Comparative Analysis of Antimicrobial Resistance and Virulence Genotypes of Escherichia coli from Poultry Meat and Young Chicks.

Recent studies indicated that some highly resistant strains of E. coli can be common contaminants of broiler meat, and resistance determinants can be of importance for the food production and human health. However, much less is known about their virulence determinants, and detailed genetic analyses of antimicrobial resistance and virulence are especially missing in E. coli from newly hatched broiler chicks. We aimed to provide a comparative description of antimicrobial resistance and virulence of E. coli isolates from young chicks from farms and from fresh broiler meat. A total of 70 E. coli isolates characterized here derived from different poultry sources: raw meat (28), young chicks from farms (represented by 11 intestinal- and 11 extraintestinal strains) and 20 E. coli isolates from newly hatched chicks. Resistance and virulence genotyping was performed using high throughput PCR microarray systems, AMR05 and Ec03 respectively. The tetra-resistant phenotype of streptomycin-nalidixic acid-sulfonamide-tetracycline commonly occured among E. coli strains from different chicken sources. The antimicrobial resistance genotype of E. coli strains from raw meet showed the highest similarity with the intestinal strains from young chicks. The high prevalence of antimicrobial resistance genes related to the flexible genome indicate the commonly high distribution of certain mobile genetic elements in poultry E. coli. Obviously, the predominance of the virulence genes in the extraintestinal E. coli strains was not surprising, however some of the virulence genes (iss, tsh, iutA) showed high prevalence in commensal isolates from newly hatched chicks and from the meat. Results indicate that E. coli from newly hathed chicks may represent a reservoir for multiresistance and virulence for both pathogenic and commensal E. coli strains of young chicks and of poultry meat. Acknowledgement: This work was supported by EU FP7 Collaborative Project PROMISE. Ama Szmolka is a holder of János Bolyai Research Scholarship of the HAS

Vaccine potential of a nonflagellated virulence-plasmid cured, (fliD-, pSEV∆) mutant of Salmonella Enteritidis for chickens

The aim of these studies was to assess residual virulence and early protective capacity of a negatively markered live attenuated vaccine candidate Salmonella Enteritidis mutant against a highly virulent S. Enteritidis strain using a day old chicken model. Nonflagellated FliD negative mutants of Salmonella Enteritidis 11 (SE11) with and without the virulence-plasmid proved to be sufficiently attenuated (limited invasiveness in vitro/vivo) without reduced ability to colonise chicken gut. The early protective activity of a nonflagellated virulence plasmid cured (fliD-, pSEV∆) mutant against organ invasion, caecal colonisation and faecal shedding by the highly virulent challenge strain S. Enteritidis 147 NalR proved to be effective and safe. The innate and adaptive immunity was demonstrable during the first 4 weeks of life, and the serological response was clearly distinguishable from the response induced by the wild parental strain. In conclusion, we provided data for the first time about virulence plasmid cured nonflagellated mutant of S. Enteritidis to serve as a basis for development of a negatively markered potential live oral vaccine against virulent S. Enteritidis in chicken

Characterization of the in vitro gene response of chicken cells to Salmonella Enteritidis

Salmonella Enteritidis (SE) is one of the most frequently reported causative agent of human gastroenteritis, originating mainly from poultry. Pathogenesis of SE infection in poultry is well-elucidated, but the complexity of the host cell response, and its relation to differring pathogenic potential of various strains is much less understood. Therefore we intended to provide a genome-wide comparative characterization of the gene expression profiles of chicken cells to wild type strains and virulence-related mutants of Salmonella Enteritidis. Freshly isolated chicken embryo fibroblast (CEF) cells co-incubated with Salmonella for 4 hrs were used to model gene response of young chickens to Salmonella infection and to measure the invasiveness of wild type strains SE147, SE11 and non-motile mutants of SE11 lacking the fliD gene and/or the virulence plasmid. Agilent custom 8×15K microarray was designed to profile the expression of 13741 chicken genes, with emphasis to those related to immune response. Significant gene expression changes with fold change ≥3 (in total of 31 genes) were verified by real-time PCR. Expression profile of infected CEF cells resulted in 314 genes significantly misregulated by the infection with the wild type strain SE147 (206 up-/108 down-regulations) while only 135 genes were significantly expressed as a result to SE11 infection (74 up-/61 down-regulations). There were 100 genes induced by both wild strains, among them CSF3 (colony-stimulating factor), IL-1β and IL-8 showing the highest upregulations. In contrast to this, infection with non-motile mutants lacking fliD gene and/or the virulence plasmid, did not cause any significant change in host gene expression. However real-time PCR results indicated that the cell cycle-related G0S2 switch-, and the enolase ENO2 genes were highly induced by the mutant strains, indicating that the reduced invasiveness of the mutants might have stimulated cell division and/or metabolism of the host cells. Results suggest that fliD gene plays a key role in the invasiveness of Salmonella strains, and could be considered as an important modulator of the chicken response to Salmonella infection. This work was supported by the EU FP6 NoE MedVetNet and OTKA 105635. Ama Szmolka is a holder of János Bolyai Research Scholarship of HAS

