4. DISCUSSION

Summary and implications of the evidence

Despite the increased global awareness regarding food safety in recent decades, FBD still cause a substantial public health burden (World Health Organization, 2021) and NTS stands among the main foodborne pathogens that contribute to this burden, particularly by the consumption of contaminated poultry and poultry-derived products. In consequence, it is fundamental to assess the prevalence and characteristics of this foodborne pathogen throughout the poultry productive chain. The current systematic review and meta-analysis provide a comprehensive synthesis of evidence regarding the prevalence, the diversity and frequency of serovars, and the AMR profiles of NTS reported in poultry samples from the Americas.
The meta-analyses showed that the prevalence of NTS was the highest in environmental samples. We performed an additional meta-analysis grouping together studies that reported internal (farm installations and infrastructure, poultry houses, eggshells, bedding, feces, dust, and litter) or external (supplies, transport cages, slaughterhouse installations, and processing facilities) sources from where the environmental samples were taken. The results showed a similar prevalence of NTS in internal (31.5%, 25.1 to 38.3) and external (29.4%, 11.1 to 51.9) sources of environmental samples and thus demonstrate that NTS is both broadly distributed and highly frequent throughout the environment related to poultry production in the Americas. This result agrees with previous studies that have demonstrated a consistent presence of NTS in environmental samples including poultry feed (Magwedere, Rauff, De Klerk, Keddy, & Dziva, 2015), farm infrastructure (Bhatia & McNabb, 1980), slaughterhouse installations and processing facilities (Rivera-Pérez, Barquero-Calvo, & Zamora-Sanabria, 2014), and leftovers such as litter or feces (Vaz, Voss-Rech, de Avila, Coldebella, & Silva, 2017).
With prevalence values ranging from 3.3 to 36.7%, SNT was isolated in the environmental samples from 8 out of 15 countries included in the study. Even though the variability was high in the estimates among the countries, this is an important result because the presence of NTS in the internal and external environment related to poultry production might facilitate the reintroduction of the pathogen to the productive chain and thus become a persistent source of exposition for further contamination (Mueller‐Doblies, Sayers, Carrique‐Mas, & Davies, 2009). In a recent longitudinal study, Voss-Rech et al. (2019) found that once NTS was detected in the litter samples during the harvest of the first flock, then the pathogen was isolated from the environment of subsequent flocks, whereas Marin, Balasch, Vega, and Lainez (2011) reported that all the different environmental samples related with poultry production were contaminated with NTS and that the prevalence of the pathogen remained high even after cleaning and disinfection. Several studies have assessed different control measures to prevent and reduce the incidence of NTS in the environment of poultry production. For instance, preventing contamination of the facilities where the animal feed is produced and killing the pathogens by pelleting the feed (Jones, 2011), inactivation of residual microorganisms in recycled litter by shallow fermentation or windrowing (Vaz et al., 2017), and cleaning and disinfection to avoid persistent contamination in the broiler houses (Davies, Breslin, Corry, Hudson, & Allen, 2001). However, the ability of NTS to persist for long periods means that every available tool must be used to control the organism, and efforts must be sustained (Jones, 2011) and constant sampling of the environment should be performed.
Additionally, the presence of NTS in the environment related to poultry production and processing increases the probability of cross-contamination of the carcasses and poultry meat during slaughtering (Volkova et al., 2010). In this regard, Carramiñana et al. (1997) reported that the percentage of poultry carcasses contaminated with NTS increased from 56.7% to 70% after the processing, whereas Tozser et al. (2019) found that the 0% positivity to NTS in the body surface of the birds before transportation increased up to 100% after the slaughter and processing stages. Given that poultry meat is one of the most frequent sources of the exposure of NTS to the human, the cross-contamination from the poultry environment to the carcasses and meat poses a serious threat to public health, thus the interventions aimed at reducing the contamination with pathogenic bacteria are fundamental and require appropriate knowledge regarding the presence and dissemination of these pathogens at various steps during the processing stage (Berrang et al., 2007). Nevertheless, more studies are needed to fully understand which interventions are effective against NTS contamination of the poultry-derived products in countries from the Americas.
