Assessing pig farm biosecurity measures for the control of Salmonella on European farms.

Salmonella spp. is a common zoonotic pathogen, causing gastrointestinal infections in people. Pigs and pig meat are a major source of infection. Although farm biosecurity is believed to be important for controlling Salmonella transmission, robust evidence is lacking on which measures are most effective. This study enrolled 250 pig farms across nine European countries. From each farm, 20 pooled faecal samples (or similar information) were collected and analysed for Salmonella presence. Based on the proportion of positive results, farms were categorised as at higher or lower Salmonella risk, and associations with variables from a comprehensive questionnaire investigated. Multivariable analysis indicated that farms were less likely to be in the higher-risk category if they had '<400 sows'; used rodent baits close to pig enclosures; isolated stay-behind (sick) pigs; did not answer that the hygiene lock/ anteroom was easy to clean; did not have a full perimeter fence; did apply downtime of at least 3 days between farrowing batches; and had fully slatted flooring in all fattener buildings. A principal components analysis assessed the sources of variation between farms, and correlation between variables. The study results suggest simple control measures that could be prioritised on European pig farms to control Salmonella.

The effect of environmental conditions on the occurrence of Campylobacter jejuni and Campylobacter coli in wastewater and surface waters.

AIMS: The purpose of the study was to evaluate the occurrence of Campylobacter jejuni and Campylobacter coli in the aquatic environment based on the water origin, seasonality and physico-chemical properties. METHODS AND RESULTS: The occurrence of C. jejuni and C. coli was determined in waste (29) or surface (56) waters in four different seasons. The air and water temperatures were measured during sampling and chemical analyses of water samples for ammonium, chloride, chlorine, nitrite, nitrate, phosphate and iron were performed. The thermotolerant Campylobacter spp. were more frequently detected in wastewater (59%; 17 positive samples) compared to surface water (38%; 21 positive samples), with the highest rate in autumn (67% of samples positive) and with a higher C. coli occurrence than C. jejuni (31% vs. 26%). Ammonium (above 0.2 mg/L) and chloride ion concentrations (above 60 mg/L) favour C. jejuni. Similarly, C. coli occurrence in water was supported by ammonium (above 0.2 mg/L), chloride (above 60 mg/L) and in addition by phosphate ion concentrations (below 0.7 mg/L). CONCLUSIONS: Campylobacter presence in water is influenced by physico-chemical parameters such as concentrations of ammonium and chloride ions. SIGNIFICANCE AND IMPACT OF THE STUDY: Water environment is an alternative source of Campylobacter. The concentration of ammonium and chloride ions can be used as a basis for successful prediction of the potential occurrence of C. jejuni and C. coli in wastewater and surface water in future.

Effect of the vaccination against Shiga toxin 2e in a farm with history of oedema disease, caused by atypical Escherichia coli producing Shiga toxin (STEC).

Oedema disease of weaned piglets is caused by shigatoxigenic Escherichia coli (STEC), typically harbouring the stx2e gene and F18 adhesins. The aim of this study was to assess the effect of a commercially available oedema disease vaccine on the zootechnical performance, mortality and individual antibiotic treatment in a herd, in which non-typical STEC strains without F18 adhesin have been identified. The zootechnical performance (average daily gain, total weight gain), mortality and individual antibiotic treatment were compared between vaccinated and non-vaccinated control piglets in a monocentric field efficacy study, which was performed using two groups in a parallel, randomised design. A significantly higher average daily gain and total weight gain were recorded in the vaccinated piglets in comparison to the controls. The lower morbidity, mortality and antibiotic treatment in piglets in the vaccine group were not statistically significant. As a conclusion, the positive effect of the vaccination was confirmed in the herd with prevalent STEC not harbouring F18 adhesin. The vaccine was, therefore, also effective against oedema disease caused by such unusual STEC isolates, under the conditions of this study.

Genomic Insights into Methicillin-Resistant Staphylococcus aureus spa Type t899 Isolates Belonging to Different Sequence Types.

