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.

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.

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.

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.

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.

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.

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.

The emergence of mcr-1-mediated colistin-resistant Escherichia coli and Klebsiella pneumoniae in domestic and imported turkey meat in the Czech Republic 2017-2018.

We investigated the occurrence of plasmid-mediated colistin resistance in Enterobacteriaceae in turkey meat produced in the Czech Republic as well as in turkey meat imported into the Czech Republic from other European Union countries. Seventeen samples of raw turkey meat from the Czech Republic (n = 4), Hungary (n = 2), Poland (n = 6) and Germany (n = 5) were cultured in peptone water at 37 °C overnight and the enriched cultures were tested for the presence of mcr-1-5 genes. PCR-positive enriched cultures were inoculated onto selective agar with colistin (3.5 mg/L). A minimal inhibitory concentration (MIC) of colistin was determined by using the broth microdilution method in PCR-positive isolates. In addition, a macrorestriction analysis was performed using XbaI endonuclease. Of 17 meat samples, 12 samples from Poland (6/6), Germany (3/5) and the Czech Republic (3/4) proved positive for the presence of the mcr-1 gene. Forty-two isolates carrying the mcr-1 gene were obtained: Escherichia coli (n = 39) revealing 32 distinct XbaI profiles and Klebsiella pneumoniae (n = 3) with 2 distinct XbaI profiles. The minimal inhibitory concentration (MIC) of the mcr-1 positive isolates was as follows: 4 mg/L (n = 28), 8 mg/L (n = 12), 32 mg/L (n = 1) and 64 mg/L (n = 1). The high prevalence (70.6%; 12/17 samples) of mcr-1-mediated colistin-resistant Enterobacteriaceae found in the turkey meat samples analysed in this study, builds on previously published evidence that poultry, and their products, represent a substantial risk for the dissemination of plasmid-mediated colistin resistance in Europe.

Prediction of Persistence of Listeria monocytogenes ST451 in a Rabbit Meat Processing Plant in the Czech Republic.

This study was focused on characterization of the genetic diversity of Listeria monocytogenes isolated from packed fresh rabbit meat obtained from one producer via retail outlets. The partial aim was to compare the characteristics of a suspect persistent strain with strains from human cases. The occurrence of L. monocytogenes in vacuum-packed rabbit meat was monitored during 2013 to 2016. All strains were characterized by serotyping, pulsed-field gel electrophoresis, and multilocus sequence typing (MLST). Selected strains, which represented each year, were analyzed using the whole genome sequencing method. L. monocytogenes was detected in 21 (38%) of 56 originally packed rabbit meat samples from one food producer during the whole monitored period. All strains showed the identical serotype (1/2a), AscI/ApaI pulsotype (735/2), and sequence type (ST451). The clonal similarity of strains from rabbit meat was also confirmed on the basis of core genome MLST (on 1,701 loci). This fact suggests the occurrence of a suspect persistent strain in the meat processing plant. Results of core genome MLST enabled us to unambiguously exclude rabbit meat as a source of listeriosis in humans caused by the indistinguishable AscI/ApaI pulsotype and sequence type, although all strains carried all genes important for the virulence of L. monocytogenes. No specific genes that may be associated with its persistence in the food processing environment were detected among the tested strains of ST451.

Lytic and genomic properties of spontaneous host-range Kayvirus mutants prove their suitability for upgrading phage therapeutics against staphylococci.

Lytic bacteriophages are valuable therapeutic agents against bacterial infections. There is continual effort to obtain new phages to increase the effectivity of phage preparations against emerging phage-resistant strains. Here we described the genomic diversity of spontaneous host-range mutants of kayvirus 812. Five mutant phages were isolated as rare plaques on phage-resistant Staphylococcus aureus strains. The host range of phage 812-derived mutants was 42% higher than the wild type, determined on a set of 186 methicillin-resistant S. aureus strains representing the globally circulating human and livestock-associated clones. Comparative genomics revealed that single-nucleotide polymorphisms from the parental phage 812 population were fixed in next-step mutants, mostly in genes for tail and baseplate components, and the acquired point mutations led to diverse receptor binding proteins in the phage mutants. Numerous genome changes associated with rearrangements between direct repeat motifs or intron loss were found. Alterations occurred in host-takeover and terminal genomic regions or the endolysin gene of mutants that exhibited the highest lytic activity, which implied various mechanisms of overcoming bacterial resistance. The genomic data revealed that Kayvirus spontaneous mutants are free from undesirable genes and their lytic properties proved their suitability for rapidly updating phage therapeutics.

Porcine pathogenic Escherichia coli strains differ from human fecal strains in occurrence of bacteriocin types.

