The effect of exposure to non-ionising radiofrequency field on Escherichia coli, Klebsiella oxytoca and Pseudomonas aeruginosa biofilms.

An environment with a higher accumulation of electromagnetic non-ionising radiofrequency (RF) emissions generated by various telecommunication, data transport and navigation devices (mobile phones, Wi-Fi, radar, etc.) may have a major impact on biological systems. This study aimed to evaluate the incidence of an electromagnetic field (EMF) on the development of bacterial biofilm. Quantification of biofilm production was done by using microtiter plate assay. Bacterial isolates of Escherichia coli, Klebsiella oxytoca and Pseudomonas aeruginosa were exposed with EMF of frequencies 1-5 and 2.4 GHz with an exposure time 3 or 24 h, respectively. Exposure of bacteria to EMF produced a statistically significant increase in biofilm production mainly at 1, 2 and 4 GHz, and in contrast, a significant inhibition of biofilm development occurred at frequencies 3 and 5 GHz, both with exception of K. oxytoca and P. aeruginosa. Wi-Fi operating at 2.4 GHz caused biofilm reduction. The results indicate that EMF exposure act on bacteria in both ways, depending on the frequency: as stressful by enhancing bacterial biofilm formation (under environmental stress, bacteria produce a polysaccharide matrix and aggregate to form biofilms to increase virulence and resistance), although some frequencies leading to biofilm damage could be caused by changes to the physicochemical properties of bacteria.

Slovak Local Ewe's Milk Lump Cheese, a Source of Beneficial Enterococcus durans Strain.

Slovak ewe's milk lump cheese is produced from unpasteurized ewe's milk without any added culture. Because of the traditional processing and shaping by hand into a lump, this cheese was given the traditional specialty guaranteed (TSG) label. Up till now, there have existed only limited detailed studies of individual microbiota and their benefits in ewe's milk lump cheese. Therefore, this study has been focused on the beneficial properties and safety of Enterococcus durans strains with the aim to contribute to basic dairy microbiology but also for further application potential and strategy. The total enterococcal count in cheeses reached 3.93 CFU/g (log 10) ± 1.98 on average. Based on a MALDI-TOF mass spectrometry evaluation, the strains were allotted to the species E. durans (score, 1.781-2.245). The strains were gelatinase and hemolysis-negative (γ-hemolysis) and were mostly susceptible to commercial antibiotics. Among the strains, E. durans ED26E/7 produced the highest value of lactase enzyme ß-galactosidase (10 nmoL). ED26E/7 was absent of virulence factor genes such as Hyl (hyaluronidase), IS 16 element and gelatinase (GelE). To test safety, ED26E/7 did not cause mortality in Balb/c mice. Its partially purified bacteriocin substance showed the highest inhibition activity/bioactivity against Gram-positive indicator bacteria: the principal indicator Enterococcus avium EA5 (102,400 AU/mL), Staphylococcus aureus SA5 and listeriae (25,600 AU/mL). Moreover, 16 staphylococci (out of 22) were inhibited (100 AU/mL), and the growth of 36 (out of 51) enterococcal indicators was as well. After further technological tests, E. durans ED26E/7, with its bacteriocin substance, can be supposed as a promising additive to dairy products.

New Insight on Antibiotic Resistance and Virulence of Escherichia coli from Municipal and Animal Wastewater.

Antibiotic resistance of the indicator microorganism Escherichia coli was investigated in isolates from samples collected during the course of one year from two wastewater treatment plants treating municipal and animal wastes in Slovakia, respectively. The genes of antibiotic resistance and virulence factors in selected resistant E. coli isolates were described. A high percentage of the isolates from municipal and animal wastewater were resistant to ampicillin, streptomycin, tetracycline, ceftiofur, ceftriaxone, and enrofloxacin. In the selected E. coli isolates, we detected the following phenotypes: ESBL (20.4% in animal wastewater; 7.7% in municipal wastewater), multidrug-resistant (17% of animal and 32% of municipal isolates), high resistance to quinolones (25% of animal and 48% of municipal samples), and CTX-M (7.9% of animal and 17.3% of municipal isolates). We confirmed an integro-mediated antibiotic resistance in 13 E. coli strains from municipal and animal wastewater samples, of which the Tn3 gene and virulence genes cvaC, iutA, iss, ibeA, kps, and papC were detected in six isolates. One of the strains of pathogenic E. coli from the animal wastewater contained genes ibeA with papC, iss, kpsII, Int1, Tn3, and Cit. In addition, one blaIMP gene was found in the municipal wastewater sample. This emphasises the importance of using the appropriate treatment methods to reduce the counts of antibiotic-resistant microorganisms in wastewater effluent.

