spread of an emerging clone of mdr, esbl-producing ......salmonella infantis (st32) from humans,...

Spread of an emerging clone of MDR, ESBL-producing Salmonella Infantis harbouring a

conjugative megaplasmid in Italy

Antonio BattistiNRL-AR, IZSLT, Rome, Italy

EURL-AR Workshop 2016Kgs. Lyngby 14 April 2016

Salmonella entericasubsp. entericaserovar Infantis hasemerged worldwide

Salmonella Infantis has been confirmed as the 4th mostcommon serovar reported in human cases, EU 2012-2014

Same %in Italy

S. Infantis: the most prevalent serovar in poultryand poultry meat (broiler meat) in EU in 2014

Increasing trend of S. Infantis in broiler meat, EU 2012-2014

Antimicrobial Resistance: main findings in EU,

MDR and ESC-R S. Infantis is emerging in humans

MDR S. Infantis is common in EU in humans, including ItalyMinimum common AMR pattern: TET+SUL+ TMP+[CIP]

Multidrug (3+) resistance rates (%) in Salmonella spp from NCP in broilers Italy, 2009-2014

10,00

15,00

20,00

25,00

30,00

35,00

40,00

45,00

50,00

55,00

60,00

65,00

2009 (N=140) 2010 (n=62) 2011 (n=29) 2012 (n=77) 2013 (n=64) 2014 (n=66)

Year

% MD

R (3+

)

Resistance rates (%) to extended-spectrum cephalosporins in broilers, from NCP Salmonellosis, Italy (2009-2014)

0

5

10

15

20

25

30

35

40

45

2009 (n=140) 2010 (n=62) 2011 (n=29) 2012 (n=77) 2013 (n=64) 2014 (n=66)

Year

% E

SC

-RTrend: Statistically significant rise

Almost all isolates in the last three years were MDR, ESC-R S. Infantis

In the National survey conducted on slaughter batches from differentepidemiological units representing >90% of broiler chicken production:

Salmonella prevalence of 12.7%

S. infantis represented 75% of allisolates (68/90), prev.: 9.5%

MDR ESC-R S. Infantis prev. 3.4%http://www.salute.gov.it/portale/documentazione/p6_2_2_1.jsp?lingua=italiano&id=2476

Materials• Salmonella Infantis isolates (n = 91) representative of national

AMR monitoring activities (2001 to 2014) were included:• -ESC-R n = 49 (2011–2014), S. Infantis isolates, • 23 were from different broiler chicken flocks, 1 from a pig holding• 6 from broiler meat, 2 from pork• 17 from unrelated human clinical cases•• -Outgroup of ESC-S n = 42 (2001 to 2014) randomly selected • 6 from different broiler chicken flocks, 1 froma guinea fowl holding• 22 from unrelated human clinical cases, • 6 from broiler meat, 5 from pork, 2 from “unspecified” meat

Methods

A variety of laboratory methods were used on ALL isolates:

-Antimicrobial susceptibility testing-Macrorestriction PFGE (XbaI digested DNA)-Detection of genes encoding beta-lactamases,

carbapenemases and PMQR-Plasmid detection (S1 Nuclease, PBRT) and genetic

environment of ESC-resistance + transferabilityA selection of isolates (based on PFGE) was Whole

Genome sequenced

• Fig 1. XbaI PFGE macrorestriction cluster analysis and antimicrobial resistance patterns of 91 ESC-susceptible and ESC-resistant Salmonella Infantis (ST32) from humans, animals and meats thereof, 2001–2014.

Franco A, Leekitcharoenphon P, Feltrin F, Alba P, Cordaro G, et al. (2015) Emergence of a Clonal Lineage of Multidrug-Resistant ESBL-Producing Salmonella Infantis Transmitted from Broilers and Broiler Meat to Humans in Italy between 2011 and 2014. PLoSONE 10(12): e0144802. doi:10.1371/journal.pone.0144802http://journals.plos.org/plosone/article?id=info:doi/10.1371/journal.pone.0144802

24 unique PFGE patterns, which wereseparated by > 90% similarity into

two defined PFGE groups I and II

Group I included 63 out of 91 (69.2%) isolates, of which 42 (46% of the total) hadindistinguishable profiles:pulsotype A

All these isolates fromhumans, broiler chickens and broiler meat (one from pork) were MDR and almostall ESC-R (2011-2014)

All FQ-R (MIC0.25 mg/L

AMR pattern and genetic basis of the MDR, ESC-R clone

• All ESC-resistant S. Infantis: blaCTX-M-1 gene, except forone human isolate with blaCTX-M-65 gene.