Methicillin-resistant Staphylococcus aureus (MRSA) presenting spa type t899 is commonly associated with sequence type 9 (ST9) but is also increasingly linked to ST398. This study provides genomic insight into the diversity of t899 isolates using core genome multilocus sequence typing (cgMLST), single nucleotide polymorphism (SNP)-based phylogeny, and the description of selected antimicrobial resistance and virulence markers. The SNP-based phylogenic tree showed that isolates sharing the same spa type (t899) but different STs highly diverged in their core and accessory genomes, revealing discriminant antimicrobial resistance (AMR) and virulence markers. Our results highlighted the idea that in a surveillance context where only spa typing is used, an additional multiplex PCR for the detection of the tet(M), sak, and seg genes would be valuable in helping distinguish ST9 from ST398 isolates on a routine basis.IMPORTANCE This study showed the genetic diversity and population structure of S. aureus presenting the same spa type, t899, but belonging to different STs. Our findings revealed that these isolates vary deeply in their core and accessory genomes, contrary to what is regularly inferred from studies using spa typing only. Given that identical spa types can be associated with different STs and that spa typing only is not appropriate for S. aureus isolates that have undergone major recombination events which include the passage of the spa gene (such as in t899-positive MRSA), the combination of both MLST and spa typing methods is recommended. However, spa typing alone is still largely used in surveillance studies and basic characterization. Our data suggest that additional markers, such as tet(M), sak, and seg genes, could be implemented in an easy and inexpensive manner in order to identify S. aureus lineages with a higher accuracy.

Escherichia coli Strains Producing Selected Bacteriocins Inhibit Porcine Enterotoxigenic Escherichia coli (ETEC) under both In Vitro and In Vivo Conditions.

Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) strains are the causative agents of severe foodborne diseases in both humans and animals. In this study, porcine pathogenic E. coli strains (n = 277) as well as porcine commensal strains (n = 188) were tested for their susceptibilities to 34 bacteriocin monoproducers to identify the most suitable bacteriocin types inhibiting porcine pathogens. Under in vitro conditions, the set of pathogenic E. coli strains was found to be significantly more susceptible to the majority of tested bacteriocins than commensal E. coli. Based on the production of bacteriocins with specific activity against pathogens, three potentially probiotic commensal E. coli strains of human origin were selected. These strains were found to be able to outcompete ETEC strains expressing F4 or F18 fimbriae in liquid culture and also decreased the severity and duration of diarrhea in piglets during experimental ETEC infection as well as pathogen numbers on the last day of in vivo experimentation. While the extents of the probiotic effect were different for each strain, the cocktail of all three strains showed the most pronounced beneficial effects, suggesting synergy between the tested E. coli strains. IMPORTANCE Increasing levels of antibiotic resistance among bacteria also increase the need for alternatives to conventional antibiotic treatment. Pathogenic Escherichia coli represents a major diarrheic infectious agent of piglets in their postweaning period; however, available measures to control these infections are limited. This study describes three novel E. coli strains producing antimicrobial compounds (bacteriocins) that actively inhibit a majority of toxigenic E. coli strains. The beneficial effect of three potentially probiotic E. coli strains was demonstrated under both in vitro and in vivo conditions. The novel probiotic candidates may be used as prophylaxis during piglets' postweaning period to overcome common infections caused by E. coli.

A Multicenter Proposal for a Fast Tool To Screen Biosecure Chicken Flocks for the Foodborne Pathogen Campylobacter.

