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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
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Salmonella entericasubsp. entericaserovar Infantis hasemerged worldwide
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Salmonella Infantis has been confirmed as the 4th mostcommon serovar reported in human cases, EU 2012-2014
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Same %in Italy
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S. Infantis: the most prevalent serovar in poultryand poultry meat (broiler meat) in EU in 2014
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Increasing trend of S. Infantis in broiler meat, EU 2012-2014
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Antimicrobial Resistance: main findings in EU,
MDR and ESC-R S. Infantis is emerging in humans
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MDR S. Infantis is common in EU in humans, including ItalyMinimum common AMR pattern: TET+SUL+ TMP+[CIP]
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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+
)
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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
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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
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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
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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
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• 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
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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
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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.
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Some isolatespulsotype A,pESI-like+ve,were ESC-S:
No CTX-Mgene(s) but allother AMRgenes
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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)
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• 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).
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
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
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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.
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• Fig 2. Different combinations of antimicrobial resistance genes in the same region of pESI-like plasmids harbored by Salmonella Infantis.
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
Compared with the Israeli megaplasmid,
dfrA1 has substitutedaadA1 or has beeninserted
Israeli
megaplasmid
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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
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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)
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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
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-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