Pathogens, Vol. 11, Pages 1446: Antimicrobial Resistance of Salmonella Strains Isolated from Human, Wild Boar, and Environmental Samples in 2018–2020 in the Northwest of Italy

1. IntroductionSalmonellosis is the second most common zoonosis reported in Europe [1]. The genus Salmonella comprises enteric bacteria hosted by humans and numerous domestic and wild animals [2]. Some Salmonella serovars, such as S. Typhi and S. Parathypi A and C, are host-adapted and responsible for typhoid fever in humans; they, in turn, act jointly, with primates as their main reservoirs [3,4]. Other serovars have a generalist behaviour, infecting a broad range of hosts, and are mainly responsible for non-typhoidal diseases in humans. Among these, S. Enteritidis, S. Typhimurium, S. Typhimurium 1,4,[5],12:i:-, S. Infantis and S. Derby have been recently reported to be the most common serotypes involved in human infections [1]. Salmonella is excreted via the faecal route and is able to disseminate in the environment and enter in the food chain. Salmonellosis in humans is generally contracted through the consumption of contaminated food of animal origin, but other types of foods, such as green vegetables irrigated with contaminated water, might also serve as a vehicle of infection [5]. Antimicrobial resistance (AMR) in foodborne pathogens are of great concern for public health safety [6], especially for the emergence of multidrug-resistant (MDR) strains, i.e., bacteria showing resistance to at least three or more antimicrobial classes [7]. Many factors may favour their development and spread, including the inappropriate use of antimicrobials in human medicine, self-medication or the early suspension of prescribed therapies, as well as their misuse in food-producing animals, aquaculture and agriculture [8]. Genes conferring resistance can be transferred among bacteria by different mechanisms [9], increasing the abundance of resistant pathogenic microorganisms that, in turn, potentially hamper the effectiveness of antimicrobial treatments. Understanding the AMR dynamics between animals and humans sometimes becomes complicated, especially when they share the same environment. The latter, alongside wild fauna, can play an important role as reservoirs of AMR, contributing to the spread of resistant bacteria to food-producing animals and humans [10]. Among wildlife species, wild boar can serve as a good indicator for the environmental spread and transmission of resistant Salmonella strains [11]. In fact, this species is increasingly expanding to more anthropized areas, leading to direct and indirect contacts with humans and their domestic animals that may also increase the chance of disease transmission [12]. Wild boar is the most widespread wild ungulate species in Liguria, northwest Italy, sometimes concentrating in high densities even near peri-urban and urban areas.Integrated surveillance of AMR in pathogenic bacteria spreading in humans, animals and the environment, is one of the top priorities for the European Union/European Economic Area (EU/EEA) [13]. In 2003, the European Parliament and the Council issued Directive 2003/99/EC to ensure that zoonoses, zoonotic agents, and related AMR were properly monitored [14]. In Italy, the surveillance of Salmonella in humans and in the environment is coordinated by Enter-Net Italia (Istituto Superiore di Sanità, Rome), while its surveillance in food products and animals is organized by the Italian Reference Centre for Salmonellosis (Enter-Vet, Istituto Zooprofilattico Sperimentale delle Venezie, Padova). In Liguria, Salmonella surveillance is carried out by the Reference Centre for Salmonella Typing (CeRTiS, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, Turin), which collects Salmonella strains isolated from human, animal, and environmental samples according to national surveillance and monitoring programs. In the view of a One Health approach, this study aimed to investigate the serotypes and related AMR profiles of Salmonella spp. strains circulating in humans, the environment and wild boar in Liguria, northwest of Italy, between 2018 and 2020. 3. DiscussionThis study highlights the great diversity of Salmonella spp. to which the human population is exposed in Liguria, northwest Italy, as well as the main serotypes circulating in its natural environment, including the wild boar population. Salmonella Typhimurium 1,4,[5],12:i was the predominant serotype identified in human infections, which is in contrast to the recent data reported about zoonoses published at the European level by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (ECDC) [1]. In 2020, S. Typhimurium 1,4,[5],12:i ranked in the third position among the most prevalent serotypes involved in human infections in Europe, with around 11% prevalence [1]. In Italy, this serotype has been frequently reported to be the most common cause of salmonellosis in humans and the main serotype isolated from animals and meat food products, especially in those from the swine production chain [15,16]. Infection by S. Typhimurium 1,4,[5],12:i has been recently recorded to exceed up to four times the infection by S. Typhimurium in humans, animals and food products [16]. This trend also emerged among the human infections occurring in our study area, where S. Typhimurium ranked in second place, with prevalence comparable to those reported at the European level [1]. Differences in the predominance of Salmonella serotypes in human infections may be explained by differences in the geographical context. For instance, S. Typhimurium seems to be the most prevalent serotype involved in human infections from southern Italy, prevailing over S. Typhimurium 1,4,[5],12:i: and S. Enteritidis [17]. Salmonella Napoli was the third serotype recorded in human infections during the study period and the second most common serotype isolated from the environment. The occurrence of this serotype is increasing in Italy but is uncommonly reported in Europe [18,19,20]. We isolated S. Napoli also from wild boar, supporting previous findings