Global Emergence of Colistin-Resistant Escherichia coli in Food Chains and Associated Food Safety Implications: A Review

Antimicrobial resistance in bacteria represents one of the most important challenges for public health worldwide. Human infections from antimicrobial-resistant bacteria can be transmitted from person to person, via the environment (especially in the hospital environment), or via handling or eating contaminated foods. Colistin is well known as a last-resort antibiotic for the treatment of human infections; a recent study performed in the People's Republic of China has revealed that colistin resistance is also conferred by the plasmid-mediated mcr-1 gene in Escherichia coli. After that discovery, further plasmid-mediated, colistin resistance genes have been detected. However, to date, only reports on E. coli carrying the mcr-1 gene (E. coli mcr-1+) in foodstuff are available. E. coli mcr-1+ has been isolated from food of animal origin and vegetables; this discovery has opened a debate among food safety experts. This review aims to provide a critical overview of the currently available scientific literature on the presence of the plasmid-mediated, colistin resistance gene E. coli mcr-1 in foodstuffs, focusing on the main implications and future perspectives for food safety

A fish market survey using a novel PCR-sequencing-based protocols for the identification of commercial significant fish species

This study developed a simple, specific, and affordable PCR-sequencing-COI gene-based protocol for the simultaneous identification of some important commercial fish species: Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasianodon hypophthalmus, Epinephelus spp. For this study, a local market survey on fish was carried out to evaluate the application of labelling laws and to detect fraudulent actions using the developed PCR protocols. Ten specimens of each fish species of interest were obtained from wholesale fishery plants and were utilized for the protocol development. DNA was extracted from the individual samples and quantified. DNA isolates were subjected to end-point PCR and the PCR products were sequenced. For the identification of fish species, novel species-specific primers were developed by the program "Primer Express 3.0" and by the software "Primer-BLAST" to amplify fragments of 200 bp, 250 bp, 300 and 562 bp, 350 bp, 400 bp and 522 bp within the COI gene for M. merluccius, L. niloticus, G. morhua, R. pretiosus, P. hypophthalmus, Epinephelus spp., respectively. Single PCR was performed using DNA isolates and developed primers for each fish species of interest. After sequencing, the isolates were compared with the selected sequences of the COI gene and showed a similarity ranging from 99 to 100%. Among 43 samples obtained for the survey, 19 (44.2%) were mislabelled, with 18 (41.9%) mislabelled samples from local fisheries and fish marketplaces and 1 (2.32%) from hypermarket stores. Among fish samples purchased at local fisheries and fish marketplaces, fraudulent actions were observed more frequently in fish slices (100%) than fish fillets (65%). Regarding fish fillets, out of four samples labelled as grouper, three were L. niloticus and one P. hypophthalmus. Two fillets marketed as cod were substituted with L. niloticus. Five samples labelled as "fillet" and two samples labelled as "perch" were identified as P. hypophthalmus. Regarding fish slices, all samples marketed as grouper (E. marginatus) were slices of R. pretiosus. The single case of mislabelling detected from fishery products purchased at hypermarket stores was a sample of "Spinycheek grouper" (Epinephelus diacanthus) that was indicated on label as "Grouper" (Epinephelus marginatus). In summary, our work highlights the need for continuous surveillance of the commercialization of fishery products, to reduce the number of fraud cases that happen in the market. Furthermore, our protocols based on PCR techniques could be useful for quality control of fresh finfish and to strengthen controls on the most frequent fraudulent actions of marketed fishery products

Prevalence of anisakid parasites in fish collected from Apulia region (Italy) and quantification of nematode larvae in flesh