Enterotoxigenic and Shiga-toxigenic Escherichia coli (i.e., ETEC and STEC) are important causative agents of human and animal diseases. In humans, infections range from mild diarrhea to severe life-threating conditions, while infections of piglets result in lower weight gain and higher pig mortality with the accompanying significant economic losses. In this study, frequencies of four phylogenetic groups, fourteen virulence- and thirty bacteriocin determinants were analyzed in a set of 443 fecal E. coli isolates from diseased pigs and compared to a previously characterized set of 1283 human fecal E. coli isolates collected in the same geographical region. In addition, these characteristics were compared among ETEC, STEC, and non-toxigenic porcine E. coli isolates. Phylogenetic group A was prevalent among porcine pathogenic E. coli isolates, whereas the frequency of phylogroup B2, adhesion/invasion (fimA, pap, sfa, afaI, ial, ipaH, and pCVD432) and iron acquisition (aer and iucC) determinants were less frequent compared to human fecal isolates. Additionally, porcine isolates differed from human isolates relative to the spectrum of produced bacteriocins. While human fecal isolates encoded colicins and microcins with a similar prevalence, porcine pathogenic E. coli isolates produced predominantly colicins (94% of isolates); especially colicins B (42.6%), M (40.1%), and Ib (34.0%), which are encoded on large conjugative plasmids. The observed high prevalence of these colicin determinants suggests the importance of large colicinogenic plasmids and/or the importance of colicin production in intestinal inflammatory conditions.

Genome Sequences of Livestock-Associated Methicillin-Resistant Staphylococcus aureusspa Type t899 Strains Belonging to Three Different Sequence Types (ST398, ST9, and ST4034).

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an emerging MRSA lineage rapidly evolving in the community. In this report, we present the draft genome sequences of nine LA-MRSA strains. These strains were isolated from meat and a human nasal swab sample and belong to one unique spa type (t899), but to three different sequence types, ST398, ST9, and ST4034.

Dissemination and Comparison of Genetic Determinants of mcr-Mediated Colistin Resistance in Enterobacteriaceae via Retailed Raw Meat Products.

The global food chain may significantly promote the dissemination of bacteria resistant to antibiotics around the world. This study was aimed at determining the prevalence and genetic characteristics of Enterobacteriaceae with mcr-mediated colistin (CT) resistance in retail meat of different origins. Bacteria of the Enterobacteriaceae family carrying the mcr-1 gene were detected in 21% (18/86) of the examined samples, especially in turkey meat and liver originating from EU and non-EU countries (19%) and in rabbit meat imported from China (2%). The examined samples of the meat and liver of chicken and other poultry and of pork and beef were negative for the presence of bacteria carrying the mcr-1 to mcr-5 genes. A huge number of isolates belonging to Escherchia coli (n = 54), Klebsiella pneumoniae (n = 6), and Citrobacter braakii (n = 1) carrying the mcr-1 gene were obtained. Despite the high heterogeneity of the tested isolates, the mcr-1 gene was localized on only three types of plasmids (IncX4, IncHI2, and IncI2). The most frequent type of plasmid was IncX4, which carried the mcr-1 gene in 77% of E. coli and K. pneumoniae isolates from turkey meat and liver samples from the Czechia, Germany, Poland, and Brazil. Our findings indicate highly probable interspecies transfer of IncX4 and IncI2 plasmids within one meat sample. The co-resistance of plasmid-mediated CT resistance encoded by the mcr-1 and ESBL genes was detected in 18% of the isolates. Another noteworthy finding was the fosA3 gene coding for fosfomycin resistance in a multidrug-resistant isolate of E. coli from rabbit meat imported from China. The observed high level of Enterobacteriaceae with plasmids carrying the mcr-1 gene in retail meat reflects the need for Europe-wide monitoring of mcr-mediated CT resistance throughout the whole food chain.

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.

Diversity of livestock associated methicillin-resistant Staphylococcus aureus.

OBJECTIVE: To evaluate the diversity and molecular characteristics of livestock associated methicillin-resistant Staphylococcus aureus in livestock animals, food of animal origin and the environment in the Czech Republic. METHODS: After having been primarily enriched in buffered peptone water, the samples were cultured on Baird-Parker agar. Presumptive colonies were sub-cultured to blood agar and assessed morphologically. Furthermore, presumptive Staphylococcus aureus colonies were confirmed by MALDI-TOF. Multiplex PCR, spa-typing, and MLST have been used to characterize the strains. Each mecA-positive Staphylococcus aureus isolates were examined against 14 different antimicrobials by using disk diffusion method. RESULTS: In this study, 13 different spa-types belonging to five sequence types (ST) were detected. Ninety four percent of tested strains belonged to CC/ST398 for which t011, t034, t2123 and t2346 were the vast major spa-types. In addition, non-ST398 clones such as CC1(t127), ST5(t3598), ST8(t064) and ST361(t315) were detected, which are known as human associated clones. CONCLUSIONS: The diversity of livestock associated methicillin-resistant Staphylococcus aureus has grown, and detecting lineages of human origin in animals and vice-versa becomes more common. Thus, livestock animal and its products will be a potential for the evolvement of methicillin-resistant Staphylococcus aureus in human population. Monitoring of pigs as well as other food-producing animal species and their products is therefore recommended.