Escherichia coli Specific Virulence-Gene Markers Analysis for Quality Control of Ovine Cheese in Slovakia.

Shiga toxin-producing and extra-intestinal pathogenic Escherichia coli (E. coli) have the potential to spread through faecal waste, resulting in contamination of food and causing foodborne disease outbreaks. With the aim of characterizing unpasteurized ovine cheese in Slovakia, a total of 92 E. coli strains were examined for eleven representative virulence genes typical for (extra-)intestinal pathogenic E. coli and phylogenetic grouping. Phylogenetic groups B1 (36%) and A (32%) were the most dominant, followed by groups C (14%) and D (13%), while the lowest incidence was recorded for F (4%), and E (1%), and 43 (47%) samples carried at least one virulent gene, i.e., potential pathogens. Isolates present in groups E, F and D showed higher presence of virulence genes (100%, 75%, and 67%), versus 55%, 39%, and 28% in commensal B1, C, and A, respectively. Occurrence of papC and fyuA (both 24%) was highest, followed by tsh, iss, stx2, cnf1, kpsII, cvaC, stx1, iutA and eaeA. Nine E. coli strains (almost 10% of all tested and around 21% of our virulence-gene-associated isolates) harboured stx1, stx2 or eae. Ovine cheeses in Slovakia are highly contaminated with E. coli including potentially pathogenic strains capable of causing intestinal and/or extra-intestinal diseases, and thus may pose a threat to public health while unpasteurized.

Cell-Free Lactobacillus casei 21L10 Modulates Nitric Oxide Release and Cell Proliferation/Cell Death in Lipopolysaccharide-Challenged HT-29 Cells.

Lactobacillus casei (L. casei) is one of the probiotic strains that may influence intestinal injury and inflammation in nonspecific intestinal diseases. We aimed to evaluate the effect of cell-free Lactobacillus casei 21L10 supernatant (LC) on the cell line HT-29 challenged with lipopolysaccharide (LPS) in order to modulate production of NO, cell proliferation, and apoptosis. Cell line HT-29 was stimulated with LPS in the presence or absence of LC. Our results showed that LC from L. casei 21L10 did not affect the viability of unstimulated HT-29 cells line. HT-29 cell line treatment with LC caused significant decrease of LPS induced NO production after 3 h, and 24 h, but not after 48 h. Proliferation activity of LPS stimulated HT-29 cell line analysed with MTT assay significantly decreased after 24 h and 48 h, but not after 3 h. The majority of LPS stimulated HT-29 cell line treated with LC showed annexin V/PI positivity at 48 h survival, which corresponded to late apoptotic/necrotic cell features. The observed differences suggest that cell-free L. casei 21L10 supernatant could participate in attenuation of LPS-induced inflammation, and may exhibit anti-proliferative and pro-apoptotic/necrotic effects. This study provides pilot data for the further development of L. casei exoproducts as an anti-inflammatory or anti-proliferative agent for the treatment of inflammatory and cancer diseases in gut. However, more data is needed before final conclusions of L. casei cell-free supernatant's efficacy can be drawn.

Antibiotic resistance and virulence of Escherichia coli strains isolated from animal rendering plant.

