297 Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 Accepted: 11.03.2020 | Available on line: 31.12.2021 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy. *Corresponding author at: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’, Teramo, Italy. E-mail: lo.sacchini@izs.it. Lorena Sacchini, Lucilla Ricci, Katiuscia Zilli, Romina Romantini, Guido Di Donato, Diana Neri, Tiziana Persiani and Elisabetta Di Giannatale* Keywords S. Typhimurium, Monophasic S. Typhimurium, Antimicrobial resistance, MLVA. Summary Salmonellosis is currently the second most common zoonosis in European Union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease trend in the yearly number of infections caused by Salmonella. In Italy, S. Typhimurium and monophasic S.  Typhimurium represent the most prevalent serotypes. In this paper, we investigated these two serovars isolated from 2012 to 2017 in Abruzzo and Molise regions. A set of 345 strains isolated from human sporadic cases, surface water, food and animals were collected and analyzed. Monophasic S. Typhimurium strains were found to be resistant to streptomycin, sulfisoxazole, ampicillin, tetracycline and nalidixic acid, while S. Typhimurium isolates showed high levels of resistance to tetracycline, sulfisoxazole and ampicillin. The 5-loci Multilocus Variable-Number Tandem Repeat Analysis (MLVA) identified 88 genotypes (GT), six of which were common for the two serovars. Several MLVA profiles were shared by human and non-human isolates. MLVA had sufficient typing resolution for epidemiological studies of S. Typhimurium but demonstrated poor discriminatory in trace-back study of monophasic S. Typhimurium. Characterization of Salmonella Typhimurium and monophasic Salmonella Typhimurium isolated in Abruzzo and Molise regions, Italy, from 2012 to 2017 in the EU, a total of 91,857 human salmonellosis cases were reported; Salmonella Enteritidis and Salmonella Typhimurium were the most common serovars, followed by monophasic S.  Typhimurium, S.  Infantis and S.  Newport (EFSA and ECDC 2018). In 2018, the number of reported confirmed human cases and the EU notification rate were at the same level as in 2017 (EFSA and ECDC 2019, EFSA and ECDC 2018). In Italy, S.  Typhimurium and monophasic S.  Typhimurium are two of the most commonly serovars isolated from humans and animals. A similar situation has also been reported in other European countries (Molbak et  al. 2014, Mueller-Doblies et  al. 2018). In Italy, over last few decades, various trends have been observed in the prevalence of different Salmonella serotypes. Between 1980 and 1988 S.  Typhimurium was the serotype most commonly isolated from humans, and in the 90’s the highest number of cases were caused by S. Enteritidis. Since 2000, and until 2012, S.  Typhimurium was once again the most prevalent serotype reported. The Introduction Salmonella spp. is one of the most common foodborne pathogens and salmonellosis is the second most frequently reported zoonosis in Europe (EFSA and ECDC 2019, EFSA and ECDC 2018). Monophasic variants of Salmonella Typhimurium-like strains, lacking the fljB-encoded second phase H antigen appear to be of increasing importance in many European Union (EU) Member States and a rapid emergence and dissemination of such strains in livestock animals, companion animals and humans has been observed (EFSA J 2010). While the number of Salmonella-positive samples collected annually is generally decreasing, the monophasic S. Typhimurium is one of the few serovars for which the opposite trend has been described both in humans and in animals and it is currently considered an emerging epidemic serovar. On the basis of genetic similarity, the monophasic strains with formula 4,[5],12:i:- are regarded as variants deriving from S.  Typhimurium that display similar virulence and antimicrobial resistance characteristics. In 2017 298 Characterization of Salmonella Typhimurium in Italy Sacchini et al. Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 Italia surveillance network. The Enternet Italia is coordinated by the Istituto Superiore di Sanità and relies on the collaboration of peripheral laboratories. It is incorporated into the European surveillance system for Food- and Waterborne Diseases (FWD) and coordinated by the ECDC. The strains isolated from animals were collected in farms. Isolates from food were obtained from the producers and from retail stores during official veterinary controls. All the strains were serotyped according to the Kauffmann-White-Le Minor schema by the slide agglutination method (Grimont et  al. 2007) using commercial antisera (Statens Serum Institute, Copenhagen, Denmark). To confirm the strains serotyped as S. Typhimurum or monophasic variant, a PCR assay was also performed, as previously described (Barco et al. 2011). Antimicrobial susceptibility testing The susceptibility to sixteen antibiotics was performed by the disc diffusion method (Kirby-Bauer) in accordance with the Clinical Laboratory Standards Institute guidelines (Patel et  al. 2017) using the following BD Sensi-DiscTM (Becton and Dickinson, Berkshire, U.K): ampicillin (A) 10  μg, amoxicillin-clavulanic acid (AmC) 20/10 μg, cefazolin (Cz) 30 μg, gentamycin (G) 10 μg, kanamycin (K) 30 μg, enrofloxacin (En) 5 μg, trimethoprim/sulfamethoxazole (Sxt) 1.25/23.75 μg, tetracycline (T ) 30 μg, ceftazidime (Caz) 30 μg, colistine (Cl) 10  μg, sufisoxazole (Su) 300 μg, nalidixic acid (Na) 30  μg, streptomycin (S) 10 μg, chloramphenicol (C) 30  μg, cephalothin (Cf ) 30 μg and ciprofloxacin (Cip) 5  μg. Escherichia coli ATCC 25922 was used as a control. Multilocus Variable‑Number Tandem Repeat Analysis (MLVA) The 345 strains were typed using 5 locus MLVA (STTR9-STTR5-STTR6-STTR10-STTR3) according to the protocol previously described by ECDC (Takkinen et  al. 2011) using multiplex PCR and capillary electrophoresis on an ABI 3500 instrument with POP 7 polymer (Thermo Fisher Scientific, MA, USA). The MLVA profiles were expressed according to the nomenclature published by Larsson and colleagues (Larsson et al. 2009). The genetic diversity of each locus was determined using the Simpson’s diversity index (SDI) calculated for a dataset of S.  Typhimurium and monophasic S.  Typhimurium, comprising of only one strain per MLVA genotype and using the online tool available at the Comparing Partitions website (http://www. comparingpartitions.info/?link=Tool). MLVA clustering was performed using the monophasic S. Typhimurium was isolated for the first time in Italy from a patient in 2003 and the number of cases has increased in the last years (Graziani et al. 2013, Dionisi et al. 2009). S.  Typhimurium was the predominant serovar isolated from veterinary samples between 2002 and 2011, however in 2012, the numbers of monophasic S.  Typhimurium-positive samples exceeded the numbers of S.  Typhimurium and a steady rise in the prevalence of the monophasic variant has been observed in the following years (https://www.izsvenezie.it/temi/malattie-patogeni/ salmonella/enter-vet/) (accessed on 22 October 2019). The emergence of a clonal group of serovar S.  Typhimurium and monophasic S.  Typhimurium R-type ASSuT (resistante to ampicillin, streptomycin, sulphonamides and tetracycline) strains in Italy, Denmark and the United Kingdom has also been described (Lucarelli et al. 2010). Multilocus variable-number tandem repeat analysis (MLVA) is a highly discriminative typing method (EFSA 2013) that has emerged as a powerful tool for subtyping of food-borne bacterial pathogens (Nadon et al. 2013) and it is used for S. Typhimurium subtyping, especially in outbreak investigations (Torpdahl et  al. 2007, Petersen et  al. 2011, Ross et al. 2011). The main aims of this study were to investigate the epidemiology of S.  Typhimurium and monophasic S.  typhimirium (4,[5],12:i:-) in Abruzzo and Molise regions, to elucidate the characteristics of the resistotypes and genotypes circulating in the environment and in animal and human populations between 2012 and 2017. Materials and methods Strains collection A set of 345 strains including 65 isolates of S.  