Background
Salmonella (
S.)
enterica is one of the most common bacterial pathogens causing foodborne infections and, thus, constitutes a major healthcare concern worldwide. For 2016, the European Food and Safety Authority (EFSA) reported over 94,000 confirmed cases of human salmonellosis in the European Union (EU) and
S. enterica was identified as the bacterial agent responsible for the most foodborne outbreaks [
1]. Poultry meat constitutes an important source of
Salmonella enterica and infections in humans are often linked to the consumption of improperly cooked poultry meat or a cross-contamination of other foodstuffs [
1]. While most non-typhoidal
Salmonella spp. infections lead to self-limiting gastrointestinal symptoms, invasive infections can occur and may be life-threatening, particularly in immunocompromised patients [
2,
3]. However, the likelihood of severe infections differs between serovars.
S. enterica serovar Typhimurium, which is among the most frequently encountered serovars in the EU, has a moderate tendency to cause invasive disease—
S. Typhimurium ST313 being a notable exception, frequently associated with invasive disease in Africa [
4]. Others, such as
S. Heidelberg and narrow host-range serovars like
S. Dublin and
S. Choleraesuis, are considerably more likely to cause severe infections requiring hospitalization [
3,
5]. Previous reports of salmonellosis outbreaks involving multiple countries have raised concerns about the role of international food and animal trade in the spread of
Salmonella serovars, especially considering the comparatively high rates of
Salmonella detection in some parts of the world [
6]. Furthermore, imported meat products have been implicated as a possible source for
S. enterica isolates resistant to clinically relevant antimicrobials [
7,
8].
In addition to consignments of meat officially imported into the EU, considerable amounts of meat products are brought into the EU illegally, circumventing any controls [
9,
10]. To date, very little data are available about serovars present in such products and their characteristics [
11,
12].
In this study, S. enterica isolates recovered from meat and meat products imported into the EU, both legally and illegally, were examined. The purpose was to identify the serovars entering the EU via these routes and to further characterize the isolates regarding their antimicrobial resistance phenotypes and genotypes.
Discussion
In recent years, the number of air travel passengers has increased continuously and is expected to rise to an estimated 7.8 billion travelers in 2036 [
34]. Consequently, the total amount of meat and meat products illegally transported in passenger luggage can be expected to rise accordingly. To date, little data are available about the characteristics of salmonellae recovered from such food items. In the present study, the two isolates from illegally imported meat belonged to the serovars Infantis or Weltevreden. Both isolates were recovered from raw meat products, in one case seasoned with a marinade of unknown composition, transported in air passenger luggage. In contrast to this, a study conducted in Spain reported the following seven different serovars among eleven isolates from food of animal origin confiscated at a Spanish airport: monophasic serovar 4, 12: d: − , Rauform, Anatum, Oranienburg, Enteritidis, Newport and Typhimurium [
11]. Notably, these were mostly of South American origin and included isolates detected in cheese samples. Counting only meat and meat products, 6 of the 122 samples examined in that study were positive for
S. enterica [
11]. In another study, a total of four isolates were detected on illegally introduced sausages of Russian origin out of 367 samples of illegally imported meat and meat products (1.1%) [
12]. Two isolates each belonged to the serovars
S. Infantis and
S. Enteritidis. In their study, as well as in the course of the sampling preceding the current study, samples were kept frozen before analyses [
12,
13]. Previous research demonstrates that freezing does not fully eliminate
S. enterica, but provides differing results on survival rates during frozen storage ranging from no significant changes in bacterial counts to minimal survival [
35,
36]. Consequently, structural injury to the bacterial cells during freezing and thawing of the samples prior to analyses may have contributed in part to the low detection of
Salmonella spp.. One study reported a relatively constant population of
Salmonella spp. isolates in artificially inoculated breaded chicken strips and nuggets during 16 weeks of storage at − 20 °C. However, enumeration on selective agar resulted in a statistically significant reduced recovery of salmonellae compared to non-selective media, indicating structural injury to the cells [
35]. In contrast to this, the authors of a study conducted in Mexico observed a considerable decline of the S
almonella population present on naturally contaminated pork during storage at − 15 °C [
36]. In a recent study, a lower detection rate of salmonellae in frozen chicken carcasses was determined compared to chilled and non-chilled samples, though the difference was not statistically significant [
37]. Due to different storage conditions and meat types used in individual studies, the comparability of the results is limited. Additionally, many further factors were suggested to influence the survival of
Salmonella spp. during frozen storage, such as pH and fat content of the meat, which were not evaluated for the samples examined prior to the current study [
35].