reported from the same area that highlighted their potential role as spreaders of this serotype in the environment [21]. Salmonella Enteritidis is the most common serotype involved in human infections in Europe [1]; however, we reported a lower prevalence, being the fourth most common serotype identified in human infections. In the veterinary field, a notable reduction in the presence of this serotype has been generally observed in recent years in Italy. The active Italian control programs implemented for the eradication of Salmonella in poultry farms may justify the minor contribution of S. Enteritidis to human infections [15,22]. Similarly, other European countries such as Greece, endowed with comparable eradication programs against Salmonella in poultry, have observed the decreasing trend of S. Enteritidis in humans, reducing its prevalence by half in the last 20 years [23]. Notwithstanding, in this country, S. Enteritidis remains the main serotype isolated in human samples. Salmonella Infantis and S. Derby are among the top five serotypes involved in human infections in Europe. The epidemiological situation observed for these serotypes in the Ligurian population is in agreement with the European average [1] and with that experienced in other Italian regions [17]. An increasing trend of infection by S. Infantis has been recently described in poultry meat [24,25], and it has also been associated with human outbreaks in Italy [26,27], while human infections by S. Derby are mostly related to the consumption of pork and poultry products [28]. S. Typhi, responsible for typhoid fever in humans, was isolated in a very low percentage of the human cases in our study. Despite typhoid fever being rare in Italy, mainly occurring in people travelling from endemic countries, important levels of AMR were observed in isolated strains [29]. The lower diversity in serotypes observed in environmental strains may be justified by the lower sampling effort employed. However, environmental factors, such as precipitation and water temperature, strongly influence the abundance and diversity of Salmonella spp. in surface waters [30]. Salmonella Veneziana was the most common serotype isolated from surface waters, followed by S. Napoli and S. Stourbridge. We also reported one isolate of S. Veneziana from human samples. This serotype has been previously reported to cause human infections [31] and has also been isolated from wild animals [15,32,33,34]. We also ascertained the presence of both S. Typhimurium and S. Typhimurium 1,4,[5],12:i in water surface samples although in low prevalence. By contrast, a previous study investigating the diversity of Salmonella strains in environmental water in France reported S. Typhimurium as the most widespread serotype in river water, marine, and freshwater sediment, probably related to the wastewater discharge from animal-rearing activities [35]. Our findings about the low presence of S. Typhimurium and its monophasic variant in the sampled surface waters could be an indicator of water contamination from not only humans but from other sources, such as animal faeces. For instance, our research group has previously reported S. Veneziana, S. Napoli, and S. Stourbridge in wild boar samples in the same area of study [21]. The S. Stourbridge has been proven to cause disease in humans [36] and its additional discovery in wild boar samples reaffirms the possible role of this animal species in the transmission of this serotype. S. Newport predominated among the serotypes identified from wild boar. Although this latter serotype was not relatively common among the human cases investigated, its pathogenicity has been proven in other countries by being the main one responsible for several human outbreaks [37,38]. We generally uncovered high AMR levels in our sample of Salmonella isolates. The highest prevalence of AMR was especially observed against sulfamides, ampicillin, tetracycline, and streptomycin, as previously described [39]. The levels of AMR reported for these antibiotics are even higher than those reported for the general Italian context [40], excepting for streptomycin, for which data are not available. Our findings are consistent with the results obtained from a recent Italian survey, in which resistance against sulfamides, ampicillin, and tetracycline were the most common type of AMR observed in human Salmonella isolates [17]. An increase in AMR against ampicillin, chloramphenicol, and trimethoprim–sulfamethoxazole—i.e., antibiotics previously considered of first line for the treatment of Salmonella infections—has been globally observed [41]. We also observed a high level of resistance against tigecycline among human isolates. This antibiotic is considered an optimal choice for the treatment of MDR Salmonella infections; however, its efficacy may be compromised due to the phenomenon of heteroresistance, which has been already observed for S. Typhimurium [42]. Despite the low resistance levels usually reported for human Salmonella strains against tigecycline [17,39,40], in some contexts these levels of resistance are higher and spread in some livestock productions, such as poultry [43,44]. Fluoroquinolones are recommended drugs to treat invasive salmonellosis or fragile patients at risk of developing it, but AMR in typhoid and non-typhoidal Salmonellae against this antibiotic is increasingly reported [45,46,47]. In 2017, the World Health Organization (WHO) listed fluroquinolones-resistant Salmonellae among the high-priority-bacteria group for which it is necessary to support the research and development of effective drugs [48]. The AMR observed against nalidixic acid in our human strains are in line with reports from other Italian and European regions, while the reported AMR prevalence against ciprofloxacin is lower [17,39,40]. Third-generation cephalosporins are defined by WHO within the category of "highest priority critically important antimicrobials". Variable degrees of AMR against this kind of antibiotics have been observed in typhoid and non-typhoidal Salmonella