Abstract Anisakis spp. and Hysterothylacium spp. are nematodes that commonly parasitize several fish species. Nematode larvae can be recovered in coelomic cavity and viscera, but also in flesh and have an important economic and public health impact. A total of 1144 subjects of wild teleosts, 340 samples of cephalopods and 128 specimens of farmed fish collected from Apulia region were analysed for anisakid larvae detection by visual inspection of coelomic cavity and viscera and by digestion of the flesh. No nematode larvae were found in farmed fish and cephalopod molluscs. All examined wild-caught fish species were parasitized, except for 5 species for each of which only a few subjects belonging to the same batch were sampled, therefore the results are just indicative. A total of 6153 larvae were isolated; among these, 271 larvae were found in the muscular portion. Larvae were identified by morphological method as belonging to the genera Anisakis (97.2%) (type I and type II) and Hysterothylacium (2.8%). Both nematodes could be found in all fish species, except for round sardinella (Sardinella aurita), infected only by Hysterothylacium spp. and for Mediterranean scaldfish (Arnoglossus laterna), little tunny (Euthynnus alleteratus) and chub mackerel (Scomber japonicus) infected only with Anisakis spp.. A sample of 185 larvae was sent to the National Reference Centre for Anisakiasis (C.Re.N.A.) of Sicily for identification at the species level: 180 larvae belonged to the species A. pegreffii and 2 larvae to A. physeteris. The remaining 3 larvae were identified at genus level as Hysterothylacium. Statistical indices such as prevalence, mean intensity and mean abundance were calculated. Chub mackerel (S. japonicus) was the species with the highest prevalence and mean intensity. Moreover, the average and the median values of larvae per 100 g of edible part for each fish species were determined to estimate the consumer exposure to Anisakis spp.. The obtained values were then recalculated by referring to the edible part of all specimens (infected and non-infected) forming a single parasitized batch, getting more realistic and objective data useful for risk assessment. Our results indicate that the consumption of raw or undercooked wild fish caught off Apulian coasts could result in the acquisition of anisakiasis; on the contrary, farmed fish and cephalopods appear to be safer for the consumer

A multicenter randomized phase 4 trial comparing sodium picosulphate plus magnesium citrate vs. polyethylene glycol plus ascorbic acid for bowel preparation before colonoscopy. The PRECOL trial

Background: Adequate bowel preparation before colonoscopy is crucial. Unfortunately, 25% of colonoscopies have inadequate bowel cleansing. From a patient perspective, bowel preparation is the main obstacle to colonoscopy. Several low-volume bowel preparations have been formulated to provide more tolerable purgative solutions without loss of efficacy. Objectives: Investigate efficacy, safety, and tolerability of Sodium Picosulphate plus Magnesium Citrate (SPMC) vs. Polyethylene Glycol plus Ascorbic Acid (PEG-ASC) solutions in patients undergoing diagnostic colonoscopy. Materials and methods: In this phase 4, randomized, multicenter, twoarm trial, adult outpatients received either SPMC or PEG-ASC for bowel preparation before colonoscopy. The primary aims were quality of bowel cleansing (primary endpoint scored according to Boston Bowel Preparation Scale) and patient acceptance (measured with six visual analogue scales). The study was open for treatment assignment and blinded for primary endpoint assessment. This was done independently with videotaped colonoscopies reviewed by two endoscopists unaware of study arms. A sample size of 525 patients was calculated to recognize a difference of 10% in the proportion of successes between the arms with a two-sided alpha error of 0.05 and 90% statistical power. Results: Overall 550 subjects (279 assigned to PEG-ASC and 271 assigned to SPMC) represented the analysis population. There was no statistically significant difference in success rate according to BBPS: 94.4% with PEG-ASC and 95.7% with SPMC (P = 0.49). Acceptance and willing to repeat colonoscopy were significantly better for SPMC with all the scales. Compliance was less than full in 6.6 and 9.9% of cases with PEG-ASC and SPMC, respectively (P = 0.17). Nausea and meteorism were significantly more bothersome with PEG-ASC than SPMC. There were no serious adverse events in either group. Conclusion: SPMC and PEG-ASC are not different in terms of efficacy, but SPMC is better tolerated than PEG-ASC. SPMC could be an alternative to lowvolume PEG based purgative solutions for bowel preparation

Investigation on Anthrax in Bangladesh during the Outbreaks of 2011 and Definition of the Epidemiological Correlations