Typhimurium and 280 isolates of monophasic S.  Typhimurium were collected in Abruzzo and Molise regions of Italy between January 2012 and December 2017. The strains of S. Typhimurium were isolated from fecal samples of chickens (n = 2), pigs (n  =  9), wild birds (n  =  6), humans (n  =  13), pork meat (n  =  6), and surface water samples (n  =  31). The strains of monophasic S.  Typhimurium were isolated from fecal samples (from 15 pigs, 2 chickens and from 145 humans), from food (21 from pork, 2 from mixed beef-pork minced meat, 1 from lamb, 1 from turkey, 8 from bivalve molluscs) and from 85 surface water samples. The strains isolated from human feces and surface water were sent to our laboratory by regional hospitals and environmental protection agency, respectively, within the Enternet 299 Sacchini et al. Characterization of Salmonella Typhimurium in Italy Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 Results The antimicrobial resistance traits of the two Salmonella serovars are shown in Table II. More than a half of tested isolates were resistant to tetracycline (61.35% for S. Typhimurium and 66.39% for monophasic S.  Typhimurium). Moreover, a large number of monophasic S. Typhimurium strains were resistant to streptomycin (81.89%), sulfisoxazole (58.49%), ampicillin (52.08%) and nalidixic acid (48.30%), while S.  Typhimurium isolates showed high levels of resistance to sulfisoxazole (38.64%) and ampicillin (36.36%). Fewer monophasic S.  Typhimurium isolates were resistant to cefazolin, kanamycin, ceftazidime, colistin, cephalotin and ciprofloxacin, while S.  Typhimurium isolates were fully susceptible to these antimicrobials (Table II). More than 30% of monophasic S.  Typhimurium belonged to the ASSuT R-type (95 strains) and only the 5.6% to the ACSSuT type (16 strains) while for S.  Typhimurium the ASSuT and ACSSuT profiles accounted for 6.87% (3 strains for each serovars). The MLVA analysis classified the 345 strains (Figure 1) into 88 GTs. Six of the MLVA profiles were common for both serovars, while 33 were specific for S. Typhimurium, and 49 for monophasic S. Typhimurium. The six common genotypes between the two serotypes were GTs 25 (3-11-9-NA-211 MLVA profile), 27 (3-12-10-NA-211), 61 (3-15-11-NA-211), 64 (3-15-12-NA-211), 70 (3-17-11-NA-211) and 75 (3-8-10-NA-211). BioNumerics software package version 7.6. (Applied-Maths, Sint-Martens-Latem, Belgium) with MST (Minimum Spanning Tree) method. Clonal complexes (CCs) were retrieved with the most stringent (conservative) definition (Spratt et al. 2004) where all members assigned to the same CC differed only by one locus with the nearest member included in the same CC. Table I shows GTs-MLVA patterns conversion. Table I. GTs-MLVA patterns convertion. Salmonella Typhimurium Monofasic Salmonella Typhimurium MLVA pattern GTs MLVA pattern GTs MLVA pattern GTs 2-10-6-6-112 2 1-9-NA-NA-210 1 3-14-9-NA-210 60 2-13-NA-NA-311 3 2-19-NA-NA-211 10 3-15-11-NA-211 61 2-16-6-12-212 4 3-10-10-NA-211 15 3-15-12-NA-210 63 2-16-6-14-112 5 3-10-11-NA-211 16 3-15-12-NA-211 64 2-17-NA-NA-105 6 3-10-12-NA-211 17 3-15-7-NA-211 65 2-17-NA-NA-211 7 3-10-15-NA-211 18 3-15-8-NA-NA 66 2-17-5-7-212 8 3-10-9-NA-112 19 3-15-8-NA-211 67 2-17-7-14-112 9 3-11-10-NA-211 21 3-15-9-NA-211 68 2-20-NA-NA-211 11 3-11-11-NA-211 22 3-17-12-NA-311 70 2-22-9-14-112 12 3-11-15-NA-211 23 3-9-12-NA-211 75 2-6-4-9-212 13 3-11-8-NA-211 24 3-9-5-NA-211 76 2-8-5-9-212 14 3-11-9-NA-211 25 3-10-9-NA-210 81 3-11-9-NA-211 25 3-12-NA-NA-211 26 3-13-NA-NA-211 82 3-12-10-NA-211 27 3-12-10-NA-211 27 3-11-NA-NA-211 83 3-12-12-12-211 31 3-12-11-NA-211 28 3-13-14-NA-211 43 3-12-11-18-NA 29 3-13-7-NA-211 44 3-12-12-NA-211 30 3-14-NA-21-311 49 3-12-13-NA-211 32 3-14-12-7-211 55 3-12-3-NA-211 33 3-14-16-17-311 57 3-12-7-NA-211 34 3-15-11-NA-211 61 3-12-8-NA-211 35 3-15-11-NA-311 62 3-12-9-NA-NA 36 3-15-12-NA-211 64 3-12-9-NA-211 37 3-17-12-NA-211 69 3-13-NA-7-211 38 3-17-12-NA-311 70 3-13-10-NA-211 39 3-19-12-NA-311 71 3-13-11-NA-211 