Genome analysis of Salmonella strains isolated from imported frozen fish in Burkina Faso


 
 Fish is an excellent source of protein and vitamins for humans, but improperly handled, fish can expose consumers to pathogenic bacteria. This study was aimed to isolate and characterize the genomes of Salmonella strains isolated from imported fish sold in the open market in Ouagadougou.
 
 
 One hundred and fifty-nine fish were collected from open markets and were cultured for Salmonella. Antimicrobial susceptibility was determined by broth microdilution. Whole-genome sequencing was done to further study antibiotic resistance genes, plasmid replicons, and MSLT types. Serotyping was done using SeqSero 2.
 
 
 Out of the 159 fish samples analyzed, 30 (18.9%) were found to be contaminated with Salmonella. Among the isolated Salmonella strains, six different serotypes, Nima, Liverpool, Kokomlemle, Teshie, Derby, and Tennessee, were found using SeqSero2. Salmonella Tennessee was the predominant serotype. All the isolates possessed at least one resistance gene. The aac6-Iaa aminoglycoside resistance gene was the most prevalent gene found in the strains. The gene fosA7 was detected in three strains. All the S. Nima isolates were of Multilocus Sequence Type (MLST) 8086, S. Teshie isolate was ST 530; Liverpool was ST 1959; Derby was ST 7880; Kokomlemle was ST 2696. The Tennessee isolates gave two different STs including ST 8395 and 8398.
 
 
 The presented results highlight the prevalence of Salmonella on imported fish purchased from the open markets. More attention should be paid regarding fish selling conditions in the country to prevent the potential health risk for consumers.



Introduction
Burkina Faso is a landlocked tropical country located in Sub-Saharan Africa. This country is characterized by a dry season from October-May with hot temperature (35-45 Celsius) and a short rainy season (June-September). In recent years, sh consumption has increased exponentially in this country with more than 96% of commercially sold sh imported from another country (1). Fish is an important source of essential amino acids and good fatty acids for humans, but sh can be contaminated by pathogenic bacteria that pose a high risk for consumer's health (2; 3). These pathogenic bacteria can contaminate ready to eat sh product through cross-contamination during sh processing (4). Salmonella has been implicated in sh outbreaks worldwide (5; 6).
Nowadays, the use of antibiotics in aquaculture practices as growth promoters or for treatment and prevention of sh diseases is increasing the risk of development of antibiotic resistant bacteria among the microbiome of sh gut and/or shing water (7). Many studies have shown widespread transmission of antibiotic resistant bacteria of the aquatic or sh to human trough environment and/or sh consumption (8,9).
According to the Centers for Disease Control and Prevention (CDC), antibiotic resistant infection is responsible for 25,000 annual deaths in the European Union and 23,000 annual deaths in the U.S (10).
The World Health Organization (WHO) report on the burden of food-borne disease clearly shows that this burden is similar to the burden of malaria, tuberculosis and even HIV AIDS (11). The report also shows that the burden of food-borne disease is disproportionately borne by the least developed countries and by children. Since imported sh is widely consumed in Burkina Faso, it is important to know the microbiological quality of these sh. Therefore, the present study aims to understand the epidemiology and antibiotic resistance of Salmonella strains isolated from sh using whole genome sequencing and phenotypic methods.

Materials And Methods
Sampling Imported sh samples were purchased from different open markets. All sh samples during collection were placed in sterile polypropylene bag, placed in polystyrene box containing crushed ice and the temperatures was 4°C during transportation. The samples were transported to the laboratory and examined on the same day for the presence of Salmonella spp.

Bacteriological analysis
Salmonella strains were isolated from sh samples following the methodologies described in the International Organization for Standardization 6579-2017 (12). The sh samples were gently removed from coolers and processed in aseptic condition. The gills, intestines parts and skin parts were removed using sterile knifes. About 10 g of samples ( sh gills, intestines parts and skin) were placed into a stomacher bag containing 90 mL of buffered peptone water (Lio lchem, Teramo, Italy) and homogenized using a stomacher (400 Circulator, Seward, London,UK) for 1 min and incubated for 24 h at 37 °C. From this non selective pre-enrichment, 0.1 mL were transferred into 10 mL of Rappaport-Vassiliadis broth (Oxoid, Basingstoke, England) and incubated for 24 h at 42°C. A loopful from the selective enrichment broth was streaked onto XLD (Oxoid, Basingstoke, England) agar and incubated for 24 h at 37 °C. Suspected colonies on selective agar plates were puri ed and bio-typed by using biochemical tests and API 20E strips (BioMerieux, Marcy l'Etoile, France).
Con rmed colonies were sent to the United States Department of Agriculture, Bacterial Epidemiology and Antimicrobial Resistance Research Unit for future analysis.
For each isolate, a nal inoculum of 5 x 10 5 CFU/ml was targeted. The panels were read after 18 h of incubation at 35°C.