We found substantial heterogeneity in the estimates at the national level. The disparate number of studies included for each country and the difference in the number of samples assessed could have increased the uncertainty of the estimations and thus cause part of the heterogeneity, this variability might reflect different husbandry practices and the absence of effective control measures across the poultry production chain (Antunes et al., 2016; Brochu et al., 2021). Despite successful control of NTS has involved actions such as culling and vaccination, the currently available vaccines have played a minor role in the control of fowl typhoid as they offer short-lived protection against clinical disease and limited or variable protection against infection with field strains (World Organization for Animal Health, 2018). Therefore, other containment methods including improved hygiene, increased biosecurity, segregated hatching, competitive exclusion treatment, and monitoring and removal of infected flocks could also be applied (EFSA Panel on Biological Hazards et al., 2019). Nevertheless, the differential implementation of these control measures caused by the variability in the poultry husbandry practices across countries from the Americas could explain part of the heterogeneous prevalence seen in these countries. In this regard, in several countries from South America the imposition of strict controls on the environment and hygiene of poultry husbandry is restricted due to high ambient temperatures (Barrow, Jones, Smith, & Wigley, 2012), thus causing differences in the incidence of NTS concerning countries with improved hygiene and management practices. Finally, national programs of poultry health, current legislation, and farmers’ perceptions are also factors capable of affecting the monitoring and control of the pathogens in poultry as well as the biosecurity measures needed to avoid their spreading (de Oliveira Sidinei, Marcato, Perez, & Bánkuti, 2021; Reis et al., 2021).
Our summary of evidence showed that Enteritidis and Typhimurium were identified in most of the countries, where they ranked among the top three serovars, thus coinciding with several studies that report Typhimurium and Enteritidis as the main serovars in poultry samples (Carramiñana, Rota, Agustin, & Herrera, 2004; El-Sharkawy et al., 2017). The serovar distribution and prevalence found in countries from the Americas differ with the pattern reported in some European countries, which report as main serovars Infantis in Hungary (Tozser et al., 2019), Hadar, Anatum, and Mbandaka in France (Le Bouquin et al., 2010), and Enteritidis, Hadar, Virchow, and Ohio in Spain (Marin et al., 2011). However, the variety and prevalence of NTS serovars are expected to differ among the studies from different regions and types of farms (Andino & Hanning, 2015). Despite the great amount of NTS isolates included in the studies, the serovar diversity was rather low because only 131 serovars were identified; therefore, more studies are necessary to increase our knowledge regarding the NTS serovar diversity and frequency present in the poultry production chain of the countries from the Americas. Especially given that many serovars are restricted to a single region of the world that generates distinct profiles both within and between regions (Galanis et al., 2006).
Enteritidis was the serovar most prevalent in birds samples (mainly laying hens) and products and sub-products (mainly eggs and carcasses), which concurs with previous reports of salmonellosis outbreaks caused by consumption of eggs and poultry meat contaminated with this NTS serovar in the USA (Jackson, Griffin, Cole, Walsh, & Chai, 2013) and Europe (EFSA, 2019). Besides Typhimurium and Enteritidis, Heidelberg and Newport have also been highlighted as NTS serovars capable of causing a great burden of FBD due to poultry and poultry-derived products (Jajere, 2019) and our results confirm this for Heidelberg because this serovar was consistently found among the top five serovars in each of the three samples assessed in our study. Even more, Heidelberg was the most prevalent serovar found in environmental samples taken from production wastes and breeding facilities. Thus, the pattern of NTS serovars found across the different sources for each type of sample demonstrates the presence of the three most common serovars frequently associated with FBD caused by poultry and poultry-derived products.
Despite the global tendency to both reduce the indiscriminate use of antibiotics and increase awareness regarding their negative effect, our results demonstrate a high and alarming prevalence of multiresistance to antibiotics in NTS isolates. The pooled prevalence of AMR to 2-3 antibiotics was 36.2% with the highest values in birds and environmental samples, whereas resistance to ≥ 4 antibiotics reached a pooled estimate of 49.6% with even a higher prevalence of 61.2% in birds. These results not only determine the magnitude of the current AMR status in the poultry husbandry in the Americas but also emphasize the major public health issue linked to the presence and dissemination of NTS multiresistant strains throughout the poultry production chain that ultimately might reach to the human. This pattern of multiresistance could be a consequence of the combined use of antibiotics, which although is prohibited in several countries, remains as a common practice in several regions and countries.