The present multicenter study aimed at assessing the performance of air sampling as a novel method for monitoring Campylobacter in biosecure poultry farms. We compared, using a harmonized procedure, the bacteriological isolation protocol (ISO 10272-1:2017) and a real-time PCR method used on air filter samples. Air samples and boot swabs were collected from 62 biosecure flocks from five European countries during the summer of 2019. For air filters, the frequency of PCR-positive findings was significantly higher (n = 36; 58%) than that obtained with the cultivation methods (P < 0.01; standardized residuals). The cultivation protocols (one with Bolton enrichment and one with Preston enrichment) were comparable to each other but returned fewer positive samples (0 to 8%). The association between type of sample and frequency of PCR-positive findings was statistically confirmed (P < 0.01; Fisher´s exact test), although no culture-positive air filters were detected using direct plating. For the boot swabs, the highest number of positive samples were detected after enrichment in Preston broth (n = 23; 37%), followed by direct plating after homogenization in Preston (n = 21; 34%) or Bolton broth (n = 20; 32%). It is noteworthy that the flocks in Norway, a country known to have low Campylobacter prevalence in biosecure chicken flocks, tested negative for Campylobacter by the new sensitive approach. In conclusion, air sampling combined with real-time PCR is proposed as a multipurpose, low-cost, and convenient screening method that can be up to four times faster and four times more sensitive than the current boot-swab testing scheme used for screening biosecure chicken production.IMPORTANCECampylobacter bacteria are the cause of the vast majority of registered cases of foodborne illness in the industrialized world. In fact, the bacteria caused 246,571 registered cases of foodborne illness in 2018, which equates to 70% of all registered cases in Europe that year. An important tool to prevent campylobacters from making people sick is good data on where in the food chain the bacterium is present. The present study reports a new test method that quadruples the likelihood of identifying campylobacter-positive chicken flocks. It is important to identify campylobacter-positive flocks before they arrive at the slaughterhouse, because negative flocks can be slaughtered first in order to avoid cross-contamination along the production line.

Campylobacter in chicken - Critical parameters for international, multicentre evaluation of air sampling and detection methods.

The present pilot study aimed at evaluating air sampling as a novel method for monitoring Campylobacter in poultry farms. We compared the bacteriological isolation of Campylobacter from boot swabs and air filter samples using ISO 10272-1:2017. A secondary aim was to evaluate the use of molecular methods, i.e. real time PCR, on the same sample set. Samples from 44 flocks from five European countries were collected, and included air samples, in parallel with boot swabs. Campylobacter spp. was isolated from seven of 44 boot swabs from three of five partners using the enrichment method. Two of these positive boot swab samples had corresponding positive air samples. Using enrichment, one positive air sample was negative in the corresponding boot swabs, but Campylobacter spp. was isolated from direct plating of the boot swab sample. One partner isolated Campylobacter spp. from six of 10 boot swabs using direct plating. Overall, 33 air filter samples were screened directly with PCR, returning 14 positive results. In conclusion, there was a lack of correspondence between results from analysis of boot swabs and air filters using ISO 10272-1:2017. In contrast, the combination of air filters and direct real-time PCR might be a way forward. Despite the use of the detailed ISO protocols, there were still sections that could be interpreted differently among laboratories. Air sampling may turn into a multi-purpose and low-cost sampling method that may be integrated into self-monitoring programs.

Detection and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Bulk Tank Milk of Cows, Sheep, and Goats.

