Monitoring of Antimicrobial Resistance of Salmonella Serotypes Isolated from Humans in Northwest Italy, 2012–2021

Salmonella enterica is among the most common causes of foodborne outbreaks in humans in Europe. The global emergence of resistance to antimicrobials calls for close monitoring of the spread and prevalence of resistant Salmonella strains. In this study, we investigated the occurrence of antimicrobial resistance of Salmonella serotypes isolated from humans between 2012 and 2021 in Piedmont, northwest Italy. A total of 4814 Salmonella strains (168 serotypes) were tested against six classes of antimicrobials. Many strains (83.3%) showed resistance to at least one antibiotic: tetracycline (85.1%), ampicillin (79.2%), quinolones (47.4%), and gentamicin (28.4%). Between the first (2012–2016) and the second study period (2017–2021), a decrease in antimicrobial resistance was noted for tetracycline (from 92.4% to 75.3%), ampicillin (from 85.3% to 71.3%), quinolones (from 49.4% to 44.6%), and cefotaxime (from 34.8% to 4.0%). Many multidrug resistant Salmonella strains (43.6%) belonged to S. ser. Typhimurium, S. ser. Infantis, and S. ser. Typhimurium 1,4,[5],12:i:-. Overall, multidrug resistance decreased from 60.7% to 26.4%, indicating a reduction in the antimicrobial resistance of Salmonella strains in Piedmont and in Europe and demonstrating the effectiveness of the measures that were put in place to reduce antimicrobial resistance.


Introduction
Salmonella enterica is the leading cause of foodborne outbreaks in the European Union (EU). In 2020 alone, 94 foodborne outbreaks of salmonellosis were reported, causing 3686 cases of illness, 812 hospitalizations, and seven deaths [1]. In 2021, 60,494 laboratory-confirmed cases of non-typhoidal salmonellosis were reported in the EU, with an increase of 14% in cases, compared to 2020. Seventy-three cases were fatal, accounting for a case fatality rate of 0.19%. The EU notification rate for salmonellosis was 16.6 cases per 100,000 population. Italy reported 3768 cases with a notification rate of 6.4 per 100,000 population [2].
Salmonella is a ubiquitous bacterium of which six subspecies and more than 2600 serotypes are known. Serotypes of the species Salmonella enterica can be classified as typhoid and nontyphoid (NTS), based on differences in host specificity, virulence, and severity of the clinical manifestations they cause in humans [3]. Typhoid Salmonella strains include S. ser. Typhi and S. ser. Paratyphi, which are human host-restricted organisms that cause enteric fever, a severe systemic syndrome with moderate to high fatality rates when untreated [4,5]. By contrast, NTS strains usually cause self-limiting gastrointestinal infections in humans. They are often acquired through the consumption of contaminated animal food products made from domestic and wild animals, which are the natural reservoirs. Furthermore, fruits and vegetables can also serve as vehicles for pathogen transmission [6,7]. In Europe, pig and poultry food products are associated with nontyphoid human infections, most Pathogens 2023, 12, 89 2 of 11 often caused by S. ser. Enteritidis, followed by S. ser. Typhimurium, S. ser. Typhimurium 1,4, [5],12:i:-, S. ser. Infantis, and S. ser. Derby [1]. Because NTS strains cause self-limiting gastrointestinal infections in humans, antimicrobial treatment is not normally required. In a small percentage of cases, however, the infection is invasive (iNTS) and antimicrobial treatment is essential and life-saving [8]. In sub-Saharan Africa iNTS infections particularly affect immunosuppressed populations, with children (<5 years) experiencing a mortality rate of 20-30%. Salmonella ser. Typhimurium (77.4%), Salmonella ser. Enteritidis (17.0%), and Salmonella ser. Dublin (0.1%) have been recorded as the main serovars involved in iNTS infections during the period from 1996 to 2016 [9]. Similarly, S. ser. Typhimurium was the most common serovar in iNTS infections from Nigeria, registering a prevalence of 39.8% during 1999-2018 and followed by S. ser. Enteritidis (29.3%) [10]. Infants, young children, the elderly, and the immunocompromised are at particular risk for bacteremia, in which multi-resistant strains are also more likely to cause invasive disease [11].
Multidrug resistant (MDR) Salmonella strains are defined as resistant to three or more antimicrobial classes [12]. The increasing resistance against first-line antimicrobials (aminopenicillins, trimethoprim-sulfamethoxazole, chloramphenicol) in the treatment of salmonellosis has led to a shift in treatment to fluoroquinolones and third-generation cephalosporins [8] and resistance has emerged with the wider use of fluoroquinolones over the last twenty years [11].
Moreover, some Salmonella serotypes have developed resistance towards broadspectrum cephalosporins through mutations in genes encoding extended-spectrum βlactamases [13]. The global emergence of resistance to antimicrobials calls for close monitoring of the spread and prevalence of resistant and multi-resistant strains and to detect possible qualitative and quantitative variations over time.
Our institute (Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, IZSPLV) supports public health agencies in human/animal outbreak investigations in northwest Italy, in addition to carrying out diagnostic testing and research activities in national surveillance programs for animal health and food safety. In 2011, IZSPLV was designated a regional reference center for Salmonella typing (Centro di Riferimento per la Tipizzazione delle Salmonelle, CeRTiS) within the national surveillance networks for enteric pathogens in human medicine (Enter-NET). CeRTiS is involved in the identification and characterization of enteric pathogens. The institute performs serotyping and investigates the antimicrobial resistance (AMR) profiles of Salmonella strains. Surveillance data on enteric pathogens generated by CeRTiS activities are regularly sent to the Italian National Institute of Health (ISS) and then to the European Centre for Disease Prevention and Control within the frame of the European Food-and Waterborne Diseases and Zoonoses Network (FWD-Net).
The aim of the present study was to investigate the prevalence of Salmonella serotypes and the occurrence of antimicrobial resistance in strains isolated from humans in Piedmont between 2012 and 2021.