Processing of animal carcasses and other animal wastes in rendering plants is a significant source of antibiotic resistant microorganisms. The main goal of this study was to investigate the resistance to 18 antibacterial agents including ß-lactams, fluoroquinolones, colistin and virulence factors (iss, tsh, cvaC, iutA, papC, kps and ibeA genes) in 88 Escherichia coli strains isolated from a rendering plant over 1 year period. ESBL (Extended-spectrum beta-lactamases) and plasmid-mediated Amp were screened by interpretative reading of MIC. ESBL phenotype was detected in 20.4% of samples and high level of resistance to fluoroquinolone was found in 27.2% of strains. Cephalosporinase CTX-M1, cephamycinase CMY-2, integrase 1 and transposon 3 genes were detected by PCR. Furthermore, there were found three CMY-2 producing E. coli with O25b-ST131, resistant to the high level of enrofloxacin and containing the gene encoding the ferric aerobactin receptor (iutA). One enrofloxacin resistant E. coli strain possessed iss, ibeA, kps and papC virulence genes also with CMY-2, integrase1 and Tn3. ST131 E. coli with CMY-2 has a zoonotic potential and presents a serious health risk to humans.

Some safety aspects of enterococci isolated from Slovak lactic acid dairy product "zincica".

In Slovakia, dairy products made from ewes' milk have a long tradition. These products include the lactic acid product called "zincica" which is a by-product occurring during the preparation of ewes' lump cheese. There is no information in the literature regarding the special properties of the microbiota, especially lactic acid Firmicutes, which can survive in "zincica." From the safety aspect, enterococci are a controversial group of bacteria, and those from "zincica" have never been tested for their properties. The "zincica" used in our study was supplied by several different agrofarms producing ewes' lump cheese in central Slovakia. The species Enterococcus faecium (strains EF30E1, EF32E1, EF34E1, EF34E5) and Enterococcus faecalis (strains EE30E4, EE35E1, E31E2, altogether 7) were detected in samples from "zincica" identified using MALDI-TOF spectrometry with secure genus identification/probable species identification and then confirmed by means of PCR. Enterococci were hemolysis-negative and the genes of the typical enterococcal virulence factors were mostly absent; the gelE gene was found in two E. faecium strains (EF30E1 and EF32E1), the agg gene was detected in E. faecalis EE35E1, and the esp gene was found in two E. faecalis strains (EE30E4 and EE31E2). No strains harbored the cytolysin A gene. Biofilm formation was detected in four strains (EF30E1, EF32E1, EF34E1, and EF34E5), indicating highly positive and low-grade positive biofilm formation. Enterococci were mostly susceptible to antibiotics tested for their phenotype. This is the first study to analyze enterococci in "zincica."

Antibiotic resistance and virulence factors of Escherichia coli from eagles and goshawks.

One of the major global problems in medicine is microbial resistance to antibiotics (antimicrobial resistance) and this has become an increasingly frequent research topic. This study focuses on antimicrobial resistance, phylogenetic and genetic characterization of Escherichia coli from wild birds: ten isolates from eagles (Aquila chrysaetos), nine from goshawks (Accipiter gentilis) and 24 from broilers in the Slovak Republic. Twenty-two strains with presence of int1 gene were selected and examined for the presence or absence of transposon gene (tn3), genes of antibiotic resistance and virulence factors. We detected sequence type (ST) in eagles ST 442 with genes iss, papC, iutA, cvaC, tsh, fyuA, iroN, kps, feoB, sitA, irp2, ireA for virulence factors and tetA, sul1, sul2, dfrA, aadA for antibiotic resistance; in goshawks ST 1011 with iss, papC, fyuA, iroN, feoB, sitA and qnrS1, tetA, sul1, sul2, dfrA, aadA, respectively. These ST types have been found in humans too and should be evaluated further for possible zoonotic potential and transfer of resistance genes from the environment.

New Insight into Genotypic and Phenotypic Relatedness of Staphylococcus aureus Strains from Human Infections or Animal Reservoirs.

Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.

Evaluation of in vitro growth-inhibitory effect of carvacrol and thymol combination against Staphylococcus aureus in liquid and vapour phase using new broth volatilization chequerboard method.

