International high-risk clonal lineages of CTX-M-producing Escherichia coli F-ST648 in free-roaming cats, South America

Accepted Manuscript International high-risk clonal lineages of CTX-M-producing Escherichia coli F-ST648 in free-roaming cats, South America

Miriam R. Fernandes, Fábio P. Sellera, Quézia Moura, Vitor C. Gaspar, Louise Cerdeira, Nilton Lincopan PII: DOI: Reference:

S1567-1348(18)30238-7 doi:10.1016/j.meegid.2018.09.009 MEEGID 3650

To appear in:

Infection, Genetics and Evolution

Received date: Revised date: Accepted date:

3 May 2018 11 September 2018 13 September 2018

Please cite this article as: Miriam R. Fernandes, Fábio P. Sellera, Quézia Moura, Vitor C. Gaspar, Louise Cerdeira, Nilton Lincopan , International high-risk clonal lineages of CTXM-producing Escherichia coli F-ST648 in free-roaming cats, South America. Meegid (2018), doi:10.1016/j.meegid.2018.09.009

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ACCEPTED MANUSCRIPT Short Communication

International high-risk clonal lineages of CTX-M-producing Escherichia coli F-ST648 in free-roaming cats, South America Miriam R. Fernandesa,1, Fábio P. Sellerab,1, Quézia Mourac, Vitor C. Gasparb, Louise

a

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Cerdeiraa, Nilton Lincopana,c,* [email protected] Department of Clinical and Toxicological Analyses, School of Pharmaceutical

Sciences, University of São Paulo, São Paulo, Brazil b

Department of Internal Medicine, School of Veterinary Medicine and Animal

Science, University of São Paulo, São Paulo, Brazil

Department of Microbiology, Institute of Biomedical Sciences, University of São

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c

*

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Paulo, São Paulo, Brazil

Corresponding author at: Department of Microbiology, Institute of Biomedical

Sciences, University of São Paulo, São Paulo, Brazil.

1

Contributed equally.

ACCEPTED MANUSCRIPT Abstract The dissemination of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli belonging to high-virulent pandemic lineages has become a global problem with serious consequences for public health worldwide. In this regard, E. coli lineages belonging to the sequence type ST648, which are mostly associated with nosocomial

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infections, have begun to be reported in animals. In this study, we report the identification and genomic characterization of international CTX-M-producing E. coli ST648/F lineages in free-roaming cats from an urban slum, in Brazil. Moreover, we have performed a comparative genomic analysis of worldwide reported E. coli ST648

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strains, highlighting an epidemiologic linkage between human and companion

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animals.

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Keywords: Companion animals, Pets, ESBL, pandemic clone, Latin America, Brazil

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In the last years, many studies have alerted on the emergence of multidrugresistant (MDR) high-risk clonal lineages of clinically significant bacteria in companion animals, raising public health concerns, since infected and colonized pets may contribute to the spread of such bacteria (Marques et al., 2017). In this respect,

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among Escherichia coli isolates, high-virulent pandemic lineages belonging to sequence types ST131, ST405 and ST648, have been associated with community acquired infection rather than nosocomial infections, being also noticed in animals (Ewers et al., 2014; Marques et al., 2017). In this study, we report the identification

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and genomic characterization of the international high-risk clone of CTX-Mproducing E. coli ST648/F in free-roaming cats, in South America. Additionally, we

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have performed a comparative genomic analysis with ST648 isolates from human hosts, highlighting an epidemiologic linkage between human and companion animals.

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Between February and April 2017, during a local surveillance study established to study the occurrence and genetic features of broad-spectrum

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cephalosporin-resistant Enterobacteriaceae in companion animals, two extended-

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spectrum beta-lactamase (ESBL)-producing E. coli isolates (ICBECG2 and ICBECG4) were recovered from faecal swab samples collected from 20 free-roaming

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cats (admitted to a veterinary clinic to vaccination purposes) inhabiting urban slums, in southeastern Brazil. In this regard, rectal samples were screened onto selective MacConkey agar plates supplemented with ceftriaxone (2 mg/L), or colistin (2 mg/L), or meropenem (2 mg/L). ESBL-producing E. coli (10%) isolates were identified by MALDI-TOF mass spectrometry (Bruker Daltonics), whereas antimicrobial susceptibility was evaluated by disc diffusion assay or broth microdilution method (http://www.eucast.org).