In 2011, in Bangladesh, 11 anthrax outbreaks occurred in six districts of the country. Different types of samples were collected from May to September in the six districts where anthrax had occurred in order to detect and type Bacillus anthracis (B. anthracis) strains. Anthrax was detected in 46.6% of the samples analysed, in particular in soils, but also in bone samples, water, animal feed, and rumen ingesta of dead animals. Canonical single nucleotide polymorphisms (CanSNPs) analysis showed that all the isolates belonged to the major lineage A, sublineage A.Br.001/002 of China and Southeast Asia while the multi-locus variable number of tandem repeats (VNTRs) analysis (MLVA) with 15 VNTRs demonstrated the presence of five genotypes, of which two resulted to be new genotypes. The single nucleotide repeats (SNRs) analysis showed 13 SNR types; nevertheless, due to its higher discriminatory power, the presence of two isolates with different SNR-type polymorphisms was detected within two MLVA genotypes. This study assumes that soil is not the only reason for the spread of the disease in Bangladesh; contaminated feed and water can also play an important role in the epidemiology of anthrax. Possible explanations for these epidemiological relationships are discussed

Biomolecular identification of fish species by PCR and analysis of microbiological risk linked to the consumption of ready to eat fishery products

The present study regarded the isolation and the characterisation of Staphylococcus aureus and Listeria monocytogenes from ready to eat (RTE) fishery products and the development and the improvement of novel PCR protocols for the identification of fish species. For the detection of S. aureus and L. monocytogenes, 99 and 135 RTE samples, respectively, were collected at local retail outlets and analysed according to ISO procedures in the laboratories of Food Microbiology of Experimental Zooprophylactic Institute of Apulia and Basilicata located in Foggia. RTE fishery products for the isolation of S. aureus and L. monocytogenes consisted of 33 and 45 samples of marinated anchovies fillets (Engraulis encrasicolus), 33 and 45 samples of smoked salmon (Salmo salar), 33 and 45 samples of seafood salad, respectively. As regards the identification of fish species, novel species-specific primers were developed by the program "Primer Express 3.0" and by the software “Primer-BLAST” to amplify fragments of 200 bp, 250 bp, 300 and 562 bp, 350 bp, 400 bp and 522 bp within COI gene for Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasius hypophthalmus, Epinephelus spp., respectively. Ten samples of each fish species of interest were obtained from wholesale fishery plants. DNA was extracted from individual sample and quantified. DNA isolates were subjected to end-point PCR analysis and PCR products were sequenced. Out of 33 samples of smoked salmon, S. aureus was isolated from one sample (3.03%). The S. aureus strains carried the icaA, seb and sec genes and were resistant to ampicillin and tetracycline. L. monocytogenes was isolated from 2 of 45 samples of smoked salmon (4.44%). The strains of L. monocytogenes, isolated from both samples, resulted to belong to the serovar 1/2a and to be susceptible to all antibiotics tested. Single PCRs were performed using DNA isolates and the developed primers for each fish species of interest. After sequencing, the isolates were compared with the selected sequences of COI gene and showed a similarity ranging from 99 to 100%. Duplex and Triplex PCR protocols were developed for the simultaneous analysis of more fish species using the designed primers with several combinations. In addition, a survey on fish products was carried out to evaluate the application of labelling laws and to detect fraudulent actions using the developed PCR protocols. Forty-three fishery products were collected, in particular 18 and 25 samples at hypermarket stores, and at local fisheries and fish marketplaces, respectively. Fishery products purchased at local fisheries and fish marketplaces consisted of 20 fish fillets and 5 fish slices. After PCR analysis and sequencing, 19 (44.2%) resulted mislabelled, with 18 (41.9%) mislabelled samples from local fisheries and fish marketplaces and 1 (2.32%) from hypermarket stores. As regards fish samples purchased at local fisheries and fish marketplaces, fraudulent actions regarded more fish slices (100%) than fish fillets (65%). Regarding fish fillets, 3 out of four samples labelled as grouper, three (75%) resulted to be Lates niloticus and one (25%) Pangasianodon hypophthalmus. Two fillets marketed as cod (100%) were substituted with Lates niloticus (100%). Five samples labelled as “fillet” and two samples labelled as “perch” were identified as P. hypophthalmus. As regards fish slices, all samples marketed as grouper slices (E. marginatus) were slices of Ruvettus pretiosus (100%). The single case of mislabelling detected from fishery products purchased at hypermarket stores regarded a sample of “Spinycheek grouper” (Epinephelus diacanthus) that was indicated on label as “Grouper” (Epinephelus marginatus). In conclusion, our work highlights the need of a continuous surveillance on the commercialisation of fishery products, in order to reduce the food-borne risk linked to the presence of S. aureus and L. monocytogenes in RTE fishery products. Furthermore, our protocols based on PCR techniques could be useful for quality controls of fresh finfish and to strengthen controls on the most frequent fraudulent actions of marketed fishery products