Conjugative IncF and IncI1 plasmids with tet(A) and class 1 integron conferring multidrug resistance in F18+ porcine enterotoxigenic E. coli

Enterotoxigenic E. coli (ETEC) bacteria are frequently causing watery diarrhea in newborn and weaned pigs. Plasmids carrying genes of different enterotoxins and fimbrial adhesins and plasmids conferring antimicrobial resistance are of prime importance in the epidemiology and pathogenesis of ETEC. Recently, the significance of the porcine ETEC plasmid pTC was revealed, carrying tetracycline resistance gene tet(B) with enterotoxin genes. In contrast the role of tet(A) plasmids in transferring resistance of porcine ETEC is less understood. Objective of the present study was to provide comparative analysis of antimicrobial resistance and virulence gene profiles of porcine post-weaning enterotoxigenic E. coli (ETEC) strains representing pork producing areas in Central-Europe and in the USA with special attention on plasmids carrying the tet(A) gene. Antimicrobial resistance phenotype and genotype of 87 porcine ETEC strains isolated from cases of post-weaning diarrhea in Austria, Czech Republic, Hungary and the Midwest USA was determined by disk diffusion and by PCR. Central-European Strains carrying tet(A) or tet(B) were further subjected to molecular characterization of their tet plasmids. Results indicated that >90% of the ETEC strains shared a common multidrug resistant (MDR) pattern of sulfamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. Tetracyclin resistance was most frequently determined by the tet(B) gene (38%), while tet(A) was identified in 26% of all isolates with wide ranges for both tet gene types between some countries and with class 1 integrons and resistance genes co-transferred by conjugation. The virulence gene profiles included enterotoxin genes (lt, sta and/or stb), as well as adhesin genes (k88/f4, f18). Characterization of two representative tet(A) plasmids of porcine F18+ ETEC from Central-Europe revealed, that the IncF plasmid (pES11732) of the Czech strain (~120 kb) carried tet(A) in association with catA1 for chloramphenicol resistance. The IncI1 plasmid (pES2172) of the Hungarian strain (~138 kb) carried tet(A) gene and a class 1 integron with an unusual variable region of 2,735 bp composed by two gene cassettes: estX-aadA1 encoding for streptothricin-spectinomycin/streptomycin resistance exemplifying simultaneous recruitment, assembly and transfer of multidrug resistance genes by tet(A) plasmid of porcine ETEC. By this we provided the first description of IncF and IncI1 type plasmids of F18+ porcine enterotoxigenic E. coli responsible for co-transfer of the tet(A) gene with multidrug resistance. Additionally the unusual determinant estX, encoding for streptothricin resistane was first reported here in porcine enterotoxigenic E. coli

Genome Sequences of Salmonella enterica subsp. enterica Serovar Infantis Strains from Hungary Representing Two Peak Incidence Periods in Three Decades

Four strains of Salmonella enterica subsp. enterica serovar Infantis isolated from humans (1980 to 1982) and broiler chickens (2016) have been sequenced. They represent the early and recent peak incidences of this serovar in Hungary. Genome sequences of these isolates provide comparative data on the evolution and rise of an endemic S Infantis clone in Hungary

Integrons and antimicrobial resistance genes of multidrug resistant Escherichia coli and coliform bacteria from foods of animal origin confiscated at the Hungarian borders

The import of contaminated food may represent a food safety risk by the spread of pathogenic and/or multidrug resistant (MDR) bacteria and their determinants for virulence and antimicrobial resistance. Here we aimed to isolate and characterize MDR E. coli and coliform bacteria from food samples from non-Schengen countries confiscated at the Hungarian borders. E. coli and coliform colonies were isolated based on their phenotype on Chromocult® Coliform selective media. Furthermore, API®, PCR and 16S rDNS sequencing were used for species identification. Resistance phenotypes were determined by disc diffusion method for 18 antimicrobials with animal and human clinical relevance. Corresponding antimicrobial resistance and virulence gene patterns were identified using PCR microarray systems AMR05 and Ec03 respectively. The gene cassette arrangements of the integrons were defined by amplicon sequencing. From the total of 207 confiscated food samples 833 coliform isolates were collected. Among them 17 (13 E. coli and 4 coliforms identified as Enterobacter spp.) showed resistance to at least three different antimicrobial classes thus were designated as MDR. The 17 strains represented 14 different food samples. Resistance genes strA, strB, sul2, blaTEM-1, tet(A) predominantly occurred, but in general the prevalence of the virulence genes was low. The identification of genes qnrB, aac(6’)-Ib, blaOXA-7 in some of the isolates indicated the presence of certain emerging antimicrobial resistance plasmids. Class 1 integrons were found in 10 of the 17 MDR isolates (9 E. coli, 1 coliform), and in the majority of them the sul1 gene was absent from their 3’ conserved segment (CS). Interestingly, in one of the pork samples we detected a non-typical class 1 integron carrying the sul3gene on its 3’CS. Above results showed that these illegal foods may frequently carry MDR E. coli and coliform bacteria with some unusual or new antimicrobial resistance traits