Carvacrol and thymol, both plant-derived volatile compounds, have extensively been studied individually as well as in combination with other agents for their antimicrobial activity in liquid phase. However, in contrast to well-established assays for testing of antimicrobial combinatory effects in liquid media, there are no standardized methods for evaluation of interactions between volatile compounds in vapour phase. The objective of this study was to verify new broth volatilization chequerboard method by testing the combination of carvacrol and thymol and to determine in vitro inhibitory effect of these compounds in liquid and vapour phase against twelve Staphylococcus aureus strains. The new method, based on combination of standard microdilution chequerboard and new broth volatilization tests allowing calculation of fractional inhibitory concentrations (FICs), was used. Combination of carvacrol and thymol produced the additive antimicrobial effect against all strains tested. In several cases, they reached ΣFIC values lower than 0.6, which can be considered as a strong additive interaction. The best result was found in vapour phase against one standard strain at combination of 128 µg/mL of carvacrol and 16-256 µg/mL of thymol (ΣFIC = 0.51) and in liquid phase against one clinical isolate at combination of 256 µg/mL of carvacrol and 256 µg/mL of thymol (ΣFIC = 0.53). The study verified that the new technique is suitable for simple and rapid high-throughput combinatory antimicrobial screening of volatile compounds simultaneously in vapour and liquid phase and that it allows determination and comparison of MIC and FIC values in both, liquid and solid media.

Small mammals as sentinels of antimicrobial-resistant staphylococci.

A total of 39 coagulase-negative staphylococci and seven Staphylococcus aureus strains were isolated from small mammal feces, i.e., the striped field mouse (Apodemus agrarius) and the yellow-necked mouse (A. flavicollis) in two sampling areas, deciduous forest and karst plains. MALDI-TOF analysis revealed five species of coagulase-negative staphylococci: S. sciuri, S. hominis, S. warneri, S. haemolyticus, and S. xylosus. All strains were susceptible to tetracycline, linezolid, vancomycin, and teicoplanin. Three MRSA strains with the mecA gene were detected. The beta-lactamase gene blaZ was detected in ampicillin-resistant staphylococci and in the high-level resistant strains (oxacillin over 2 mg/L) mecA gene. The mecC gene was not detected by PCR. Erythromycin-resistant staphylococci harbored the ermC gene and/or the efflux gene msrA. There were no detectable dfr genes in trimethoprim-resistant staphylococci and the rifampicin-resistant strains were without mutation in the rpoB gene. In summary, wild small mammals may serve as sentinels of mecA-positive S. aureus with erythromycin resistance genes ermC and efflux msrA. Small mammals appear to be useful indicators of antibiotic resistance.

Resistance in bacteria and indirect beta-lactamase detection in E. coli isolated from Culex pipiens detected by matrix-assisted laser desorption ionization time of flight mass spectrometry.

The aim of this study was detections of antibiotic resistance and resistance mechanism in bacteria isolated from mosquitos (Culex pipiens) living near humans. Therefore, antibiotic resistance in bacteria isolated from Culex pipiens was investigated by disk diffusion test and MIC E-test in this study. MALDI-TOF mass spectrometry was used for detection of resistant mechanism. In this study, hydrolytic breakdown products after a few hours of incubation of the bacteria isolated from Culex pipiens were detected. Results show that enzymatic destruction of ampicillin by beta-lactamases is able to be detected by MALDI-TOF mass spectrometry from wild strains of potential pathogens. The MALDI-TOF mass spectrometry is useful method for routine detection of beta-lactamases resistant mechanism, but overnight incubation of pure culture is necessary. The results are important for proper and fast intervention to limit the spread of beta-lactamase-producing wild bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.

Extended spectrum beta-lactamases in Escherichia coli from municipal wastewater.

INTRODUCTION AND OBJECTIVE: Over the past decades, awareness of the environmental load of resistant organisms has increased. The presented paper focuses on antibiotic resistance and detection of resistance genes in environmental E. coli and on the evaluation of biofilm formation in ESBLs (extended spectrum beta-lactamase) producing E. coli isolated from an urban wastewater treatment plant. MATERIALS AND METHOD: Wastewater samples and artificially added polystyrene pellets were used as the source for E. coli isolation. Minimal inhibitory concentrations of 19 antibiotics were determined according to CLSI (2013). Biofilm formation was investigated by crystal violet or resazurin methods. CTX-M, carbapenemases, qnrS, mobile elements and virulence factors were determined by PCR. Clonal relatedness of strains was detected by principal component analysis by a Maldi biotyper. RESULTS: ESBL phenotype was detected in 26% of environmental strains. CTX-M, CMY-2 and qnrS genes of antibiotic resistance were detected. IMP gene together with integron 1 in one ertapenem resistant E. coli was also recorded. There was no evident correlation between antibiotic resistance, virulence and biofilm production. CONCLUSIONS: The results showed that the wastewater is a source of ESBLs, carbapenemases and plasmid fluoroquinolone resistance. Strains with biofilm production, antibiotic resistance of CTX-M group, CMY-2, qnrS genes and virulence factors present a potential environmental health risk.