strains involved in human infections worldwide [49,50,51]. The origin of AMR against third-generation cephalosporins mainly resides in extended-spectrum β-lactamase genes [52]. In Europe, an increase of this kind of AMR has been recently reported, especially in Salmonella strains isolated from humans [52]. Notwithstanding, the average prevalence of AMR against third-generation cephalosporins (ceftazidime and cefotaxime) remains at least relatively low (0.8%). For the Italian context, the AMR prevalence against ceftazidime and cefotaxime are similar, averaging around 2% for both antibiotics [40]. We have recorded higher AMR levels against ceftazidime, while a lower prevalence was observed for cefotaxime. However, contrasting results have been reported in other regions. For instance, in the Netherlands, there have been observed higher levels of resistance against cefotaxime and lower AMR levels against ceftazidime, recording a maximum prevalence of 2.1% [39]. In the south of Italy, resistance levels were analysed, targeting specific Salmonella serotypes: S. Derby, S. Infantis, and S. Typhimurium, and recorded the highest levels of AMR against ceftazidime and cefotaxime, ranging from 19.0% to up to 40.0% of prevalence, however, the greatest levels of AMR were generally recorded when these serotypes were exposed to cefotaxime [17]. We observed resistance against cephalosporins of the third generation in 12 different strains, but they most frequently occurred in S. Typhimurium 1,4,[5],12:i:-. An important finding of this study is the absence of resistance against carbapenem in all of the Salmonella strains isolated; they are considered critical antibiotics of last resort. Carbapenem-resistant Salmonella have been previously isolated from animals, animal-food products, and humans [53,54,55]. In Europe, only a few official reports related to carbapenem resistance have been hitherto published, recording a very low level of AMR in the human infections from Spain (0.1%) in 2019 and from Denmark (0.4%) and Belgium (0.1%) in 2020 [40]. We also observed noteworthy AMR levels against azithromycin that exclusively involved almost all environmental strains isolated in 2020. Our results, however, cannot be directly compared with other studies, since our sample size was very low. In Europe, azithromycin resistance has been reported in human isolates from 2020, with an average prevalence of 0.8%; in Italy, this prevalence reached levels up to 0.3% during the same period. Even so, records from southern Italy have reported 36.3% of AMR prevalence against azithromycin in Salmonella strains isolated from humans and a lower percentage in samples from food of animal origin (7.3%) between 2017 and 2021 [17]. In 2020, azithromycin was overused during the COVID-19 pandemic [56,57,58], so it would be interesting to monitor the trend of the AMR against this antibiotic to evaluate its impact in the near future. We observed that the phenomenon of MDR more often occurred among human isolates; however, simultaneous resistance against ampicillin, streptomycin, tetracycline, and sulfamides (AMP-STR-SSS-TET) was experienced by S. Typhimurium 1,4,[5],12:i: isolated from both humans and the environment. This co-resistance is increasing worldwide, and it has also been recently reported in Italy, especially in Salmonella strains collected from human and animal samples [59,60].The spread of Salmonella strains, concurrently resistant to third-generation cephalosporines and fluoroquinolones, is an important therapeutic issue in many parts of the world [61,62]. Unfortunately, we also observed co-resistance to ciprofloxacin and ceftazidime in two isolates of S. Typhimurium and in a single S. Othmarschen of human origin. Although moderate and high levels of AMR were observed against the combination trimethoprim + sulfamethoxazole and sulfamides, the AMR prevalence observed in environmental strains was generally lower compared with that detected in human samples. High AMR prevalence against sulfamides and streptomycin have been reported in urbanized areas [63], as has the presence of environmental strains resistant against ciprofloxacin and cefotaxime [48,59]. In our environmental strains, no evidence for resistance against third-generation cephalosporines was observed, while the AMR levels against fluoroquinolones were very low. The role of the environment in the spread of AMR is known and has been recently reviewed [64]. Most of the AMR and MDR strains detected from our environmental isolates are probably related to water contamination, for example, as a consequence of wastewater discharge from anthropized areas and/or agricultural activities, but also by the entry of Salmonella strains from wildlife that may host AMR strains of variable degree. Many actions have been taken by the European Union and the Italian government to face the AMR phenomenon. The reduction in the use of antimicrobials in animals has been one of the main targets of these initiatives, in particular in livestock [65]. In our geographic context, livestock activity is very limited; hence, its contribution to the spread of AMR pathogens can be considered low. This situation may explain the lower presence of AMR in the environmental samples and also the high susceptibility observed in Salmonella isolates from wild boar. Conversely, the role of wild boars as reservoir and spreaders of MDR bacteria had been already observed in the same study area [15,66] but also in other parts of Italy [67,68] and Europe [69]. Herein, we report lower levels of AMR in wild boar compared to previous studies conducted in the same study area [21]. These contrasting results may be explained by the lower sample size analysed in the present study, making it difficult to draw a general picture about AMR in this animal population. More in-depth studies should be carried out in order to better characterize AMR strains circulating in wild boar populations and to assess the role of the environment in the release of resistant bacteria.

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