S. Weltevreden is a common serovar in Southeast Asia and the isolate in the present study was recovered from illegally imported rabbit meat from Vietnam [
38,
39]. The isolate did not show resistances to any of the antimicrobial agents included in the test panel. This is in accordance with previous studies that found a low prevalence of antimicrobial resistances for this serovar [
40,
41]. Of the 11
S. enterica isolates from illegally imported food products of animal origin examined in the study by Rodríguez-Lázaro and collegues, most isolates did not show phenotypic resistances to any of the antimicrobial agents tested, either. The remaining isolates were each resistant to a single class of antimicrobials [
11].
In contrast to this, the
S. Infantis in the present study was resistant to substances belonging to five different classes of antimicrobials. In recent years, resistances to multiple antimicrobial agents have been observed comparatively frequently in
S. Infantis. Together with
S. Kentucky, this serovar currently accounts for a significant proportion of multiresistant non-typhoidal
S.
enterica isolates in the EU [
42].
The isolates from legally imported chicken meat in the current study included one non-flagellated isolate. However, this isolate appeared to be closely related to the two
S. Heidelberg isolates recovered from legal imports, based on the high similarity in PFGE band patterns and the shared O-group. Flagella play an important role as virulence factors and enhance adhesion to and invasion of host cells. However, this mostly affects the early stages of infection and non-flagellated strains do not appear notably less virulent if an infection reaches the systemic phase [
43,
44].
S. Heidelberg is not commonly associated with human cases of salmonellosis in the EU and did not rank among the 20 most frequent serovars in an EFSA report covering the years 2014–2016 [
1]. Data on national cases of human salmonellosis, released by the Robert Koch Institute, also indicate a low prevalence of
S. Heidelberg infections in Germany [
45]. According to these data, the four serovars accounting for the vast majority of cases in 2016 were
S. Enteritidis and
S. Typhimurium, followed by
S. Infantis and
S. Derby. Particularly in North America, however,
S. Heidelberg is often the cause of human salmonellosis and was reported to be the sixth most frequent serovar in the US in 2015 [
46,
47].
Notably, all isolates from legal imports in this study were recovered from fresh poultry meat. Regarding
Salmonella, commission regulation (EC) 2073/2005, amended by commission regulation (EU) 1086/2011, only specifies the absence of
S. Enteritidis and
S. Typhimurium (including its monophasic variant) as food safety criteria for fresh poultry meat. Other serovars are not covered, even though they might be more common in other countries such as Brazil – the largest exporter of chicken meat supplying the EU [
8,
48‐
50].
All three isolates from legally imported poultry meat in this study were AmpC producers carrying
blaCMY-2 and they were additionally resistant to ciprofloxacin. Like third-generation cephalosporins, fluoroquinolones are important choices for the treatment of severe cases of salmonellosis [
51]. Consequently, co-resistance to both is of particular concern. The overall prevalence of ESBL- and AmpC-β-lactamases among
Salmonella enterica isolates is still low in the EU, however, the AmpC phenotype in particular has been comparatively often seen in
S. Heidelberg isolates [
52,
53].
S. Heidelberg carrying
blaCMY-2 have previously been detected on imported poultry meat of Brazilian origin and such imports were suggested to be involved in the increase of third-generation cephalosporin-resistant
S. Heidelberg in the Netherlands [
7,
8]. The
blaCMY-2 genes of the isolates in the present study were located on large conjugative plasmids belonging either to IncA/C or IncI1. Plasmids of these incompatibility groups have previously been associated with AmpC-producing
S. Heidelberg from various sources and likely play an important role in the spread of resistance to third-generation cephalosporins in
S. Heidelberg and other
Salmonella serovars [
7,
8,
47,
54].
Authors’ contributions
AM prepared the manuscript, performed the experiments and analyzed the data. CK coordinated the project, analyzed the data and revised the manuscript. WJ and NG analyzed results and reviewed the manuscript. All authors read and approved the final manuscript.