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

Abstract

We report the spread of a clone of multidrug-resistant (MDR), ESBL-producing (blaCTX-M-1) Salmonella enterica subsp. enterica serovar Infantis, in the Italian broiler chicken industry and along the food-chain. This was first detected in Italy in 2011 and led to human infection in Italy in 2013-2014.A set (n = 49) of extended-spectrum cephalosporin (ESC)-resistant (R) isolates of S. Infantis (2011-2014) from humans, food-producing animals and meat thereof, were studied along with a selected set of earlier and more recent ESC-susceptible (ESC-S) isolates (n = 42, 2001-2014). They were characterized by macrorestriction-PFGE analysis and genetic environment of ESC-resistance. Isolates representative of PFGE-patterns and origin were submitted to Whole Genome Sequencing. The emerging ESC-R clone, detected mainly from broiler chickens, broiler meat and humans, showed a minimum pattern of clinical resistance to cefotaxime, tetracycline, sulfonamides, and trimethoprim, beside ciprofloxacin microbiological resistance (MIC 0.25 mg/L). All isolates of this clone harbored a conjugative megaplasmid (~ 280-320 Kb), similar to that described in ESC-susceptible S. Infantis in Israel (pESI-like) in 2014. This megaplasmid carried the ESBL gene blaCTX-M-1, and additional genes [tet(A), sul1, dfrA1 and dfrA14] mediating cefotaxime, tetracycline, sulfonamide, and trimethoprim resistance. It also contained genes conferring enhanced colonization capability, virulence (fimbriae, yersiniabactin), resistance and fitness (qacE1, mer) in the intensive-farming environment. This emerging clone of S. Infantis has been causing infections in humans, most likely through the broiler industry. Since S. Infantis is among major serovars causing human infections in Europe and is an emerging non-typhoidal Salmonella globally, further spread of this lineage in primary productions deserves quick and thorough risk-management strategies.

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.

Abbreviations: ESC-R: Extended-spectrum cephalosporin resistance; AMP: ampicillin; CTX: cefotaxime; CHL: chloramphenicol; CIP: ciprofloxacin; NAL: nalidixic acid; STR: streptomycin; KAN: kanamycin; GEN: gentamicin; TET: tetracycline; SMX: sulfamethoxazole; TMP: trimethoprim

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

A) Multiplex alignment of the region where drfA1 and aadA1 genes are located in the pESI-like plasmid, in different combinations, in the isolates of this study; in the pESI plasmid (ASRF01000099); and in the reference sequence for the drfA1 gene (JQ690541). The arrows represent the genes: integrase I (orange), drfA1 (blue), aadA1 (green), multidrug efflux pump (pink). B) Pairwise comparison of the pESI-like megaplasmid in the 12037823/11 isolate, and the pESI plasmid (ASRF01000099) where the “substitution” of aadA1 by drfA1 in the isolate of this study is represented. C) Pairwise comparison of the pESI-like megaplasmid in the 13002124/34 isolate, and the pESI megaplasmid (ASRF01000099) where the “insertion” of drfA1 in the isolate of this study is represented.

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).