Detection, molecular characterization, and antimicrobial susceptibility, of Campylobacter spp. isolated from shellfish
Introduction
Campylobacteriosis refers to a disease caused by Campylobacter spp. infections. Reported symptoms include diarrhea, abdominal pain, fever, malaise and headaches, and serious complications like Guillain-Barré syndrome (Ternhag et al., 2005; Van Dyke et al., 2010). Campylobacter spp. are the most common cause of acute bacterial enteritis in humans worldwide, and are the leading cause of foodborne disease in Europe with more than 200,000 cases in Europe in 2015, and the second most important foodborne causing disease in the United States with more than 6000 cases (CDC, 2015; EFSA and ECDC, 2017). Three specific species are included in the group of so called thermotolerant campylobacters that account for the majority of the infections: C. coli, C. jejuni, and C. lari (Josefsen et al., 2010; Lubeck et al., 2003). Each year approximately 1% of Europeans suffer campylobacteriosis (Humphrey et al., 2007), costing approximately 2.4 billion € to the public health system ((BIOHAZ), 2010). The majority of these cases are caused by C. jejuni, to a lesser extent C. coli and last by C. lari (He et al., 2010; Laprade et al., 2016; Mayr et al., 2010).
Since the coastal environment is a receptacle of agricultural and urban wastewater effluents, Campylobacter spp. could be present in these coastal areas. Because of their filter-feeding activities, shellfish can concentrate and retain pathogenic microbes present in their environment. The farming of mussels Mytilus galloprovincialis in the Rias of Galicia (NW Spain) constitutes a very important industry. This region produces more than 95% of the mussels in Spain (Eurofish, 2016) and the country itself is the largest European producer of mussels with more than 200,000 tons per year (FAO, n.d.). The risk to public health associated with the consumption of shellfish, traditionally consumed raw or undercooked, has been well documented (Feldhusen, 2000; Potasman et al., 2002), and it is assumed that these incidental reports are only the tip of the iceberg. Pathogenic microorganisms such as Salmonella, or marine bacteria such as pathogenic Vibrio species, were implicated in shellfish foodborne diseases in Galician Rias (Lozano-León et al., 2003; Martinez-Urtaza et al., 2004). In Europe, to protect consumer's health from enteric microbial pathogens, shellfish harvesting areas were classified on the basis of the shellfish microbial contamination using E. coli as the fecal indicator bacteria (91/492/EEC, 1991). This regulation determined the level of treatment, such as depuration or relay, which needs to be applied prior to the commercialization of shellfish depending on the levels of the fecal indicator.
In order to establish an optimal risk assessment plan, it is critical to know the potential threats to which the population can be exposed. In this sense, previous studies, along with the outbreaks mentioned, have already highlighted the presence of different pathogens in the Galician Rias, such as Vibrio spp. and Shiga Toxin-producing E. coli (STEC) (Garrido-Maestu et al., 2016; Rodríguez-Souto et al., 2017). Additionally, it is worth highlighting that the presence of Campylobacter spp. in seafood has also been previously described (Endtz et al., 1997). For this reason, the aims of this study were to determine the occurrence of Campylobacter spp. in Mytilus galloprovincialis harvested in the Ria of Arosa, Galicia, Spain (which is the area's most important mussel producer) from February to June 2015. We also evaluated their pathogenic potential and determined their antimicrobial resistance profile. Lastly, we performed whole genome sequencing of all the isolated strains.
Section snippets
Sampling
Between February and June 2015, a total of 91 mussel-samples were collected from different harvesting areas of the Ria of Arosa. This location was selected for the study as it is the Ria with the highest number of rafts for mussel farming in Galicia. The number and distribution of the samples analyzed, as well as the geographical locations included in the present study, are detailed in Table 1 and Fig. 1.
Microbiological analysis
The samples were collected from the mussel farming rafts, placed in sterile bags with
Incidence and molecular characterization
During the period of study, 7 out of 91 samples analyzed by qPCR tested positive for Campylobacter spp. (8%) (Table 1 and Fig. 1). A single strain was recovered from each positive sample. The species identification by MALDI-TOF indicated that the isolated strains were C. lari, with scores ≥ 2.0. In order to confirm that the strains isolated from the positive samples were indeed C. lari, the genome of each strain was determined by whole genome sequencing (WGS).
Discussion
In the current study the presence and characterization of Campylobacter spp. in an economically important mussel harvesting area was assessed. The sampling area covered 19 harvesting zones, and the study was conducted over 5 months which included late winter until summer (February to June) to get a glimpse of the possible seasonality of these pathogens. In this sense, over the period of study neither C. jejuni, nor C. coli, which are the most important etiological agents of campylobacteriosis,
Conclusions
In the current study we showed the presence of Campylobacter spp., being more specific C. lari, in mussel samples in Galicia (Spain) one of the most important shellfish harvesting areas in Europe. No specific virulence genes were detected in the isolates recovered, but the antimicrobial profile showed the presence of multi-resistant strains which is of high importance in the context of the One Health approach. Furthermore, WGS indicated that some of the STs reported in this study match the STs
CRediT authorship contribution statement
Antonio Lozano-León: Conceptualization, Writing - review & editing. Rafael R. Rodríguez-Souto: Formal analysis, Writing - review & editing. Narjol González-Escalona: Formal analysis, Data curation, Resources, Writing - review & editing. José Llovo-Taboada: Formal analysis, Writing - review & editing. José Iglesias-Canle: Formal analysis, Writing - review & editing. Ana Álvarez-Castro: Formal analysis, Writing - review & editing. Alejandro Garrido-Maestu: Data curation, Writing - original draft.
Declaration of Competing Interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We would like to Eleanor S. Henning for editing support. NGE was supported by the FDA Foods Program Intramural Funds.
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