Conventional and molecular methods to detect bacterial pathogens in mussels (original) (raw)
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The control of the microbiological quality of bivalve molluscs assumes particular importance because they are among the most produced seafood products and mostly consumed as a whole, raw, or lightly cooked. The composition of the bacterial community associated with bivalves depends mostly on the microbiology of the surrounding environment at growing sites. Once the relationship between microbiology of bivalves and environment is established, a better classification and monitoring of the shellfish beds and evaluation of depuration strategies can be achieved. In this work, we tested if the methods of DNA extraction commonly used for the culture-independent microbiological analysis of sediment and water could be used directly, or with modifications, for the analysis of bacteria in mussels. The commercial kits Genomic DNA Purification Kit (MBI Fermentas, Vilnius, Lithuania), UltraClean TM Soil DNA Isolation Kit (MOBIO Laboratories, Inc., Carlsbad, CA) and the method described by Griffiths and collaborators for DNA/ RNA co-extraction were compared. The efficiency of extraction was assessed by DNA fluorescence and the denaturing gradient gel electrophoresis gel patterns of 16S ribosomal RNA gene fragments were used to compare the reproducibility and representativeness of the extraction methods. Results showed that the DNA/RNA co-extraction method with modifications was the most suitable. However, the results must be interpreted in the light of the purpose of the study and the relevance of maximizing extraction yield or diversity estimate, without compromising reproducibility. To our knowledge, this was the first attempt to transpose the procedure currently used for DNA extraction from sediments or waters, to the analysis of whole mussels.
Multiresistant Bacteria: Invisible Enemies of Freshwater Mussels
SSRN Electronic Journal, 2021
Freshwater mussels are among the most endangered groups of fauna anywhere in world. The indiscriminate use of antibiotics has led to the emergence of resistant strains. These antibiotic-resistant bacteria play a key role in increasing the risk allied with the use of surface water and in spread of resistance genes. Two endangered freshwater mussel species, Margaritifera margaritifera and Potomida littoralis, were sampled at 4 sampling sites along a 50 km stretch of River Tua. Water samples were taken at same sites. Of the total of 135 isolates, 64.44% (39.26% from water and 25.19% from mussels) were coliform bacteria. Site T1, with the lowest concentration of coliform bacteria, and site T2 were the only ones where M. margaritifera was found. No E. coli isolates were found in this species and the pattern between water and mussels was similar. P. littoralis, which was present at T3/T4 sites, is the one that faces the highest concentration of bacterial toxins, which are found in treated wastewater effluents and around population centers. Sites T3/T4 have the isolates (water and mussels) with the highest resistance pattern, mainly to β-lactams. Water and P. littoralis isolates (T3/T4) showed resistance to penicillins and their combination with clavulanic acid, and to cephalosporins, precisely to a fourth generation of cephalosporin antibiotics. The analysis provides important information on the risk to water systems, as well as the need to investigate possible management measures. It is suggested that future studies on the health status of freshwater bivalves should incorporate measures to indicate bacteriological water quality.
Detection of Vibrionaceae in mussels and in their seawater growing area
Letters in Applied Microbiology, 2001
Aims: The seasonal trend and frequency of detection of Vibrionaceae in seawater samples and in molluscs collected in the Adriatic Sea was measured. Methods and Results: Over a 2-year period, 726 bacterial strains were isolated, of which 46á9% belonged to the Vibrio genus, 29á8% to the Aeromonas genus and the remaining 23á3% was made up of the Pseudomonas, Flavobacterium, Pasteurella, Agrobacterium and Ochrobacterium genera. Many of the isolated strains were shown to produce toxins. Conclusions: The Vibrio genus, which was isolated more often than the other genera, was particularly prevalent in summer (54á4% of the total number of bacteria isolated during this season), while it was scarce in the winter months.
Agricultura, 2020
The present study was carried out to assess the bacteriological quality of the cultured mussel "Mytilus galloprovincialis" and its impact on public health and to study the influence of the three physicochemical variation (environmental) factors: medium temperature (TS), pH and salinity (SL). The exploration of the bacteriological quality of the mussels "Mytilus galloprovincialis" was carried out by research and enumeration of Total flora (TF), Total coliform (TC), Fecal coliform (FC), Escherichia coli (EC), Fecal streptococci(FS) and Staphylococcus aureus (SA), using specific techniques and methods. Likewise, for environmental factors, they were evaluated by typical instruments. The results of the bacterial count revealed that the means of the isolated bacteria were 2.27 ± 0.24 log10 cells / 100ml, 3.34 ± 0.51 log10 cells / 100ml, 2.64 ± 0.72 log10 cells / 100ml, 0.94 ± 1.63 log10 cells / 100ml, 1.85 ± 1.61 log10 cells / 100ml and 0.63 ± 1.09 log10 cells / 100m...
Journal of Food Quality, 2016
To assess bacteriological safety of mussels Perna perna, harvested in Itaipu, Niter oi, RJ and purchased at São Pedro fishmarket at the same city, fecal coliform counts and detection of Escherichia coli, Salmonella spp. and Vibrio spp. was performed in 27 mussel and 9 seawater samples collected between March 2012 and April of 2013. The pathogens isolated were submitted to antimicrobial susceptibility test. The majority (74%) of the mussel samples were unfit for human consumption and 89% of the seawater samples had unsatisfactorily high levels of contamination according to Brazilian laws. From the 77 E. coli and 51 Vibrio spp. isolated, 13% and 68.6% showed multiresistance and 15.6% and 72.5% showed multidrug resistant, respectively. The only Salmonella strain isolated was susceptible to all antimicrobial tested. Serotypes of Salmonella spp. and V. cholerae were not detected from the enrichment broths. Water pollution and food manipulation, throughout the food production chain, were factors which influenced the contamination of mussels. PRACTICAL APPLICATIONS This is the first report studying microbial quality of mussels along their process before arriving at the market; including characteristics of virulence genes and antimicrobial resistance of pathogens. Our study demonstrated that after collection, the manipulation realized by mussel's pickers is an important source of contamination, warning to the necessity of a better monitoring in the commercialization of this seafood.