This study is aimed at detecting and characterizing methicillin-resistant Staphylococcus aureus (MRSA) from bulk tank milk samples of cows, sheep, and goats collected from dairy farms in the Czech Republic. All MRSA isolates were identified using PCR detection of the Staphylococcus aureus-specific fragment SA442 and mecA gene. The staphylococcal chromosomal cassettes mec (SCCmec), spa, and multilocus sequence types (MLST) were determined. The presence of genes encoding enterotoxins (ses), Panton-Valentine leukocidin (pvl), exfoliative toxins A, B (eta, etb), and toxic shock syndrome toxin (tst) were assessed. To differentiate human and animal origin, the presence of staphylokinase (sak) gene, ϕSa3 prophage, and susceptibility to tetracycline was tested. Out of 49 bulk tank milk samples examined, 14 (28.6%) were MRSA-positive. Eleven positive samples came from cow's milk (38%) and the remaining three from goat's milk (33%). All samples of ewe's milk were negative. In MRSA isolates three sequence types containing seven spa types were identified. Twelve isolates (85.7%) belonged to ST398 spa types t011/SCCmec IVa, t011/SCCmec V, t034/SCCmec V, t1456/SCCmec IVa, t1255/SCCmec V, and t2346/SCCmec V. Another two isolates belonged to ST5/t3598/SCCmec IVa and ST8/t064/SCCmec IVNT. In six isolates, one or more ses genes (seb, sed, seg, sei, and sej) were confirmed. One isolate from cow's milk harbored the tst gene. Another two isolates (ST398/t1456/SCCmec IVa and ST5/t3598/SCCmec IVa) harbored the sak gene and ϕSa3 prophage, and the latter was the only tetracycline-susceptible isolate in this study. However, none of the isolates was positive for pvl or eta, etb. These results suggest that there is the wide geographical spread of ST398 across different regions of the Czech Republic with no host preference among dairy cattle and goats. Therefore, when evaluating the occupational and foodborne risks, MRSA carriage and infection should be taken into account.

[Zoonotic significance of Escherichia coli strains isolated from cattle and pigs].

OBJECTIVES: The aim of this study was to monitor characteristics of Shiga toxin-producing Escherichia coli (STEC) obtained from animals according to the serogroup they belonged to, Shiga toxin type and subtype and adhesion factor intimin. Then, based on the results, to evaluate the occurrence of Shiga toxin subtypes and their possible significance for humans. MATERIALS AND METHODS: The study included 131 STEC strains isolated from rectal swabs from cattle (80) and pigs (51) sampled on farms in the Czech Republic from 2000 to 2017. Selected strains differed in origin and serogroup. The presence of Shiga toxins, intimin and the Shiga toxin subtypes stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e, stx2g was determined by PCR. RESULTS: The stx1 gene was confirmed in 33 % of the strains (43/131), the stx2 gene in 55 % (72/131) and 16 strains carried the genes for both toxins simultaneously (12 %). Strains harboring the eae gene were detected in 46 (35 %) cases, mostly in rectal swabs from cattle. STEC from cattle belonged to 21 different serogroups. The presence of Shiga toxin 1 (55; 69 %) predominated in these strains, with subtypes stx1a (54) and stx1d (1). Shiga toxin 2 was confirmed in 39 of the bovine strains (49 %), with the following subtypes: stx2a (9), stx2e (6), stx2g (3), stx2a, stx2c (5), stx2a, stx2b (1) and stx2c, stx2d (1). Also combinations of stx1a, stx2a (12) and stx1a, stx2c (2) were detected. STEC from pigs belonged to 5 different serogroups. Shiga toxin 2 was most frequently detected (49; 96 %), with subtypes stx2e (42) and stx2a (7). Shiga toxin 1 was detected in 4 strains (8 %), as subtypes stx1a (1) and stx1c (1) and also in the combination stx1a, stx2a (2). CONCLUSION: STEC strains isolated from cattle, compared to those from pigs, belonged to a larger spectrum of serogroups, they more often carried adherence factor intimin and the diversity of Shiga toxin subtypes was higher, including those associated with serious human diseases. In the set of isolates from pigs, the stx2e gene predominated; its significance for human health has not been fully clarified yet.

Occurrence and characterization of food-borne pathogens isolated from fruit, vegetables and sprouts retailed in the Czech Republic.

Food of non-animal origin is a major component of the human diet and has been considered to pose a low risk from the point of view of bacteriological safety. However, an increase in the number of outbreaks of illness caused by such pathogens and linked to the consumption of fresh fruit and vegetables have been reported from around the world recently. Salmonella spp., STEC (Shiga toxin producing Escherichia coli) and Listeria monocytogenes are among the most frequently identified agents. Additionally, the transmission of antibiotic resistant strains including also the methicillin resistant S. aureus (MRSA) to humans via the food chain is one of the greatest public health problems being confronted today. Therefore, we focused on the bacterial safety of fruit, vegetables and sprouts on sale in the Czech Republic. One strain (0.3%) of Salmonella Enteritidis phage type PT8, one strain (0.3%) of MRSA and 17 strains (5.0%) of L. monocytogenes were isolated from a total of 339 collected samples. The most problematic commodities were frozen fruit and vegetables (packed and unpacked) and fresh-cut vegetables. Our findings indicate deficiencies in hygiene practices during harvesting, processing and distribution of these commodities. Although sprouts and berries are the most likely to be contaminated by human pathogens, only two samples were positive for the presence of L. monocytogenes.