40 3-19-NA-NA-311 72 3-13-12-NA-211 41 3-8-10-NA-211 73 3-13-13-NA-211 42 3-9-NA-NA-210 74 3-13-7-7-211 45 3-9-12-NA-211 75 3-13-8-NA-211 46 4-12-9-7-211 77 3-13-9-NA-210 47 4-13-9-22-311 78 3-13-9-NA-211 48 6-24-15-22-11 79 3-14-10-NA-211 50 6-9-13-9-211 80 3-14-11-NA-211 51 6-19-15-18-NA 84 3-14-11-9-211 53 2-19-6-14-112 85 3-14-12-NA-211 54 2-17-NA-NA-210 86 3-14-13-NA-211 56 3-10-NA-NA-210 87 3-14-3-NA-211 58 2-20-5-13-112 89 3-14-8-NA-211 59 Table II. Antibiotic resistance rates (%) of S. Typhimurium and S. 4,[5],12:i:- isolated between 2012 and 2017 in Abruzzo and Molise regions. Antibiotic % Resistant S. Typhimurium S. 4,[5],12:i:- Ampicillin (A) 10μg 36.36 52.08 Amoxicillin/Clavulanic acid (Amc) 20/10 μg 11.36 20.38 Cefazolin (Cz) 30 μg 0,00 3.02 Gentamicyn (G) 10 μg 4.55 3.02 Kanamycin (K) 30 μg 0,00 5.66 Enrofloxacin (En) 5 μg 6.82 12.08 Trimethoprim/sulfamethoxazole (Sxt) 1.25/23.75 μg 4.55 9.81 Tetracycline (T) 30 μg 61.36 66.79 Ceftazidime (Caz) 30 μg 0,00 0.01 Colistine (Cl) 10 μg 0,00 1.89 Sufisoxazole (Su) 300 μg 38.64 58.49 Nalidixic acid (Na) 30 μg 9,09 48.30 Streptomycin (S) 10 μg 22.73 81.89 Chloramphenicol (C) 30 μg 6.82 6.04 Cephalothin (Cf ) 30 μg 0.00 6.79 Ciprofloxacin (Cip) 5 μg 0.00 0.00 300 Characterization of Salmonella Typhimurium in Italy Sacchini et al. Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 (3-15-11-NA-311) included human and pig samples, GT72 (3-19-NA_NA-311) and GT61 (3-15-11-NA-211) contained strains isolated from humans and the surface water. GT27 (3-12-10-NA-211) included S.  Typhimurium isolated from humans and surface water and belonged to CC2 that contained isolates from pig. CC5 included only isolates from surface water, while CC3, CC4 and CC7 comprised of strains from pig and surface water. GT85 (2-19-6-14-112) group included only poultry isolates and clustered closely with GT5 (2-16-6-14-112) samples from surface water in CC9. We identified four genotypes that were shared between the human and environmental or animal samples. The genotype that contained the higher number of isolates was G62 (MLVA profile 3-15-11-NA-311) that was identical for seven pigs and 1 human isolate. GT61, with a MLVA pattern 3-15-11-NA-211, was assigned to isolates from four surface water samples and from one human sample. Genotype 27 (3-12-10-NA-211) was found in one water, two wild birds and 1 human isolates, and G72 (3-19-12-NA-311) was shared between one strain collected from surface water and two strains isolated from humans. For monothasic S.  Typhimurium, clustering analysis retrieved only one major CC which grouped almost all the MLVA genotypes (45 out of 55) (Figure 3). Several genotypes, i.e. GT28 (3-12-11-NA-211) and GT37 (3-12-9-NA-211) included, beside human isolates, samples from more than one source (pig, turkey, poultry, water, etc.). Three MLVA profiles, 3-11-10-NA-211 (GT21), 3-12-3-NA-211 (GT33) and 3-11-15-NA-211 (GT23), were shared between humans and bivalve molluscs; while, 3-11-11-NA-211 (GT22) included monophasic variant strains isolated from humans and sheep. Fifteen genotypes were retrieved from human isolates only. GT51 (MLVA profile 3-14-11-NA-211) was the most common genotype identified in monophasic S.  Typhimurium (85 human feces and 25 surface water strains), followed by GT41 (3-11-10-NA-211) assigned to 14 surface water and 3 human isolates. Other frequently identified MLVA pattern included The degree of polymorphism of MLVA genotypes associated with the two serovars was quantified by calculating the diversity index of each locus (Table  III). For S.  Typhimurium, STTR9 showed the lowest number of alleles (n = 4) with a SDI of 0.622, STTR5 resulted as the most variable locus (15 allele) with SDI of 0.934. For monophasic S.  Typhimurium only STTR5 and STTR6 were highly discriminating, other loci showed an SDI below to 0.325 (Table III). Ten CCs were assigned using MST analysis of S.  