococcal infections in both humans and animals (Guardabassi et al., 2004; Bagcigil et al., 2007). Subsequently, it is not surprising that MLS resistance is common among staphylococci in the community (Aslantas et al., 2013; Gandolfi-Decristophoris et al., 2013; Garbacz et al., 2013; Wedley et al., 2014; Couto et al., 2016; Han et al., 2016). Of note, combined resistance to erythromycin and clindamycin is the most common MLS pattern among CoPS isolates (Gomez-Sanz et al., 2013a,b), however, most MRCoNS isolates here were either resistant only to erythromycin or to clindamycin. This pattern reflects the potential differential ability to acquire different resistance genes between CoPS and MRCoNS populations.