Mupirocin-mucin agar for selective enumeration of Bifidobacterium bifidum.

Bifidobacterium bifidum is a bacterial species exclusively found in the human intestinal tract. This species is becoming increasingly popular as a probiotic organism added to lyophilized products. In this study, porcine mucin was used as the sole carbon source for the selective enumeration of B. bifidum in probiotic food additives. Thirty-six bifidobacterial strains were cultivated in broth with mucin. Only 13 strains of B. bifidum utilized the mucin to produce acids. B. bifidum was selectively enumerated in eight probiotic food supplements using agar (MM agar) containing mupirocin (100 mg/L) and mucin (20 g/L) as the sole carbon source. MM agar was fully selective if the B. bifidum species was presented together with Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, and Bifidobacterium longum subsp. longum species and with lactic acid bacteria (lactobacilli, streptococci). Isolated strains of B. bifidum were identified using biochemical, PCR, MALDI-TOF procedures and 16S rRNA gene sequencing. The novel selective medium was also suitable for the isolation of B. bifidum strains from human fecal samples.

Isolation and characterization of bifidobacteria from ovine cheese.

Animal products are one of the niches of bifidobacteria, a fact probably attributable to secondary contamination. In this study, 2 species of the genus Bifidobacterium were isolated by culture-dependent methods from ovine cheeses that were made from unpasteurized milk without addition of starter cultures. The isolates were identified as Bifidobacterium crudilactis and Bifidobacterium animalis subsp. lactis using matrix-assisted laser desorption/ionization time-of-flight analysis and sequencing of phylogenetic markers (16S rRNA, hsp60, and fusA).

Genome Sequence of Lactobacillus plantarum 19L3, a Strain Proposed as a Starter Culture for Slovenska Bryndza Ovine Cheese.

The genome sequence of Lactobacillus plantarum isolated from ovine cheese is presented here. This bacterium is proposed as a starter strain, named 19L3, for Slovenská bryndza cheese, a traditional Slovak cheese fulfilling European Food Safety Authority (EFSA) requirements.

Species identification and antimicrobial resistance of coagulase-negative staphylococci isolated from the meat of sea fish.

UNLABELLED: Seventy-eight isolates of staphylococci obtained from the meat of Theragra chalcogramma, Scomber scombrus, and Clupea harengus were identified and tested in this study. 16S rDNA sequence specific for the genus Staphylococcus was detected in all isolates with the help of PCR method. All of 78 isolates were coagulase-negative, and DNAse activity was only confirmed in 4 of them. The following species of staphylococci were identified using MALDI-TOF mass spectrometry: S. warneri (52%), S. epidermidis (33%), S. haemolyticus (6.4%), S. pasteuri (3.8%), S. sciuri (1.2%), S. capitis (1.2%), and S. hominis (1.2%). Antimicrobial resistance to 7 antibiotics was determined in each isolate with the help of agar dilution method. In general, resistance against ampicillin was observed in majority of isolates (87%). On the contrary, the best sensitivity of CoNS was determined to gentamicin (96%). Only 1 S. warneri strain showed resistance to cefoxitin. Furthermore, 83% of staphylococcal isolates were simultaneously resistant to 2 or more antibiotics. PRACTICAL APPLICATION: This study confirmed the need of monitoring antimicrobial resistance in coagulase-negative staphylococci not only in the meat of slaughter animals but also in retail marine fish. The results showed that MALDI-TOF mass spectrometry is useful, accurate, and rapid method for species identification of food pathogens including Staphylococcus spp.