Typhimurium based on MLVA typing results (Figure 2). The major clonal complex (CC1) included 8 genotypes, three of which were associated with human and non-human isolates. GT62 Figure 1. Minimum Spanning Tree (MST) of 65 S. Typhimurium and 280 S. 4,[5],12:i:- analyzed between 2012 and 2017 in Abruzzo and Molise regions. The node labels represent the unique genotype (GT). The diameter of each node is proportional to the number of isolates. The branch length is proportional to the loci differences. Table III. Number of partition, Simpson’s diversity index (SDI) and confidence interval (CI 95 %) for the five loci of the MLVA panel for S. Typhimurium and S. 4,[5],12:i:-. The calculation of confidence intervals for Simpson's index was performed by use of the large sample approximation, thereby improving the objective assessment of the discriminatory power of typing techniques. Locus S. Typhimurium S. 4,[5],12:i:- # partitions SDI CI (95%)* # partitions SDI CI (95%)* STTR9 4 0.622 (0.536-0.708) 3 0.072 (1.000-0.168) STTR5 15 0.934 (0.903-0.964) 9 0.868 (0.843-0.893) STTR6 13 0.899 (0.853-0.944) 11 0.894 (0.868-0.921) STTR10 11 0.725 (0.580-0.869) 4 0.173 (0.038-0.308) STTR3 8 0.777 (0.684-0.871) 5 0.325 (0.168-0.481) #Number of different repeats present at this locus; *Precision of the Diversity Index, expressed as 95% upper and lower boundaries. 301 Sacchini et al. Characterization of Salmonella Typhimurium in Italy Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 possible implication of environmental sources was suspected (Cito et al. 2016). The MLVA protocol designed for Salmonella Typhimurium routine typing (Takkinen et  al. 2011) is also commonly used for the characterization of monophasic S.  Typhimurium and it has facilitated the successful detection and investigation of multiple food-borne disease outbreaks (Cito et  al. 2016, Barco et  al. 2014, Raguanaud et  al. 2012). For routine surveillance of Salmonella, MLVA seems to have notable advantages over Pulsed field gel electrophoresis (PFGE) method. The protocol is fast and can be completely automated and the generated data are easily analyzed and compared between laboratories. Moreover, MLVA has been shown to have a higher discriminatory power than PFGE (Torpdahl et  al. 2007, Hopkins et  al. 2007, Best et  al. 2009). In our study, the analysis of the MLVA profiles of S.  Typhimurium and S.  4,[5],12:i:- isolates showed that, in spite of the high similarity and close relationship between the two serovars (Switt et  al. 2009), only six MLVA profiles were in common between the two serovars. Moreover, the genetic resolution power of three MLVA loci was much lower for monophasic S.  Typhimurium than for S.  Typhimurium. Cluster analysis performed for monophasic variant retrieved only one major clonal complex that grouped almost all the MLVA profiles, 3-11-10-NA-211 (G21) assigned to humans (n  =  4), pigs (n = 4), surface water (n = 3) and bivalve molluscs (n = 3). 3-12-11-NA-211 MLVA profile (GT28) found in human (n = 1), surface river (n = 1), pigs (n = 1), cattle (n = 1) and chikens (n = 2) strains and 3-12-3-NA-211 (GT33) assigned to human (n = 4),pigs (n = 3),surface river (n  =  2) and strains isolated from bivalve molluscs (n = 2). Discussion In Italy, S.  Typhimurium and monophasic S.  Typhimurium are two of the most commonly reported serovars isolated from human and veterinary samples. While, in general, the total number of yearly Salmonella isolation have been decreasing, the opposite trend has been observed for a few serovars including monophasic S.  Typhimurium (EFSA and ECDC 2019). Our data confirmed this observation as were reported four times greater number of isolated monophasic variant than S.  