Whole genome sequencing
Genomic DNA was isolated using the GenElute bacterial genomic DNAkit (Sigma-Aldrich, St. Louis, MO, USA) following instructions for Gram-negative bacteria, from 5 mL of overnight cultures grown in Luria-Bertani Broth, Miller (Difco™, Becton Dickinson and Company, Sparks, MD) at 37°C with shaking. The extracted DNA quality was read using NanoDrop 2000c spectrophotometer (Thermo, Fisher Scienti c, USA). DNA was stored at -20°C prior to library preparation.
Extracted DNA was quanti ed using the Qubit double-stranded DNA (dsDNA) high-sensitivity (HS) assay kit according to the manufacturer's instructions (Life Technologies, Inc., USA). The Illumina libraries were prepared using the Nextera XT DNA library preparation kit and Nextera XT index primers (Illumina, USA). The library fragment size distribution was checked using the Bioanalyzer 2100 with an Agilent HS DNA kit (Agilent Technologies, USA) and quanti ed using a Qubit DNA HS assay kit in a Qubit uorometer (Thermo, Fisher Scienti c, USA). The generated libraries were then sequenced using a MiSeq version 2 reagent kit with 500 and 300 cycles. The paired-end read length of 2 X 250 bp was used for 500 cycles and 2 X 150 bp for 300 cycles on the Illumina MiSeq platform. The quality metrics of the reads were performed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The sequence data were assembled using the A5-miseq assembler (14), and the genome sequence was annotated via the NCBI Prokaryotic Genome Annotation Pipeline (15).
The Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession XXXXXX000000000. The version described in this paper is version XXXXXX010000000.
Identi cation of antibiotic resistance genes, chromosomal mutations, serotypes, MLST and plasmid from total genome sequence Antibiotic resistance genes and chromosomal mutations were identi ed using ResFinder 3.2 (16).
SeqSero 2 was used to determine the serotypes of salmonella strains from genome assembly data. MLST sequence type was identi ed using MLST database from the center of genomic epidemiology (17). The PlasmidFinder were used to detect plasmid from the strains (18).

Results
Out of the 159 sh samples analysed, 28 (17.61%) were found to be contaminated with Salmonella. Among the isolated Salmonella strains, 6 different serotypes such as Nima, Liverpool, Kokomlemle, Gaminara, Derby and Tennessee were found using SeqSero2 with some being N/A. S. Tennessee was the predominant serotype. All the isolates possessed at least one resistance gene. The non-functional aac6-Iaa and aac6-Iy conferring resistance to aminoglycosides was the most prevalent gene found in the strains. The gene fosA7 conferring resistance to Fosfomycin was detected in two strains. The isolates were susceptible to all drugs tested. Four S. Nima isolates were identi ed from the 28 isolates and were all MLST Sequence Type (ST) 2258. One S. Gaminara isolate was identi ed and was ST 5197; two S. Liverpool isolates were detected and were identi ed as MSLT ST 1959; one S. Derby isolate was found and was ST 3997; One S. Kokomlemle was detected and was MLST ST 2696. Thirteen S. Tennessee isolates were found with different sequence types including ST 3763, ST 3997, ST 3135, and ten were unknown. Two strains possessed plasmid replicons: one IncFII(S) and one IncFII(pCRY) ( Table 1). The amino acid substitution Thr57Ser and Ser80Ile in ParC were detected in 25 isolates. Point mutations in the quinolone resistance-determining regions (QRDRs) were detected in 25 (89.28%) isolates at positions 57 (Thr57Ser) and 80 (Ser80Ile) for ParC (Table 1). Accession numbers generated during the current study are assigned in Table 1.