ACCEPTED MANUSCRIPT Both E. coli isolates displayed a MDR profile to amoxicillin/clavulanic acid, ceftiofur, ceftriaxone, cefotaxime, ciprofloxacin, enrofloxacin, gentamicin, nalidixic acid, trimethoprim/sulfamethoxazole and tetracycline. Moreover, the two E. coli isolates were confirmed as extended-spectrum beta-lactamase (ESBL) producers, displaying cefotaxime and ceftriaxone MICs >32 mg/L, and remaining susceptible to

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carbapenems and colistin (http://www.eucast.org/clinical_breakpoints/). Indeed, blaCTX-M-2 and blaCTX-M-15 ESBL genes were identified by PCR and Sanger sequencing in E. coli ICBECG2 and ICBECG4, respectively.

Total DNA of both ESBL-producing E.coli strains was extracted and

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sequenced using an Illumina NextSeq500 platform. De novo assemblies were accomplished by CLC Genomic Workbench 10.0. Resistome, MLST, serotype,

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plasmidome and virulome were evaluated using bioinformatics tools available from the Center for Genomic Epidemiology (http://genomicepidemiology.org/), whereas

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genotyping data [phylogroup, whole genome MLST (wgMLST), core genome MLST (cgMLST) and ribosomal MLST (rMLST)] were further analyzed using EnteroBase

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(http://enterobase.warwick.ac.uk/species/index/ecoli).

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Main results from whole-genome sequencing analysis are summarized in Table 1. In both isolates, resistome revealed the presence of genes conferring

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resistance to aminoglycosides, β-lactams, macrolides, quinolones, sulfonamides, tetracyclines and trimethoprim. Although, both ICBECG2 and ICBECG4 strains harboured IncF plasmids, in silico analysis revealed that blaCTX-M genes were located into

chromosomal

contigs,

as

distinguished

by

BLASTn

(https://blast.ncbi.nlm.nih.gov/Blast.cgi) searching against the GenBank nucleotide database (Orlek et al., 2017). Additionally, attempts to demonstrate plasmid transfer

ACCEPTED MANUSCRIPT by broth mating and electroporation methods, using E. coli C600STR and E. coli TOP10 as recipient lineages, were unsuccessful. Regarding virulence determinants, the presence of air (enteroaggregative immunoglobulin repeat protein), iss (increased serum survival), mchF (ABC transporter protein MchF), cba (colicin B), cma (colicin M), ipfA (long polar

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fimbriae), gad (glutamate decarboxylase), ireA (siderophore receptor), eilA (Salmonella HilA homolog), tsh (temperature-sensitive hemagglutinin) and iroN (enterobactin siderophore receptor protein) virulence genes was confirmed in E. coli strain ICBECG2; whereas only the gad, ipfA and eilA genes were identified in strain

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ICBECG4. Both strains belonged to the high-virulent phylogenetic group F (Johnson et al., 2017).

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Interestingly, both ICBECG2 and ICBECG4 E. coli isolates belonged to the international high-risk clone sequence type ST648, which has contributed to the

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spread of CTX-M-, CMY-2-, NDM-, OXA-48-, and MCR-1-type encoding genes (Poirel et al., 2018). This E. coli lineage has emerged as a pandemic clone, being

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globally reported in healthy and diseased humans and companion animals (Ewers et

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al., 2014; Gonçalves et al., 2016; Marques et al., 2017; Sellera et al., 2018; Shaik et al., 2017; Solgi et al., 2017; Toleman et al., 2015). On the other hand, the association

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between ST648 and CTX-M-type β-lactamases has been reported in companion animals in Asian, European, American and Oceania countries (Ewers et al., 2014; Huber et al., 2013; Karkaba et al., 2017; Li et al., 2017; Marques et al., 2017), so far; highlighting the convergence of broad-spectrum cephalosporin resistance and high virulence background. A phylogenetic tree was constructed in Enterobase using the minimum spanning tree (MSTree V2) algorithm based on the whole genome MLST (wgMLST),