Typhimurium strains. The high dissimilarity of number of sampling between the two serogroups was partly due to an epidemic outbreak (MLVA 3-14-11-NA-211) of monophasic S. Typhimurium observed in Abruzzo region during 2013 and 2014 (67.80% of the strains) where a Figure 2. Minimum spanning tree (MST) of 65 strains of S. Typhimurium isolated in Abruzzo and Molise regions between 2012 and 2017. The node labels represent the unique genotype (GT) and are colored according to the source of isolation. The diameter of each node is proportional to the number of isolates. The branch length is proportional to number of different loci. Clonal complexes (CCs) are shaded in grey. Figure 3. Minimum spanning tree (MST) of strains of 280 monophasic S. Typhimurium:- isolated in Abruzzo and Molise regions between 2012 and 2017. The node labels represent the unique genotype (GT) and are colored according to source of isolation. The diameter of each node is proportional to the number of isolates. The branch length is proportional to number of different loci. Clonal complexes (CC) are shaded in grey. 302 Characterization of Salmonella Typhimurium in Italy Sacchini et al. Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 2017 approximately 40% of human S. Typhimurium isolates, and 80% of monophasic S.  Typhimurium were resistant to multiple antimicrobials. In pigs, multi-drug resistance (MDR) was observed in approximately 60% of S.  Typhimurium and 77% of the monophasic variant isolates (EFSA and ECDC 2019). In recent years, there has been an overall decline in the level of antimicrobial resistance in serovar S. Typhimurium in several European countries related to a decrease in the number of isolates of multiresistent DT104 (Meakins et al. 2008). To some extent this reduction has been counterbalance by the increase in prevalence of monophasic S.  Typhimurium that expresses resistance to ampicillin, streptomycin, sulphonamides and tetracyclines (Mueller-Doblies et  al. 2018, Lucarelli et  al. 2010). In our study, we found that more than 30% of S.  Typhimurium were resistant to four antibiotics (ASSuT resistance type) and only 5.6% were additionally resistant to chloramphenicol (ACSSuT resistance type). Overall, the majority of monophasic S.  Typhimurium isolates appeared to show a multidrug resistance phenotype while the majority of S.  Typhimurium isolates were resistant to only a few antimicrobial drugs (data not showed). In conclusion, based on the characterization of human and non-human S.  Typhimurium and monophasic S.  Typhimurium strains obtained over a 6-year surveillance at the regional level, we found antimicrobial resistance results comparable to those reported by the last EFSA report from 2017. The MLVA 5-loci scheme resulted suitable for S.  Typhimurium typing but the discriminatory power observed for monophasic S.  Typhimurium was insufficient when used as an exclusive typing method in epidemiological studies. Use of powerful discrimination typing method based on Whole Genome Sequencing (WGS) such as SNPs analysis or core genome MLST are recommended to identify epidemiological relationship of monophasic S. Typhimurium isolates. confirming the lack of discrimination of STTR3, STTR10 and STTR9 loci, as previously demonstrated by Barco and colleagues (Barco et  al. 2015) On the contrary, for S.  Typhimurium, the diversity of MLVA profiles was conferred by all the five loci (SDI > 0.6). Hopkins and colleagues (Hopkins et  al. 2007) reported that since MLVA is a highly discriminatory method, some minor changes in targeted loci can be found in outbreak-related strains. Therefore, for Salmonella the authors proposed a difference of no more than two repeats at one single locus to identify isolates that are part of the same outbreak. As the MLVA protocol for typing of monophasic S.  Typhimurium includes three out of five highly stable loci, the correct interpretation of MLVA profiles can be critical especially for large scale epidemiological investigations and for source attribution studies. In our work, MLVA analysis of S.  Typhimurium assigned only four genotypes in common between human and non-human isolates (pig or surface water), while, for monophasic S.  Typhimurium about half of genotypes were shared by human isolates and the isolates from other sources (pig, turkey, poultry, water, etc.). These findings support the conclusion of Barco and colleagues (Barco et al. 2015) that 5-loci MLVA scheme applied to monophasic S.  