[A rare mechanism of resistance to colistin in Escherichia coli isolated from raw poultry meat].

Plasmid-mediated resistance to colistin is a recently described phenomenon. The study reports this new type of colistin resistance in food isolates of Escherichia coli in the Czech Republic. Strains with phenotypically determined colistin resistance were studied for presence of the mcr-1 and mcr-2 genes. A positive finding of E. coli harboring the mcr-1 gene was confirmed in a sample of raw minced turkey meat imported from Poland. Two different strains of E. coli carrying the mcr-1 gene were detected in the same sample. This is the first reported case of this type of resistance in E. coli strains isolated from foods at retail in the Czech Republic.

A novel mutation leading to a premature stop codon in inlA of Listeria monocytogenes isolated from neonatal listeriosis.

The study objective was to investigate whether the strain of L. monocytogenes serotype 1/2c isolated from neonatal listeriosis carries a premature stop codon (PMSC) mutation in the inlA gene. The strain was characterized by serotyping, macrorestriction analysis after digestion with the restriction enzyme AscI, and sequencing of the inlA gene. The tested strain of serotype 1/2c and pulsotype 1 possesses a new type of point mutation leading to a PMSC in the inlA gene and production of truncated internalin A. The case of early onset form of neonatal listeriosis caused by serotype 1/2c with a PMSC mutation in the inlA gene confirmed the transplacental transmission potential of this strain.

[Prevalence and characteristics of MRSA strains isolated from pigs on farms, at slaughterhouses and in pork meat at retail sale in the Czech Republic]. / Výskyt a vlastnosti kmenu MRSA izolovaných u prasat na farmách, na jatkách a ve veprovém mase v trzní síti CR.

OBJECTIVES: To detect and characterize strains of methicillin-resistant Staphylococcus aureus (MRSA) on pig farms, at slaughterhouses and meat samples at retail sale. MATERIAL AND METHODS: A total of 890 samples were examined in the years 2013-2015, comprising 59 samples from primary production (pig farms), 463 swabs from processing plants (slaughterhouses) and 368 samples of pork meat and liver collected at retail sale. The culture method was used for the detection of MRSA. After homogenization, the samples were enriched in buffered peptone water at 37 °C for 18-24 hours and two-stage selective enrichment was performed. Five milliliters of PPV were transferred to Mueller-Hinton medium with addition of 6.5 % NaCl and after incubation at 37 °C for 18-24 hours, 1 ml was inoculated into tryptone soy broth with cefoxitin and aztreonam. The suspension was plated onto Baird-Parker and BrillianceTM MRSA 2 agar on the next day. Suspected colonies were confirmed by PCR, the specific S. aureus fragment SA442, the mecA gene encoding resistance to methicillin and relation to the clonal complex CC398 were detected. Further, resistance test to a panel of 11 antimicrobial agents using the disk diffusion method was performed. RESULTS: Within this study, 51 MRSA strains were obtained, of which 15 originated from live pigs, 31 isolates were from slaughterhouses and 5 were detected in retail samples. Forty-seven (92.2 %) MRSA strains belonged to the clonal complex CC398. Four non-CC398 isolates were obtained from two slaughterhouses and came from three farms. The strains often showed multiple resistance. In some MRSA isolates, resistance to erythromycin (36; 70.6 %), tetracycline (29; 56.9 %), fluoroquinolones (7; 13.7 %), co-trimoxazole (6; 11.8 %) and aminoglycosides (4; 7.8 %) was detected. CONCLUSION: MRSA isolates of the clonal complex CC398 dominate in foods of animal origin. These strains originate from livestock and are often characterized by multiple resistance to antimicrobials. The role of the food chain in the spread of LA-MRSA is not yet clearly understood.