Methicillin-resistant Staphylococcus aureus (MRSA) in slaughtered pigs and abattoir workers in Italy

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen present in the hospital environment (HA-MRSA), in the community (CA-MRSA) and in livestock, including pigs (LA-MRSA). MRSA may enter the human food chain during slaughtering and may infect humans coming into direct contact with pigs or pork products. This study aimed to determine the prevalence and characteristics of MRSA isolated from pigs and workers at industrial abattoirs in southern Italy. A total of 215 pig nasal swabs were screened for the presence of MRSA using PCR. An MRSA isolate was detected from each mecA/. nuc PCR-positive sample and characterized by spa-typing, Multi-Locus Sequence Typing, SCC-. mec and Panton-Valentine Leukocidin (PVL), and also tested for the production of staphylococcal enterotoxins (SEs).Eighty-one MRSA isolates (37.6%) were obtained from the 215 pig nasal swabs; 37 of these isolates were further characterized, and showed 18 different spa-types and 8 different STs. The most frequently recovered STs were ST398 (CC398-t034, t011, t899, t1939 - 43.2%) followed by ST8 (CC8-t008, t064, t2953, t5270 - 24.3%) and ST1 (CC1-t127, t174, t2207 - 10.8%).Nine MRSA isolates were obtained from the 113 human swabs; the isolates showed 5 different spa-types and 5 different STs, including the novel ST2794 (t159). The most representative STs recovered were ST1 (CC1-t127) and ST398 (CC398-t034) (33.3%). None of the MRSA isolates showed the ability to produce SEs and PVL and all resulted resistant to two or more classes of antimicrobials. This study shows the great genetic diversity of MRSA strains in slaughtered pigs and in abattoir employees in Italy, and clearly demonstrates the need for improved hygiene standards to reduce the risk of occupational and food-borne infection linked to the handling/consumption of raw pork containing MRSA

Efficiency of the Q3 lab-on-chip platform in detecting protozoan pathogens in bivalve mollusks

Cryptosporidium, Giardia and Toxoplasma have been recorded worldwide in economically important edible shellfish, and are thus likely to present a significant public health risk. The development of an innovative, user-friendly diagnostic tool is required to improve food safety control. The Q3 system, a miniaturized platform which integrates amplification and detection process for realtime-PCR on a Lab-on-chip, was developed by STMicroelectronics for the detection of zoonotic protozoans in three shellfish species according to realtime-PCR protocols previously set up in our lab. Q3’s efficiency and applicability were investigated and compared with results obtained by standard realtime-PCR. Tanks of saltwater containing acclimated pathogen-free Mytilus galloprovincialis, Tapes semidecussatus and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 103, 104 and 105). Untreated control shellfish were included in each test. At 24h and 72h post-contamination (p.c.), thirty specimens of each shellfish species were collected from each group. Haemolymph, gills and gastric glands were removed and stored in pools at -20°C. After DNA extraction, all samples were tested by standard realtime-PCR and Q3, and we evaluated the sensitivity, specificity, predictive values, repeatability and the concordance between standard realtime-PCR and Q3-system. A significant concordance between standard realtime-PCR and Q3-system CT (Threshold Cycle) values was registered for all the shellfish species at the highest parasite load (i.e. 105) at 72h p.c. for Toxoplasma and Giardia in M.galloprovincialis, for Toxoplasma and Giardia in O.edulis and for Cryptosporidium in T.semidecussatus (P<0.05). No significant differences were registered between the anatomical sites. Q3 demonstrated an ability to detect all investigated pathogens, that was similar to standard realtime-PCR, and a very high level of ability to detect Toxoplasma in M.galloprovincialis and Toxoplasma and Giardia in O.edulis. Currently, the Q3-system can be used efficiently to detect Toxoplasma from whole mussels and oysters, and to a lesser extent also from clams

Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh.

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country