Journal of Applied Microbiology, 1994
Mussels (Perna perna) harvested on the coast of Ubatuba, in three different stations in the State of São Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g-1) were: Vibrio alginolyticus (<3–24000), V. parahaemolyticus (<3–24000), V. fluvialis (<3–1100), V. cholerae non-O1 (<3–23), V. furnissi (< 3–30), V. mimicus (< 3–9) and V. vulnificus (< 3–3). The highest incidence was observed for V. alginolyticus (92–100%), followed by V. parahaemolyticus (67–92%), V. fluvialis (34–67%), V. vulnificus (8–17%), V. furnissii (0–17%), V. mimicus (0–17%) and V. cholerae non-O1 (0–8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18–3300 MPN 100 ml-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.
Bacterial Loads in Moroccan Mussels from Harvest to Sale
Journal of Food Protection, 1995
This study concerns the seasonal variation of bacteria in seawater and mussels harvested from a Moroccan coastal area subject to clandestine shellfishing. The changes in the level of bacterial counts of mussels from harvest to sale are also presented. Both seawater and mussels showed regular increases in bacterial loads from fall to summer. Freshly harvested mussels and market mussels were the most contaminated, while freshly shucked mussels, obtained by removing shells after heating shellstock, were the least contaminated. Heating, traditionally used to remove shells, was found to reduce the initial bacterial loads by 72%. However, the storage of shucked mussels for 6 to 8 h at ambient temperatures prior to marketing resulted in an increase in the number of bacteria either due to recontamination or by growth of survivors. Thus, market mussels were 20 to 86 times more contaminated than shellstock and shucked mussels, respectively. No human pathogens were found, but several species o...
International Journal of Food Microbiology, 2009
Vibrio parahaemolyticus is a marine bacterium with a worldwide distribution and is frequently associated with human outbreaks of infection. Detection and isolation of V. parahaemolyticus from natural sources is often problematical because of limitations in the analytical procedures. We evaluated a combination of conventional and molecular protocols previously described for the investigation of V. parahaemolyticus, with the aim of identifying the best procedures for improved detection of this organism in environmental matrixes. A total of 259 samples of zooplankton (103), mussels (48) and seawater (108) were investigated by an Absence-Presence method (A/P), whereas 118 samples of zooplankton (70) and mussels were analyzed by the Most Probable Number (MPN) method. All samples were processed by a two-step enrichment procedure, firstly with APW broth and then with SPB as selective secondary broth. Detection of V. parahaemolyticus was by direct-PCR and by plate culture on TCBS and CHROMagar Vibrio, after sample enrichment in APW and SPB. With the A/P method, V. parahaemolyticus was detected in 23.6% samples by direct-PCR, whereas only 11.2% samples were positive with the plate culture method. With the MPN method, V. parahaemolyticus was detected in 54.2% and 27.1% of the samples by direct-PCR and plate culture respectively; this indicated the existence of 31% false negative results with the A/P method. No significant differences between the use of a single (APW) or two-step enrichment (APW+SPB) were observed by direct-PCR with A/P or MPN, although a significant higher presence of V. parahaemolyticus was detected by plate culture in both protocols with the two-step enrichment procedure. In conclusion, direct-PCR after sample enrichment in APW broth was the most successful method for detection of V. parahaemolyticus with the A/P procedure and enumeration by MPN. Better detection was obtained with MPN than with the A/P protocol. Conversely, the plate culture procedure showed better results with the two-step enrichment protocol in which CHROMagar Vibrio was used as the selective agar.
Dreissenid mussels as sentinel biomonitors for human and zoonotic pathogens
2014
Zebra mussels (Dreissena polymorpha) are recognised biomonitors in determining the presence and viability of the human waterborne pathogens Cryptosporidium parvum, C. hominis, Giardia intestinalis and microsporidia in surface waters. This study investigated whether the size of zebra mussels is a significant factor in the concentration of protozoan Cryptosporidium oocysts, Giardia cysts and microsporidian spores. Zebra mussels were collected in Lough Arrow, a small Irish lake, which is utilized for drinking water abstraction and is subject to agricultural and human wastewater pollution drivers, both recognised risk factors for human waterborne pathogens. Zebra mussels were cleaned, divided into size (5 mm) interval classes based on their shell length and made up to 150 g samples (wet weight with shell). Combined fluorescence in situ hybridization (FISH) and immunofluorescent antibody (IFA) techniques were utilized as biomolecular techniques to assess the presence and concentration of the pathogens. PCR analysis provided source-tracking information on human and animal pollution sources. There was no significant relationship between the size of D. polymorpha and pathogen loads in similar sized samples, indicating that different sites in the same or different waterbody can be compared in terms of relative concentrations of human waterborne parasites irrespective of the zebra mussels' size. Cryptosporidium was the most abundant species, with lower counts of Giardia and the microsporidian Encephalitozoon hellem, respectively. Cryptosporidium oocysts and Giardia cysts were detected in zebra mussel samples at all three lake water abstraction points. A lake transect showed a decline in Cryptosporidium with increasing distance from a stream discharging sewage. Samples from agricultural sites indicated faecal inputs contaminated with these pathogens. Species identification implicated both human and animal faecal inputs to the lake from treated effluent, septic tanks, and agriculture. The research demonstrates the efficacy of zebra mussels as sentinels of water quality i rrespective of their size.