In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves.

A total of 73 chicken and calves isolates were diagnosed using matrix-assisted laser desorption ionization-time-of flight mass spectrometry (Maldi-Tof MS). After a preliminary subtractive screening based on the high acid tolerance at pH 2.5 and bile resistance at 0.3% oxgall, twenty isolates belonging to the species Lactobacillus salivarius, Lactobacillus agilis, Lactobacillus reuteri, Lactobacillus murinus and Lactobacillus amylovorus were in vitro screened for the safety assessment and probiotic properties, including antibiotics susceptibility patterns, biochemical activity and potential for competitive exclusion of biofilm producing pathogens determined by crystal violet and/or quantitative Fluorescent in situ Hybridisation (FISH) assays utilizing 5'Cy 3 labelled probe Enter1432 for enteric group. Antibiotic susceptibility testing was performed according to the ISO norm 10932. The sixteen strains were susceptible to certain antimicrobial agents, except for two chicken (L. salivarius 12K, L. agilis 13K) and two calves (L. reuteri L10/1, L. murinus L9) isolates with the presence non wild-type ECOFFs (epidemiological cut-off) for gentamicin (≥256 µg ml(-1)), tetracycline (≥128 µg ml(-1)), kanamycin (≥256 µg ml(-1)) and streptomycin (≥96 µg ml(-1)). The two referenced chicken isolates gave positive aac(6')Ie-aph(2″)Ia and tet(L) PCR results. The wild-type ECOFFs isolates were subjected to the apiZYM analysis for enzyme profile evaluation and amino acid decarboxylase activities determined by qualitative plate method and multiplex PCR for the detection of four genes involved in the production of histamine (histidine decarboxylase, hdc), tyramine (tyrosine decarboxylase, tyrdc) and putrescine (via eithers ornithine decarboxylase, odc, or agmatine deiminase, agdi). From examined strains only two chicken isolates (L. reuteri 14K; L. salivarius 15K) had no harmful ß-glucuronidase, ß-glucosidase activities connected with detrimental effects in the gastrointestinal tract and together no amino acid decarboxylase activities and no genes associated with biogenic amines production though only chicken L. salivarius 15K whole cells and acid supernatants shown strong suppressive potential against biofilm-forming Klebsiella and Escherichia coli. Our results highlight that above-mentioned isolate L. salivarius 15K fulfils the principle requirements of a qualified probiotic and may be seen as a reliable candidate for further validation studies in chicken.

Virulence and antibiotic resistance of Escherichia coli isolated from rooks.

With regard to antibiotic resistance studies in various model animals in the urban environment, the presented study focused on the rook, many behavioural and ecological aspects of which are important from an epidemiological point of view. A total of 130 Escherichia coli strains isolated from rook faeces during a two-year period (2011-2012) were investigated for antibiotic resistance and virulence. Resistance to ampicillin (60%) and streptomycin (40%) were the most frequent, followed by resistance to fluoroquinolones (ciprofloxacin-22% and enrofloxacin-24%), tetracycline (18%), cotrimoxazol (17%) and florfenicol (14%). Ceftiofur resistance occured in 10.7% of strains and cefquinom resistance in 1.5% of strains. Twenty-five E.coli strains with a higher level of MICs of cephalosporins (over 2mg/L of ceftazidime and ceftriaxon) and fluoroquinolones were selected for detection of betalactamase genes (CTX-M, CMY), plasmid-mediated quinolone resistance qnrS, integrase 1, and for APEC (avian pathogenic E.coli) virulence factors (iutA, cvaC, iss, tsh, ibeA, papC, kpsII). Genes of CTX-M1, CMY-2, integrase 1, papC, cvaC, iutA were detected in one strain of E.coli, and qnrS, integrase 1, iss, cvaC, tsh were detected in another E.coli. DNA microarray revealed the absence of verotoxin and enterotoxin genes and pathogenicity islands. The results show that rooks can serve as a reservoir of antibiotic-resistant E. coli with avian pathogenic virulence factors for the human population, and potentially transmit such E.coli over long distances.