ACCEPTED MANUSCRIPT which consists of 25,002 loci from 71 E. coli ST648 genomes (accessed at the time of January

2018;

https://bitbucket.org/enterobase/enterobase-

web/wiki/Escherichia%20Statistics). Based on Patristic Distance matrix, the difference between ICBECG2 and ICBECG4 strains, resulting from the sum of branch length, was 1862.60, which was represented by clades E and H (Figure 1).

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Therefore, although these strains belong to the same ST and phylogenetic group, differences related to serotype, and virulence and resistance genes (i.e., presence of blaCTX-M-2 or blaCTX-M-15), might contribute for the genetic distance between them; even though they derive from a common ancestor (Spratt and Maiden, 1999). On the

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other hand, ICBECG2 was grouped together with human and livestock E. coli ST648 strains from France, United States, Australia and Denmark, whereas ICBECG4 was

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arranged in a restrict human cluster represented by American, Australian and Colombian E. coli isolates (Figure 1).

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The presence of the pandemic high-risk lineage of E. coli CTX-M-F-ST648 in healthy stray cats is a public health concern, since it could indicate an important and

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silent dissemination of this clinically significant clone in the human-animal-

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environment interface. Therefore, homeless dogs and cats should be considered as an important source of MDR pathogens, and a risk factor for dissemination of these

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bacteria to other companion animals and/or other animal species in contact with humans.

Although, the origin of the pandemic clone CTX-M-F-ST648 could not be identified in this study, this clone could have been acquired from the environment or human beings. More recently, raw pet food consumption has become another risk factor for shedding of ESBL-producing Enterobacteriaceaeto household cats, underscoring a new source of transmission of MDR bacteria to domestic cats (Baede

ACCEPTED MANUSCRIPT et al., 2017). Furthermore, it is noteworthy that free-roaming cats may be active predators of urban passerines and, in this sense, E. coli CTX-M-ST648 was also reported in free-living passerines, in Germany (Guenther et al., 2010), which could be another interesting influence to be considered. In summary, we report the identification of the international high-risk E. coli

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lineage ST648 recovered from free-roaming cats in South America. Therefore, monitoring free-roaming companion animals as potential sentinels is essential for a better understanding of potential transmission routes of MDR bacteria between

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humans and animals.

Acknowledgements

de

o

esen ol imento

a lo

ient ico e ecnol gico

is a PhD fellowship of

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stado

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This work was funded by research grants from

nda o de

m aro

nda o de -9),

m aro

Conselho

es

isa do

Nacional

de

462042/2014-6). M.R. Fernandes es

isa do

stado de

o a lo

PT

(FAPESP 2015/13527-2). N.L. is a research grant fellow of CNPq (312249/2017-9).

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We thank Cefar Diagnóstica Ltda. (Brazil) for kindly supplying antibiotic discs for

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susceptibility testing; and CEFAP-Genial facility for sequencing.

Conflict of interest The authors have no conflict of interest to declare.

ACCEPTED MANUSCRIPT REFERENCES Baede, V.O., Broens, E.M., Spaninks, M.P., Timmerman, A.J., Graveland, H., Wagenaar, J.A., Hordijk J., 2017. Raw pet food as a risk factor for shedding of extended-spectrum beta-lactamase-producing Enterobacteriaceae in household cats. PLoS One 12, e0187239.

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Clermont O, Christenson JK, Denamur E, Gordon DM. The Clermont Escherichia coli phylo-typing method revisited: improvement of specificity and detection of new phylo-groups. Environ Microbiol Rep. 2013, 5:58-65.

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Doi, Y., Zong, Z., Kola, A., Schaufler, K., Semmler, T., Fruth, A., Wieler, L.H., Guenther, S., 2014. CTX-M-15-D-ST648 Escherichia coli from companion

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animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J. Antimicrob. Chemother. 69, 1224-1230.

