+

Clonal expansion of ciprofloxacin-resistant Campylobacter jejuni

Jasna Kovač, Alison J. Cody, James E. Bray, Kate E. Dingle, Sonja Smole Možina, Martin C.J. Maiden

+

Oxford, United Kingdom

Ljubljana, Slovenia

+Campylobacter

!

+Importance of Campylobacter

Food safety aspect

23.6% broiler meat on the market contaminated, in some EU countries up to 80% !

Public health aspect

#1 bacterial cause of gatroenteritis in western world

+Antibiotic resistance in Campylobacter

Most of the human campylobacteriosis cases are caused by Campylobacter jejuni

Disease is usually self-limiting, but severe cases need clinical treatment with antibiotics

Quinolones (ciprofloxacin) and macrolides (erythromycin) are the first drugs of choice for campylobacteriosis treatment

High prevalence of antibiotic resistance is becoming a serious problem

www.med.uottawa.ca

+Prevalence of antibiotic resistance in Campylobacter

Ciprofloxacin Co-amoxiclav Erythromycin Gentamicin Tetracyclines0

2000

4000

6000

8000

10000

12000

14000

Resistant Susceptible

Isola

tes (

N)

EFSA, 2015

54.6% !

+Antibiotic resistance mechanisms

Multidrug resistance (cmeABC)

porA, omp50

Tetracycline (tetO) Quinolones (gyrA)

Aminoglycosides (aphA-3 …)

β-lactams (Cj0299)

porA, omp50

Iovine, 2013

Plasmid encoded Plasmid encoded

+Prevalence of antibiotic resistance - Studied population

Campylobacter strains isolated from patients in Oxfordshire

The hospital covers population of 600,000, which is about 1% of UK population the results are representative for UK

Time frame: 2 years, 1714 isolates Year 1 (June 2011 - June 2012): 839 isolates Year 2 (June 2012 - June 2013): 875 isolates

  Total Year 1 Year 2

STs 293 204 174

CCs 33 31 31

rSTs 596 377 330

+Whole genome sequencing

Illumina HiSeq 2000

76-bp paired-end reads generated

Assembled with Velvet version 1.2.01

Established automatized genome assembly and annotation pipeline

+PubMLST database

Consists of two separate databases that interact:

Sequence and profile definitions (allele seq. definitions)

PubMLST Isolate Database (metadata)

www.pubmlst.org/campylobacter

+BIGS

Bacterial Isolate Genome Sequence Database (BIGSdb)

Pubmlst.org/campylobacter database runs on BIGS

1,667 loci defined in the database, including: MLST loci Ribosomal (rMLST) loci porA antigen-encoding genes etc.

BIGSdb autotagger automatically: Identifies loci Assigns alleles Tags the sequences for future reference

Automatic report of the MLST alleles, sequence types (STs), clonal complexes and porA alleles, genome comparison…

+Genome comparator module in BIGS

Calculates the distance matrix based on the differences in allelic types (number of loci that differ between each pair of isolates)

The distance matrix is generated in NEXUS format

NEXUS format is used as an input for SplitsTree phylogenetic analysis

SplitsTree generates the phylogenetic network based on the split decomposition neighbor-net method Advantage of networks over trees is that they account for

the recombination events

+Antibiotic resistance prevalence

  Gene % Resistant

  Year 1 Year 2

Quinolones gyrA 38 38.5

Tetracyclines tetO 41.2 37.6

β-lactams Cj0299 79.5 81.4Aminoglicosides

aphA-3, aadE, sat4 0.7 0.7

MDRgyrA, tetO, Cj0299 21.3 22.1

 Unique variants

gyrA 243tetO 222Cj0299 43aphA-3 4aadE_B 1aadE_Ef 2sat4 4cmeB 267porA 442

MOMP 227omp50 222

The phenotypic resistance to QN was determined in a subset of 685 isolates from year two over 97% congruence between phenotypic and genotypic methods of resistance determination

+Association of quinolone resistance with MLST genotypes

21

*50

*5136

*464

*354

*572

0

2

4

6

8

10

Sequence type

Pro

port

ion o

f is

ola

tes (

%)

21

828

48

*464

UA

206

353

*354

0

5

10

15

20

25

30

Clonal complex

Pro

port

ion o

f is

ola

tes (

%) Green colour (*) represents

statistically significant association of genotype with with quinolone resistance.

+Increased prevalence of ST-464 over the years

+How does the quinolone resistance spread?

By many individual events of resistance mutation

or

with increased survival fitness of specific resistant clones?

+

1501 core loci

Genome comparator (distance matrix)SplitsTree (wgs phylogenies)

Blue = susceptible genotypeRed = resistant genotype

+ Clonal expansion of quinolone resistance

1515 core loci

1525 core loci

Blue = susceptible genotypeRed = resistant genotype

+ Co-resistance to quinolones and tetracycline – chicken associated clonal complexes

Co-resistance to CIP and TETYear 1: 221/839 (26.3%)Year 2: 217/875 (24.8%)

Co-resistance to CIP, TET and BLYear 1: 179/839 (21.3%)Year 2: 193/875 (22.1%)

CIP … ciprofloxacinTET … tetracyclinBL … β-lactams

Isolates from year 1 and 2

TET R is chromosomally encoded in ST-464 and ST-354

Alignment, NJ tree produced in MEGA

+Why are these two genotypes so successful?Random forest analysis in Orange data mining tool to identify genes that are associated with this genotype.

http://orange.biolab.si

+Conclusions

High prevalence of quinolone resistance in Campylobacter in UK (38%)

MLST clonal complexes ST-464 and ST-354 are highly associated with quinolone resistance

Quinolone resistance is expanding clonally in ST-464 and ST-354

High level of co-resistance to quinolones, tetracyclines and β- lactams (21-22% MULTIDRUG RESISTANCE)

New approaches to managing antibiotic resistance and preventing the proliferation of resistance along the food production chain are needed

Thank you for your attention!

Now…

Thanks to ICGEB, BIOS, and Genetic Society of Slovenia for travel grant.