Discussion
The present study was initiated to determine the microbiological quality of imported and local sh consumed in the city of Ouagadougou, Burkina Faso. The prevalence of Salmonella strains was 17.41% from imported sh. This result could be explained by the fact that imported sh are exposed to several stages of handling including packaging at the farm in the origin country, transport to Burkina Faso, reception at wholesalers, delivery to semi-wholesalers, and delivery to different retailers. All these steps undoubtedly favor the contamination by bacteria like Salmonella. However, the consumption of imported sh is very high in Burkina Faso because it is very accessible and inexpensive in all the localities of the country. In these localities, the imported sh is cut into small pieces by small traders and sold at a minimum price of 50 FCFA (about 1 cent of dollar). This necessitates permanent monitoring of the prevalence of germs that can affect the health of consumers as well as chemicals. The population of Burkina Faso is over 80% illiterate, which will undoubtedly lead to an increase in contamination of raw sh and the possibility of cross-contamination due to a lack of training and information on the causes and consequences of foodbornes diseases (19). The prevalence of Salmonella in sh in this study is higher than those reported by Broughton and Walker, (20) from sh in China (5%) and by Heinitz et al. (21) in U.S.-imported raw seafood from several Asian countries (10%). These variations in prevalence can be explained by differences in farming methods, and in the food safety regulations of each country. For example, in Burkina Faso, many researchers demonstrated that good hygienic practices are not respected yet by food sellers and domestic food safety regulation and / or training program still missing (22; 23; 24).
Salmonella Tennessee was the most prevalent serotypes among sh samples. This serotype of Salmonella was detected in different types of samples and in the stools of patients with diarrhea in other studies from Burkina Faso (25). S. Tennessee has also been implicated in outbreaks in the United States due to contaminated peanut butter; powdered milk products and infant formula (26; 27). These facts show us that the Tennessee serotype is not necessarily linked to a speci c food or environment. Depending on the handling of food, this serotype can contaminate humans through any contaminated food.
We also have the presence of S. Liverpool, which is a pathogenic serotype and has not been identi ed in our previous studies carried out in Burkina Faso in diarrheal patients, chickens, the environment, or animals (25; 28; 29). S. Derby identi ed was the most dominant in our previous studies in chickens and slaughter animals (Kagambèga et al., 2013). S. Nima and S. Kokomlemle also have been isolated in chicken and beef previously in Burkina Faso (30). S. Gaminara has been identi ed in a patient suffering from diarrhoea in Burkina Faso (28).
The presence of these serotypes in sh shows that chicken, slaughter animals, the environment and humans share the same pathogens that circulate in our country.
Twelve different MLST sequence type were nd from the strains. A diversity of MSLT type was detected in our study with S. Tennessee. This may show that S. Tennessee has genetic diversity within its population. We can say that the other serotype with a unique MSLT type retained their genetic characteristic during their evolution while keeping the same type of MLST. On the other hand, S. Tenessese population structure has changed during evolution.
All the Salmonella strains found in this study possessed aminoglycoside resistance genes, encoding acetyltransferases (aac(6')-Iaa; aac(6')-Iy). While these genes were not functional in this study and are commonly non-functional in Salmonella, mutations in the promoter of the gene can lead to expression and phenotypic resistance (31). Rather et al. (32) demonstrated that aminoglycoside resistance in Salmonella strains is usually secondary to increased gene expression following regulatory mutations.
Point mutations in the quinolone resistance-determining regions (QRDRs) were detected in 25 (89.28%) isolates at positions 57 (Thr57Ser) and 80 (Ser80Ile) for ParC with known acquired antibiotic resistance as Nalidixic acid and Cipro oxacin. However, all isolates were susceptible to both nalidixic acid and cipro oxacin.
In this study, two Salmonella strains Kokomlemle and one unknown serotype possessed IncFII-type plasmids, which have been important in spreading resistance genes such as bla NDM−1 and bla CTX−M−15 (Xavier et al., 2016). Both strains did not harbour any beta-lactamase resistance genes. More investigation into these plasmid sequences are needed to determine any bene t they provide the strains.

Conclusion
This study has shown that widely consumed sh in Burkina Faso are contaminated with pathogenic bacteria of the genus Salmonella. The microbiological quality of sh sold in Burkina Faso must be improved to reduce the risks of contamination to consumers. Improved food safety will lead to reduced losses, better access to markets and hence better incomes. The modern molecular biology technique used in this study as whole genome sequencing is a technique that is not yet available in the developing country. An urgent action is needed by decision-makers in Burkina Faso, other developing countries, and those around the world for collaboration in the regulation and monitoring of foodborne pathogens.