Detection, molecular characterization, and antimicrobial susceptibility, of Campylobacter spp. isolated from shellfish

Highlights

• Total of 91 mussel samples were analyzed being 8% positive for Campylobacter spp.

• Higher prevalence observed in january-march.

• Bacterial isolates were resistant to 4 antibiotics.

• All the isolates were identified as Campylobacter lari.

• No specific virulence genes were detected by PCR or NGS.

Abstract

Campylobacteriosis is one of the most important reported zoonosis worldwide. Besides poultry other sources of infection have been described. In the current study, the incidence of Campylobacter spp. was assessed over a five-month period in mussel samples harvested from one of the most important producing areas (Galicia, NW Spain) in Europe. Out of 91 samples screened, 8% were positive and identified as C. lari by MALDI-TOF and whole genome sequencing. All were detected during the colder months (February and March). The antimicrobial resistance and virulence genes analysis indicated that all were multi-resistant to at least 4 antimicrobials. They were negative for the presence of 5 virulence-related genes. This is the first report of this zoonotic pathogen in mussels from one of the most important shellfish producing regions in Europe. The genomes of these 7 C. lari isolates were released to the genome public database at NCBI.

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.