[Potential use of mass spectrometry for subtyping of Campylobacter]. / Moznosti vyuzití hmotnostrí spektrometrie k subtypizaci kampylobakteru.

OBJECTIVES: Molecular epidemiology is a field that uses results of typing techniques to obtain information on detailed characterization of bacterial strains for determining the identity, similarity or difference in bacteria of the same genus, species or serotype. Nowadays, the most commonly used methods are based on monitoring differences in bacterial genotypes. However, most of these techniques are time-consuming and costly. A method increasingly used in routine microbiological testing is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is based on analysis of the bacterial proteome. It is mainly used for rapid and accurate classification of bacteria into genera and species. The aims were to assess the potential use of this method for typing of Campylobacter below the species level and to apply these results in epidemiological investigations. MATERIAL AND METHODS: The study comprised 39 strains of Campylobacter jejuni isolated from food (16) and humans (23). Macrorestriction fragment profiling by pulsed-field gel electrophoresis (PFGE) and simultaneous protein profile analysis using MALDI-TOF MS were performed for all tested strains. RESULTS: Similar pulse profiles were found among isolates originating from the same outbreak or repeatedly collected from a single patient. The same pulse profiles were also detected in strains of unknown relationship but sharing the same place of origin and year of isolation. The comparison of dendrograms from both analyses showed that strains identified as identical by PFGE appeared in the same subgroups in dendrograms obtained by MALDI-TOF MS, the only exception being isolates repeatedly collected from a single patient. CONCLUSION: The results suggest that confirmation of the identity or similarity of strains in accordance with the established epidemiological facts has not been clearly demonstrated using MALDI-TOF MS.

[Dogs as a possible source of human Campylobacter infecfions]. / Psi jako mozný zdroj jampylobakterových infekcí cloveka.

OBJECTIVES: The aims of this study were to obtain current information on the prevalence and species representation of bacteria of the genus Campylobacter in dogs in Moravia and to evaluate the risk factors affecting their occurrence with respect to possible transmission to the human population. MATERIAL AND METHODS: Rectal swabs of dogs obtained in the routine practice of veterinarians in the South Moravian and Olomouc -regions were examined from May 2013 to December 2014. The basic tests were performed in laboratories of the State Veterinary Institute in Olomouc and the University of Veterinary and Pharmaceutical Sciences Brno. To detect Campylobacter spp., the samples were cultured on mCCDA (modified charcoal-cefoperazone-deoxycholate agar). Suspected colonies were confirmed by MALDI-TOF MS (Biotyper Microflex, Bruker) or using specific PCR which allows to distinguish between the species C. jejuni, C. coli, C. lari and C. upsaliensis. A detailed history was obtained from questionnaires completed by the dog owners. RESULTS: From a total of 258 rectal swabs examined, 41 samples were positive (16 %). The most frequently detected species was C. jejuni, followed by C. upsaliensis a C. coli. There was only one sample of C. lari. The evaluation of the questionnaire data showed that the frequency of Campylobacter spp. and their species representation depended on the age of the animals, the composition of feed and the clinical signs of the disease. CONCLUSION: Young dogs on a homemade diet and with diarrhea may be considered a risk group in terms of possible transmission of Campylobacter infections from pets to humans. Households with young children are the most affected group in the Czech Republic and EU countries. As such, they should be given a high priority with respect to the basic hygiene rules if they breed dogs, especially puppies.

[Occurrence of Shiga toxigenic Escherichia coli strains in pigs and cattle at slaughterhouses in the Czech Republic in 2013]. / Vyskyt shigatoxigennich kmenu Escherichia coli u prasat a skotu na jatkach v CR v roce 2013.