• MDR was a constant feature of the ESC-R isolates (and of Group I): Minimum common resistance pattern:CTX-[AMP]-TET-SMX-TMP-NAL-CIP-[STR]

-MICs to NAL (128 mg/L), and CIP (0.25 mg/L) not in the range of clinical resistance. NO PMQR gene

Genetic Environment of MDR ESBL+ve S Infantis

• Sixty isolates, including all the 49 ESC-R isolates,harboured a huge (280-320 Kb) plasmid that tested positive for the IncP incompatibility group using a PBRT.

• But it contained IncI1 ! (ardA, pilL, sogS, trbA) pMLSTgenes, except the IncI1 replicase gene repl1 (it was anIncP…).

• A conjugative “mosaic megaplasmid”, named “pESI-like”similar to that described in Israel (pESI, Aviv et al. 2013) of was identified in the end.

Some isolatespulsotype A,pESI-like+ve,were ESC-S:

No CTX-Mgene(s) but allother AMRgenes

WGS analysis of the core genomesSNP-based phylogeny on selected recent and older isolates (human; broilers, pigs and their meats)demonstrated that this ESBL-producing, pESI-likepositive clone:

• Has been transmitted along the broiler chickenindustry (animals and meat thereof) to humansand caused disease

• Is different from the earlier clone(s) which weredominant up to 2008… (Dionisi et al, 2011) and whichcarried other plasmids, either incH1 (or IncI1)

• Fig 3. Single-nucleotide polymorphism (SNP)-based phylogeny of 12 selected ESC-resistant and ESC-susceptible Salmonella Infantis from poultry, meats, and humans, in Italy (2006–2014).


9 to 29 SNP difference foundbetween isolates of broiler chicken and human origin(2012-2014): Within SNP difference range found in outbreak isolates!

Two clusters separated by 119 SNPsSNP-tree on

547 informative SNPs

pESI-like megaplasmid content• Beside blaCTX-M-1: acquired resistance genes:

tet(A), sul1, dfrA1& dfrA14, and aadA1 genes(vs drugs most used in Italy and in the EU)• Fimbriae (ipf, K88-like usher feaD) and the

yersiniabactin siderophore system genes, associatedwith increased ability for colonization and virulence;

• Genes associated with resistance and enhanced fitness(e. g. qacEΔ1, mer operon)advantageous in the intensive-farming environment.

• Fig 2. Different combinations of antimicrobial resistance genes in the same region of pESI-like plasmids harbored by Salmonella Infantis.


Compared with the Israeli megaplasmid,

dfrA1 has substitutedaadA1 or has beeninserted

Israeli

megaplasmid

Conclusions

• WGS analysis approach may facilitate sourceattribution studies of human cases(supported by epidemiological data)

• Particularly useful for certain infections– whose pattern is not outbreak-associated or– whose epidemic nature goes unrecognised

Conclusions• S. Infantis is among serovars of major concern in

EU• The “Italian” emerging clone was able to acquire

and maintain in the intensive broiler farming system a plasmid which confers:-MDR and traits of major concern (ESBL);-increased pathogenicity, and enhanced fitness

• Further concern arises from Co-R to FQ (mutation)

Conclusions

• Collaborative studies across the EU would bevery useful to investigate similarities anddifferences within this serovar

• Thorough risk-management strategies forquick action to be taken are needed, to stopthe spread and mitigate human exposure

• Input / collaboration from all stakeholders isnecessary for its control

-NRL-AR & Department

Alessia Franco

Fabiola Feltrin

Patricia Alba

Gessica Cordaro

Manuela Iurescia

Rita Tolli

-The ITAVARM network

-The National HealthInstitute (ISS, Ida Luzzi)

-EURL-AR

WHO Collaborating Centre forAR in Foodborne pathogens

Rene Hendriksen

Pimlapas Leekitcharoenphon

spread of an emerging clone of mdr, esbl-producing ......salmonella infantis (st32) from humans,...

Documents