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Gonçalves, L.F., de Oliveira Martins-Júnior, P., de Melo, A.B.F., da Silva, R.C.R.M., de Paulo Martins, V., Pitondo-Silva, A., de Campos, T.A., 2016. Multidrug dissemination

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β-lactamase-producing

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ACCEPTED MANUSCRIPT Johnson, J.R., Johnston, B.D., Gordon, D.M., 2017. Rapid and specific detection of the Escherichia coli sequence type 648 complex within phylogroup F. J. Clin. Microbiol. 55, 1116-1121. Karkaba, A., Grinberg, A., Benschop, J., Pleydell, E., 2017. Characterisation of extended-spectrum

β-lactamase

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AmpC

β-lactamase-producing

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Increase in antimicrobial resistance and emergence of major international highrisk clonal lineages in dogs and cats with urinary tract infection: 16 year

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D., Woodford, N., Walker, A.S., Phan, H., Sheppard, A.E., 2017. Plasmid

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ACCEPTED MANUSCRIPT clone with other epidemiologically successful extraintestinal pathogenic Escherichia coli (ExPEC) lineages. MBio8, e01596-17. Solgi, H., Giske, C.G., Badmasti, F., Aghamohammad, S., Havaei, S.A., Sabeti, S., Mostafavizadeh, K., Shahcheraghi, F., 2017. Emergence of carbapenem resistant Escherichia coli isolates producing blaNDM and blaOXA-48-like carried on IncA/C

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ACCEPTED MANUSCRIPT TABLE 1Genomic analysis of CTX-M-producing Escherichia coli ICBECG2 and ICBECG4 strains isolated from free-roaming cats E. coli ST648 strain ICBECG2

ICBECG4

Source

Cat rectal swab

Cat rectal swab

Genome size (bp)

5,334,675

5,158,044

G + C content (%)

50.3

50.6

tRNA (n)

47

40

rRNA (n)

2

Non-conding RNA (n)

5

No. total of genes

5,595

Pseudogenes

273

No. of CDS

5,539

Serotype

O153:H9

fimH-type

H58

Phylogroup

F

b

cgMLST

5

5,322 145

5,273

O153:H6

648/648 c

wgMLST

57,956

e

62,362

Virulome

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ST/CC

3

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a

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Characteristics

air, iss, mchF, cba, cma, ipfA, gad,

H27 F

648/648 57,939 62,363 gad, ipfA, eilA

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ireA, eilA, tsh, iroN Resistome β-lactams

aadA5

blaCTX-M-2

blaTEM-1B, blaCTX-M-15

-

ermB, mphA

qnrB19

qnrB19, gyrA (Ser83Leu; Asp87Asn), parC (Ser88Ile)

sul1, sul2,

sul1

Tetracyclines

tetB

tetB

Trimethoprim

-

dfrA17

Inc-type

ColE, IncFIB, ColpVC, IncFIC

IncFIA, IncFIB, IncFII

PEDQ00000000.1

PEDR00000000.1

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Sulphonamides

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Macrolides Quinolones

aac(3)-VIa, aadA1

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Aminoglycosides

GenBank accession Number a

CDS, coding sequences. ST, sequence type; CC, clonal complex. c cgMLST, core genome multilocus sequence typing. d rMLST, ribosomal multilocus sequence typing. e wgMLST, whole genome multilocus sequence typing. b

ACCEPTED MANUSCRIPT FIGURE 1 Minimum spanning tree of seventy-one worldwide distributed E. coli strains belonging to ST648, based on the wgMLST constructed by the MSTree V2 algorithm

from

Enterobase.

The

figure

was

generated

with

iTOL

v.4

(https://itol.embl.de). Feline illustrations represent ICBECG2 (G2S) and ICBECG4

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(G4S) strains.

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FIGURE 1

ACCEPTED MANUSCRIPT Highlights 

Occurrence of ESBL-producing bacteria in free-roaming cats has been investigated. Presence of CTX-M-producing Escherichia coli is highlighted.

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Genomic analysis revealed presence of international high-risk lineage ST648.

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Dissemination of MDR pathogens among sentinel animals has been discussed.

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