OBJECTIVES: This study was performed in cooperation with the State Veterinary Administration (SVA) in order to monitor the occurrence of Shiga toxin-producing Escherichia coli isolates in swabs from the carcasses of pigs and cattle at slaughterhouses. MATERIAL AND METHODS: From June to August 2013, SVA staff took 168 swabs from cattle and 318 from pigs at 157 different slaughters in the Czech Republic. Basic processing of the samples was carried out in the State Veterinary Institutes (SVIs) in Prague, Jihlava and Olomouc according to the methodical process coordinated by the National reference laboratory (NRL) for Escherichia coli (Czech Ministry of Agriculture). The procedure was based on the guideline ISO TS 13136. RESULTS: Out of the 486 swabs, twenty-two positive samples were detected. There were a total of 22 isolates of Shiga toxigenic E. coli (STEC) and 1 strain with the characteristic of enterohemorrhagic E. coli (EHEC). Genes typical for enteroaggregative E. coli (EAggEC) were not found in any of the isolates. Most STEC strains originated from pigs. The stx1 gene was detected twice (stx1a, stx1d) and the stx2 gene 13 times (12 times stx2e, once stx2a). Seven STEC isolates were detected from samples of cattle origin. One strain was stx1 (stx1a) -positive, the stx2 gene was found 6 times (4 stx2e, 1 stx2a and 1 stx2c). One isolate carried simultaneously both stx1a and stx2a. Each of the serogroups O91, O113 and O146 described as etiological agents of severe disease in humans were detected only once. None of these strains harbored additional virulence factors typical for strains causing serious illness. CONCLUSION: RESULTS of this study show the overall prevalence of Shiga toxigenic E. coli of 4.5 % and 0.2 % of enterohemorrhagic strains in the studied samples. Raw meat originating from local farms does not currently represent an important source of STEC for humans.

Fate of fluoroquinolones in field soil environment after incorporation of poultry litter from a farm with enrofloxacin administration via drinking water.

The practice of incorporating animal manure into soil is supported within the European Circular economy as a possible substitute for mineral fertilizers and will become crucial for the sustainability of agriculture. However, this practice may indirectly contribute to the dissemination of antibiotics, resistance bacteria, and resistance genes. In this study, medicated drinking water and poultry litter samples were obtained from a broiler-chick farm. The obtained poultry litter was incorporated into the soil at the experimental field site. The objectives of this research project were first to develop analytical methods able to quantify fluoroquinolones (FQs) in medicated drinking water, poultry litter, and soil samples by LC-MS; second to study the fate of these FQs in the soil environment after incorporation of poultry litter from flock medicated by enrofloxacin (ENR); and third to screen the occurrence of selected fluoroquinolone resistance encoding genes in poultry litter and soil samples (PCR analysis). FQs were quantified in the broiler farm's medicated drinking water (41.0 ± 0.3 mg∙L-1 of ENR) and poultry litter (up to 70 mg∙kg-1 of FQs). The persistence of FQs in the soil environment over 112 days was monitored and evaluated (ENR concentrations ranged from 36 µg∙kg-1 to 9 µg∙kg-1 after 100 days). The presence of resistance genes was confirmed in both poultry litter and soil samples, in agreement with the risk assessment for the selection of AMR in soil based on ENR concentrations. This work provides a new, comprehensive perspective on the entry and long-term fate of antimicrobials in the terrestrial environment and their consequences after the incorporation of poultry litter into agricultural fields.

Epiphytic microflora and mycotoxin content in meadows-Is plant biodiversity affecting fungal contamination?