Human Sample Collection and Salmonella Serotyping
CeRTiS receives samples of enteric pathogens isolated from symptomatic human patients presenting at health care centers. A total of 22 clinical laboratories located in Piedmont (northwest Italy) sent CeRTiS Salmonella strains isolated from cases of human infections. These Salmonella strains are previously identified using the Vitek ® 2 System (Biomerieux, Marcy l'Étoile, France) or Wellcolex Color Salmonella Test (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturers' instructions. A total of 4814 S. enterica strains isolated from 2012 to 2021 were analyzed. The isolates were obtained from samples of feces (n = 4518), urine (n = 111), blood (n = 129), purulent exudate (n = 6), cerebrospinal fluid (n = 1), and other biological fluids (n = 49). The strains were subcultured on Columbia Blood Agar (Becton&Dickinson, Franklin Lakes, NJ, USA) at 37 • C for 24 h and then serotyped according to the Kaufmann-White and Le Minor scheme [14] using O and H antisera (Statens Serum Institut, Artillerivej, Denmark).

Statistical Analysis
Data analysis was performed using Stata 16 [19]. The binomial exact test was applied to calculate the prevalence of the Salmonella serotypes identified and to summarize the prevalence of AMR for each class of antibiotics and their combinations. We evaluated temporal patterns in prevalence and AMR of Salmonella strains with the non-parametric Wilcoxon-type test for trend [20]. Comparisons of infection prevalence between serotypes according to the source of the samples were made using Pearson's chi-squared test. We also applied this test to compare AMR against single drug and MDR patterns among Salmonella serotypes during the first study period (2012-2016) and the second (2017-2021). Statistical significance was set at p < 0.05.

Figure 2.
Prevalence and 95% CI of AMR in the most common serotypes recovered in the two halves of the study period. Prevalence is expressed as the number of isolates that showed resistant phenotypes against at least one drug. Asterisks denote significant differences in AMR between the two time periods (Pearson's chi-squared test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5.
Prevalence and 95% CI of MDR in the most frequent serotypes recorded in the two halves of the study period. Asterisks denote significant differences in the prevalence of MDR between the two study periods (Pearson's chi-squared test, * p < 0.05; ** p < 0.01; *** p < 0.001).

Discussion
Global monitoring of the emergence of antimicrobial resistance of Salmonella strains is essential for protecting public health. In developing countries, for example, the spread of antimicrobial resistant Salmonella strains can cause more acute and invasive infections, in addition to treatment failure and greater risk of mortality [21].
Here we analyzed the AMR of Salmonella serotypes isolated from human biological samples between 2012 and 2021 against six antimicrobial classes and 52 antimicrobial combinations. We found highly diverse resistance pattern phenotypes. Among the 168 Figure 5. Prevalence and 95% CI of MDR in the most frequent serotypes recorded in the two halves of the study period. Asterisks denote significant differences in the prevalence of MDR between the two study periods (Pearson's chi-squared test, * p < 0.05; ** p < 0.01; *** p < 0.001).