Typhimurium may not provide a sufficient resolution for source attribution studies. In the definition of an outbreak caused by monophasic S.  Typhimurium, Cito and colleagues (Cito et  al. 2016) reported the PFGE profile and the R-type. Another study confirmed the insufficient resolution of MLVA scheme in the characterization of S.  4,[5],12:i:- isolates, given that isolates with considerable diversity in terms of single-nucleotide polymorphisms (SNPs) were indistinguishable by their MLVA profiles (Petrin et al. 2019). The EFSA-ECDC report from 2017 (EFSA and ECDC 2018) describing the prevalence of antimicrobial resistance in EU included data on Salmonella isolated from humans, fattening pigs and calves. In 303 Sacchini et al. Characterization of Salmonella Typhimurium in Italy Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 Barco L., Barrucci F., Cortini E., Ramon E., Olsen J.E., Luzzi I., Lettini A.A. & Ricci A. 2015. Ascertaining the relationship between Salmonella Typhimurium and Salmonella 4,[5],12:i:- by MLVA and inferring the sources of human salmonellosis due to the two serovars in Italy. Front Microbiol, 6, 301. Barco L., Lettini A.A., Ramon E., Longo A., Saccardin C., Pozza M.C.D. & Ricci A. 2011. A rapid and sensitive method to identify and differentiate Salmonella enterica serotype Typhimurium and Salmonella enterica serotype 4,[5],12:i:- by combining traditional serotyping and multiplex polymerase chain reaction. Foodborne Pathog Dis, 8, 741-743. Barco L., Ramon E., Cortini E., Longo A., Dalla Pozza M.C., Lettini A.A., Dionisi A.M., Olsen J.E. & Ricci A. 2014. Molecular characterization of Salmonella enterica serovar 4,[5],12:i:- DT193 ASSuT strains from two outbreaks in Italy. Foodborne Pathog Dis, 11, 138-144. Best E.L., Hampton M.D., Ethelberg S., Liebana E., Clifton-Hadley F.A. & Threlfall E.J. 2009. Drug-resistant Salmonella Typhimurium DT 120: use of PFGE and MLVA in a putative international outbreak investigation. Microb Drug Resist, 15, 133-138. Cito F., Baldinelli F., Calistri P., Di Giannatale E., Scavia G., Orsini M., Iannetti S., Sacchini L., Mangone I., Candeloro L., Conte A., Ippoliti C., Morelli D., Migliorati G., Barile N.B., Marfoglia C., Salucci S., Cammà C., Marcacci M., Ancora M., Dionisi A.M., Owczartek S., Luzzi I. & outbreak investigation group. 2016. Outbreak of unusual Salmonella enterica serovar Typhimurium monophasic variant 1,4 [5],12:i:-, Italy, June 2013 to September 2014. Euro Surveill, 21 (15), 10.2807/1560-7917.ES.2016.21.15.30194. https://doi. org/10.2807/1560-7917.ES.2016.21.15.30194. Clinical and Laboratory Standards Institute (CLSI). 2017. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100. Wayne, PA. Dionisi A.M., Graziani C., Lucarelli C., Filetici E., Villa L., Owczarek S., Caprioli A. & Luzzi I. 2009. Molecular characterization of multidrug-resistant strains of Salmonella enterica serotype Typhimurium and Monophasic variant (S. 4,[5],12:i:-) isolated from human infections in Italy. Foodborne Pathog Dis, 6, 711-717. European Centre for Disease Prevention and Control (ECDC). 2011. Laboratory standard operating procedure for MLVA of Salmonella enterica serotype Typhimurium. ECDC, Stockholm. European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC). 2018. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA Journal, 16 (12), e05500. European Food Safety Authority (EFSA) and European Centre for Disease Prevention and Control (ECDC). 2019. The European Union One Health 2018 Zoonoses Report. EFSA Journal, 19 (2), e06406. EFSA Panel on Biological Hazards (BIOHAZ). 2010. Scientific Opinion on monitoring and assessment of References the public health risk of “Salmonella Typhimurium-like” strains. EFSA Journal, 8, 1826. EFSA Panel on Biological Hazards (BIOHAZ). 2013. Scientific Opinion on the evaluation of molecular typing methods for major food-borne microbiological hazards and their use for attribution modelling, outbreak investigation and scanning surveillance: Part 1 (evaluation of methods and applications). EFSA Journal, 11 (12), 3502. Graziani C., Mughini-Gras L., Owczarek S., Dionisi A., Luzzi I. & Busani L. 2013. Distribution of Salmonella enterica isolates from human cases in Italy, 1980 to 2011. Euro Surveill, 18 (27), 20519. Grimont P.A.D. & Weill F.