Ecosystem services are an important aspect of grasslands utilization; however, they are often contradictory to their main purpose, which is a production of good quality and safe feed. In this study, we evaluated the difference between grass monocultures and species-rich mixtures in terms of epiphytic microflora and mycotoxin contamination levels. We hypothesized that higher species diversity would lead to higher microbial counts, which could lead to higher mycotoxin contamination risk. Differences in epiphytic fungal, yeast and total amount of microorganisms (CFU g -1) depending on the species diversity in the field has been evaluated by cultivation method. Concentration of deoxynivalenol (DON), zearalenone (ZEN) and aflatoxin B1 (AFB1) was measured by ELISA. Results are suggesting that higher total amount of microorganisms were found in monocultures, however, fungal and yeast counts were higher in species-rich mixtures. Higher species diversity of grasses was related to higher total microbial count (TMC) and yeast colonization of phyllosphere. Our results suggest higher risk of fungal phyllosphere colonization of species-rich mixtures with higher biodiversity and therefore higher risk of mycotoxin contamination of such feed.

Virulence Properties of mcr-1-Positive Escherichia coli Isolated from Retail Poultry Meat.

The great plasticity and diversity of the Escherichia coli genome, together with the ubiquitous occurrence, make E. coli a bacterium of world-wide concern. Of particular interest are pathogenic strains and strains harboring antimicrobial resistance genes. Overlapping virulence-associated traits between avian-source E. coli and human extraintestinal pathogenic E. coli (ExPEC) suggest zoonotic potential and safety threat of poultry food products. We analyzed whole-genome sequencing (WGS) data of 46 mcr-1-positive E. coli strains isolated from retail raw meat purchased in the Czech Republic. The investigated strains were characterized by their phylogroup-B1 (43%), A (30%), D (11%), E (7%), F (4%), B2 (2%), C (2%), MLST type, and serotype. A total of 30 multilocus sequence types (STs), of which ST744 was the most common (11%), were identified, with O8 and O89 as the most prevalent serogroups. Using the VirulenceFinder tool, 3 to 26 virulence genes were detected in the examined strains and a total of 7 (15%) strains met the pathogenic criteria for ExPEC. Four strains were defined as UPEC (9%) and 18 (39%) E. coli strains could be classified as APEC. The WGS methods and available on-line tools for their evaluation enable a comprehensive approach to the diagnosis of virulent properties of E. coli strains and represent a suitable and comfortable platform for their detection. Our results show that poultry meat may serve as an important reservoir of strains carrying both virulence and antibiotic resistance genes for animal and human populations.

Detection and molecular characterisation of methicillin-resistant Staphylococcus aureus isolated from raw meat in the retail market.

OBJECTIVES: This study aimed to detect and characterise methicillin-resistant Staphylococcus aureus (MRSA) from retail meat in the Czech Republic. METHODS: Isolates were identified by PCR detection of the S. aureus-specific fragment Sa442 and mecA gene. spa typing, MLST, detection of genes encoding staphylococcal enterotoxins, Panton-Valentine leukocidin (pvl), exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin (tst) and staphylokinase (sak), detection of φSa3 prophage and antimicrobial susceptibility testing were performed. RESULTS: Of 65 raw meat samples examined (poultry, beef, pork and rabbit), 23 (35.4%) were positive for MRSA. Twelve positive samples originated from poultry (12/33; 36.4%), while the remaining eleven came from pork (9/9; 100%) and pork/beef mixed minced meat (2/5; 40.0%). Eight spa types belonging to five different sequence types (STs) were identified. ST398 was the most frequent (28/36; 77.8%), presenting spa types t011, t034, t2576, t4132, t588 and t899. Other livestock-associated MRSA STs (ST9-t899, ST5-t002, ST692-t8646 or the newly described ST4034-t899) were also sporadically identified. In seven isolates (19.4%), one or more staphylococcal enterotoxin genes were detected, with sea, seg and sei prevailing. Three isolates from turkey [ST398-t899 (n = 2) and ST398-t011] harboured the sak gene, and the latter also harboured the sea gene. Seven isolates from poultry harboured the φSa3 prophage and were resistant to tetracycline. CONCLUSION: Specific kinds of meat appear to be a possible source of MRSA, although the risk to humans is hard to define. Therefore, surveillance of MRSA in meat as well as hygienic practices should be improved.