Discussion
Global monitoring of the emergence of antimicrobial resistance of Salmonella strains is essential for protecting public health. In developing countries, for example, the spread of antimicrobial resistant Salmonella strains can cause more acute and invasive infections, in addition to treatment failure and greater risk of mortality [21].
Here we analyzed the AMR of Salmonella serotypes isolated from human biological samples between 2012 and 2021 against six antimicrobial classes and 52 antimicrobial combinations. We found highly diverse resistance pattern phenotypes. Among the 168 different Salmonella serotypes involved in human infections, the three most frequent were S. ser. Typhimurium 1,4, [5],12:i:-, S. Typhimurium, and S. ser. Enteritidis, which were detected over almost the entire study period, as previously reported by global studies [1,22,23]. In addition, 3.6% and 3.4% of the isolates were S. ser. Napoli and S. ser. Derby, respectively. The most detected serotype in Piedmont was S. ser. Typhimurium 1,4, [5],12:i:-, which is consistent with previous reports published at national level [24]. By contrast, S. ser. Enteritidis is the serovar that is more frequently identified in EU, however an increase in prevalence has been observed for S. ser. Typhimurium 1,4, [5],12:i:-while S. ser. Enteritidis is following a decreasing trend [1].
Investigation of the susceptibility of Salmonella serotypes toward drugs indicated that most strains were resistant to at least one antibiotic and that the highest levels of resistance were against tetracycline, ampicillin, quinolones, and gentamicin. The high levels of resistance we observed is shared by previous reports and is of particular concern, since these antibiotics are commonly used in the first-line treatment of human and animal infections [12]. For instance, fluoroquinolones are the gold standard in treatment against invasive salmonellosis in human medicine, and ampicillin and tetracycline are widely used in veterinary medicine as first-line treatments [32]. The resistance to third-generation cephalosporines we noted was moderate, albeit higher than that reported previously [22,33]. In the present study, chloramphenicol had the lowest level of resistance, likely due to its use in veterinary medicine having been prohibited in Europe since January 1997 [34]. Though antimicrobial resistance to chloramphenicol is still present, it appears to be decreasing [35]. To compare antibiotic resistance patterns, we divided the study into two time periods. Resistance rates significantly decreased during the latter half of the study period, especially for S. ser. Typhimurium 1,4, [5],12:i:-, S. ser. Typhimurium, S. ser. Napoli, S. ser. Derby, S. ser. Brandenburg, and S. ser. Infantis. During both periods, we observed a reduction in resistance to tetracycline, ampicillin, quinolones, and cefotaxime. Similarly, a decline in resistance to tetracyclines and ampicillin in Salmonella from humans was observed in other European countries (nine and ten countries, respectively) over the period from 2015 to 2019, which was particularly true for S. ser. Typhimurium [12]. Decreasing trends of resistance were more commonly observed for ampicillin in S. ser. Typhimurium (nine countries) and for tetracycline in Salmonella spp. (eleven countries), S. ser. Typhimurium (nine countries), S. ser. Typhimurium 1,4, [5],12:i:-(six countries), and S. ser. Infantis (two countries). Despite the decline, resistance to these antibiotics remains high in bacteria isolated from humans and animals [12]. These resistance data are of particular concern for clinicians who use antimicrobials (e.g., fluoroquinolones and cephalosporins) in the treatment of children and in the early treatment of severe gastroenteritis or invasive infections in adults.
Worldwide actions have been undertaken to prevent the emergence of drug-resistant bacteria and to promote food safety and public health through plans to ban or reduce the use of certain antimicrobials. The development of AMR can be slowed by restricting inappropriate use of antimicrobials and by improving hygiene conditions and practices in healthcare settings or in the food chain to reduce the transmission of resistant microorganisms, where more than one cause may play a role. Furthermore, the application of European regulations concerning the utilization of antibiotics in veterinary medicine has had a positive impact on slowing the spread of AMR. The JIACRA report also identifies links between antimicrobial consumption in animals and AMR in bacteria from food-producing animals, which is associated with antimicrobial resistance in bacteria from humans. Data also show that the use of antibiotics has decreased and is now lower in food-producing animals than in humans [36].
Regarding MDR strains, our data showed that almost 50% of Salmonella strains are resistant to more than three classes of antibiotics. According to European data [9], MDR is highest in S. ser. Typhimurium, S. ser. Infantis, and S. ser. Typhimurium 1,4, [5],12:i:-. In our study, the number of MDR isolates was far higher in the first half than the second half of the study period, in which the significant reduction in the occurrence of S. ser. Typhimurium 1,4, [5],12:i:-, S. ser. Typhimurium, S. ser. Enteritidis, S. ser. Napoli, S. ser. Derby, and S. ser. Brandenburg may be ascribed to the implementation of Italian and European control programs for the eradication of Salmonella in poultry [37].
This study has benefited from the well-structured and coordinated surveillance health system implemented in the region. Collaboration among different health services has made possible the collection of a significant amount of data on human salmonellosis in the long term, enabling the evaluation of temporal trends of Salmonella strains and identify the main AMR patterns occurring in human infections. Notwithstanding, our study is limited to only six antibiotic classes because of the monitoring of AMR involving other antimicrobials was sometimes interrupted, thus preventing comparisons over time. Moreover, our survey was restricted to a specific geographical context, not allowing our findings to be generalized to other Italian or European regions.

Conclusions
Our study focused on the occurrence and the antibiotic resistance of Salmonella spp. detected in humans in northwest Italy over a 10 year period. High AMR levels were uncovered among Salmonella strains toward tetracycline, ampicillin, and quinolones in particular. These data provide supplementary information about AMR in human strains of Salmonella. The decreasing trend of AMR experienced by Salmonella in Piedmont is consistent with data from the European Union and demonstrates the effectiveness of measures implemented in human and veterinary medicine.
Institutional Review Board Statement: Salmonella strains, isolated from patients' samples, examined for this study were submitted to CeRTiS for surveillance purposes, within the framework of the Directive 2003/99/EC on the monitoring of zoonoses and zoonotic agents. In addition, information and consent to medical intervention was obtained from patients visited by medical doctors, in accordance with the current Italian legislation. All data on humans were treated in anonymized modes and were used solely for the purposes of scientific research.
Informed Consent Statement: Patient consent was waived due to the anonymity of the samples used and personal data were not used.