Kentucky, Heidelberg, Typhimurium, Enteritidis, and Infantis were the top five NTS serovars with the highest prevalence of AMR in the poultry samples from the Americas, which partially coincide with several studies in which the highest prevalence of AMR was found in the serovars Enteritidis, Typhimurium, and Infantis (Carramiñana et al., 2004; El-Sharkawy et al., 2017; Kunadu, Otwey, & Mosi, 2020). The presence of these isolates with AMR in the poultry production chain is a concern given that several actors from this husbandry and supply chain frequently handle equipment, animals, and products without the necessary protections and thus increase the risk of resistance transfer and spread to commensal bacteria (Reis et al., 2021). Besides, even though the serovar Heidelberg is not frequently reported among the main resistant serovars, we found that Heidelberg was the 2ndtop-ranked serovar with AMR in the Americas and this is an important result for public health given that Heidelberg is one of the serovars that can induce systemic complications in people, especially children, the elderly or immunocompromised people (Silva, Milbradt, Zamae, Andreatti Filho, & Okamoto, 2016).
Cephalosporins, penicillins, tetracyclines, and aminoglycosides concentrated 71.9% of the resistant isolates and thus were the four main groups of antibiotics to which NTS was resistant in poultry samples from the Americas. This profile partially contrast with recent secondary studies, which reported that quinolones and beta-lactams in Europe (Antonelli et al., 2019) and sulphonamides, quinolones, and tetracyclines in Brazil (Voss-Rech et al., 2017) were the main groups of antibiotics to which NTS isolated from poultry and poultry-derived products were resistant. Even though the specific pattern of AMR was distinct among the poultry samples, tetracycline was consistently the 1st top-ranked, whereas ampicillin varied between the 2nd and 5th top-ranked. Such a result should probably reflect the fact that tetracyclines and penicillins were the largest selling antimicrobials, but their use gradually reduced while cephalosporins increased (Kim, Seo, Jeon, Lim, & Lee, 2018), thus causing an increase in AMR to cephalosporins in NTS from poultry as have been reported in NTS serovar Typhimurium, though with concurrent resistance to ciprofloxacin and ceftriaxone increasing in other serovars (Wong, Zeng, Liu, & Chen, 2013). AMR to ceftriaxone and ciprofloxacin in NTS isolates from poultry is of major interest to public health, because these two antibiotics in conjunction with azithromycin are the key drugs of choice for the treatment of invasive NTS infections. However, according to our profile of AMR, except for the samples from birds in which ceftriaxone was 8thtop-ranked, none of these three antibiotics were among the top 10 antibiotics to which NTS was resistant.
4.2 Limitations
We detect several limitations in our study: 1) unpublished studies were not included to maintain a comparable level of methodological quality among the studies, which could introduce bias because only published studies were searched and included, 2) despite several databases were searched, we only found studies for 15/35 countries from the Americas, in consequence the epidemiological landscape found might not be representative for the countries that were not included, 3) our study only included poultry samples from broilers, laying hens, and reproducers and thus the epidemiological landscape for NTS in ducks, turkeys, quails, and ostriches still needs to be assessed in countries from the Americas, and 4) to provide an overall summary of the studies, we grouped a broad variety of poultry samples into three discrete categories that avoided the meta-analysis of the specific sources of NTS contamination within each particular stage or condition.
4.3 Conclusion
Our systematic review and meta-analysis both confirm the presence of NTS throughout the poultry productive chain in countries from the Americas and determines the magnitude of the prevalence of this pathogen that causes a high burden of FBD. Despite we found a reduced diversity of NTS serovars among the three types of poultry samples, the presence of zoonotic serovars capable of causing outbreaks was consistent across countries. Besides, the results demonstrated a high and alarming prevalence of AMR in the NTS isolates from poultry samples, which in addition showed an increasing trend towards higher prevalence of multiresistance. Furthermore, we found that the serovars with the higher prevalence of AMR were those commonly reported in salmonellosis outbreaks associated with poultry. Taken together, these results confirm the growing concern of NTS as a public health problem caused by the consumption and exposure to contaminated poultry and poultry-derived products. Additionally, our results highlight the need for appropriate control measures against NTS in the entire poultry productive chain to prevent further emergence and dissemination of multiresistant serovars to the human, which threatens the successful treatment of invasive infections caused by this pathogen.