-X. 2007. Antigenic formulae of the Salmonella serovars. 9th ed. Institut Pasteur, Paris, France. Hopkins K.L., Maguire C., Best E., Liebana E. & Threlfall E.J. 2007. Stability of multiple-locus variable-number tandem repeats in Salmonella enterica serovar typhimurium. J Clin Microbiol, 45, 3058-3061. Larsson J.T., Torpdahl M., Petersen R.F., Sorensen G., Lindstedt B.A. & Nielsen E.M. 2009. Development of a new nomenclature for Salmonella typhimurium multilocus variable number of tandem repeats analysis (MLVA). Euro Surveill, 14 (15), 19174. Lucarelli C., Dionisi A.M., Torpdahl M., Villa L., Graziani C., Hopkins K., Threlfall J., Caprioli A. & Luzzi I. 2010. Evidence for a second genomic island conferring multidrug resistance in a clonal group of strains of Salmonella enterica serovar Typhimurium and its monophasic variant circulating in Italy, Denmark, and the United Kingdom. J Clin Microbiol, 48, 2103-2109. Meakins S., Fisher I.S.T., Berghold C., Gerner-Smidt P., Tschäpe H., Cormican M., Luzzi I., Schneider F., Wannett W. Coia J., Echeita A., Threlfall E.J & Enter-net participants. 2008. Antimicrobial drug resistance in human non typhoidal Salmonella isolates in Europe 2000-2004: a report from the Enter-net International Surveillance Network. Microb Drug Resist, 14, 31-35. Mølbak K., Simonsen J., Jørgensen C.S., Krogfelt K.A., Falkenhorst G., Ethelberg S., Takkinen J. & Emborg H.D. 2014. Seroincidence of human infections with nontyphoid Salmonella compared with data from public health surveillance and food animals in 13  European countries. Clin Infect Dis, 59 (11),1599-1606. Mueller-Doblies D., Speed. K.C.R., Kidd S. & Davies R.H. 2018. Salmonella Typhimurium in livestock in Great Britain - trends observed over a 32-year period. Epidemiol Infect,164 (4), 409-422. Nadon C.A., Trees E., Ng L.K., Møller Nielsen E., Reimer A., Maxwell N., Kubota K.A. & Gerner-Smidt P. 2013. MLVA Harmonization Working Group Development and application of MLVA methods as a tool for inter-laboratory surveillance. Euro Surveill, 18, 20565. Petersen R.F., Litrup E., Larsson J.T., Torpdahl M., Sørensen G., Müller L. & Nielsen E.M. 2011. Molecular characterization of Salmonella Typhimurium highly 304 Characterization of Salmonella Typhimurium in Italy Sacchini et al. Veterinaria Italiana 2021, 57 (4), 297-304. doi: 10.12834/VetIt.2060.12083.1 tools in the epidemiological investigation of Salmonella enterica serovar Typhimurium clusters. Curr Microbiol, 62, 1034-1038. Spratt B.G., Hanage W.P., Li B., Aanensen D.M. & Feil E.J. 2004. Displaying the relatedness among isolates of bacterial species - the eBURST approach. FEMS Microbiol Lett, 241, 129-134. Switt A.I.M., Soyer Y., Warnick L.D. & Wiedmann M. 2009. Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i:-. Foodborne Pathog Dis, 6, 407-415. Torpdahl M., Sørensen G., Lindstedt B.-A. & Nielsen E.M. 2007. Tandem repeat analysis for surveillance of human Salmonella Typhimurium infections. Emerging Infect Dis, 13, 388-395. successful outbreak strains. Foodborne Pathog Dis, 8, 655-661. Petrin S., Longo A., Barco L., Cortini E., Peruzzo A., Antonelli P., Ramon E., Cibin V., Lettini A.A. & Ricci A. 2019. Different resolution power of multilocus variable-number tandem repeat analysis and whole-genome sequencing in the characterization of S.  1,4,[5],12:i:- Isolates. Foodborne Pathog Dis, 16, 558-561. Raguenaud M.E., Le Hello S., Salah S., Weill F.X., Brisabois A., Delmas G. & Germonneau P. 2012. Epidemiological and microbiological investigation of a large outbreak of monophasic Salmonella Typhimurium 4,5,12:i:- in schools associated with imported beef in Poitiers, France, October 2010. Euro Surveill, 17, 20289. Ross I.L., Davos D.E., Mwanri L., Raupach J. & Heuzenroeder M.W. 2011. MLVA and phage typing as complementary