Vaccine potential of a nonflagellated, virulence-plasmid-cured (fliD–, pSEVΔ) mutant of Salmonella Enteritidis for chickens

The aim of these studies was to assess residual virulence and early protective capacity of a negatively markered live attenuated vaccine candidate Salmonella Enteritidis mutant against a highly virulent S. Enteritidis strain using a dayold chicken model. Nonflagellated FliD negative mutants of Salmonella Enteritidis 11 (SE11) with and without the virulence plasmid proved to be sufficiently attenuated (limited invasiveness in vitro/in vivo) without reduced ability to colonise chicken gut. The early protective activity of a nonflagellated, virulence-plasmidcured (fliD–, pSEVΔ) mutant against organ invasion, caecal colonisation and faecal shedding by the highly virulent challenge strain S. Enteritidis 147 NalR proved to be effective and safe. The innate and adaptive immunity was demonstrable during the first four weeks of life, and the serological response was clearly distinguishable from the response induced by the wild parental strain. In conclusion, we provided data for the first time about a virulence-plasmid-cured, nonflagellated mutant of S. Enteritidis to serve as a basis for development of a negatively markered potential live oral vaccine against virulent S. Enteritidis in chicken

Molecular epidemiology of the endemic multiresistance plasmid pSI54/04 of Salmonella Infantis in broiler and human population in Hungary

Salmonella Infantis (SI) became endemic in Hungary where the PFGE cluster B, characterized by a large multiresistance (MDR) plasmid emerged among broilers leading to an increased occurrence in humans. We hypothesised that this plasmid (pSI54/04) assisted dissemination of SI. Indeed, Nal-Sul-Tet phenotypes carrying pSI54/04 occurred increasingly between 2011-2013 among SI isolates from broilers and humans. Characterization of pSI54/04 based on genome sequence data of the MDR strain SI54/04 indicated a size of ~277 kb and a high sequence similarity with the megaplasmid pESI of SI predominant in Israel. Molecular characterization of 78 representative broiler and human isolates detected the prototype plasmid pSI54/04 and its variants of together with novel plasmid associations within the emerging cluster B. To test in vitro and in vivo pathogenicity of pSI54/04 we produced plasmidic transconjugant of the plasmid-free pre-emergent strain SI69/94. This parental strain and its transconjugant have been tested on chicken embryo fibroblasts (CEFs) and in orally infected day old chicks. The uptake of pSI54/04 did not increase the pathogenicity of the strain SI69/94 in these systems. Thus, dissemination of SI in poultry could be assisted by antimicrobial resistance rather than by virulence modules of the endemic plasmid pSI54/04 in Hungary

Cefotaximase (CTX-M) and quinolone resistance genes (qnr) with additional antimicrobial resistance mechanisms in commensal Escherichia coli from healthy pigs

Concerning the importance of food producing animals as potential reservoirs of enteric bacteria with clinically relevant antimicrobial resistance traits, we tested the prevalence of extended-spectrum β-lactamase (ESBL)-producing and fluroquinolone resistant E. coli from pigs in order identify multiple resistance mechanisms circulating in pig farms in Hungary and Croatia with special regards to plasmid mediated genes encoding cefotaximases (CTX-M) and quinolone resistance (qnr). For this purpose, faecal samples were collected from pigs representing three farms from Hungary and six farms from Croatia with 45 and 60 samples respectively. Farms were located in separate regions of the countries. Cefotaxime or nalidixic acid resistance were used as prime markers for the isolation of multiresistant E. coli strains. A second selection was based on resistance to additional antimicrobials (i.e. gentamicin) aiming to reduce the collection to isolates with representative multiresistance phenotypes. In several cases more than two different multiresistance phenotypes have been isolated from the same pig, which were considered as independent E. coli isolates. This collection of multidrug resistant E. coli contained 139 strains and was tested for the presence of blaCTX-M and qnr genes by PCR. Selected isolates carrying genes blaCTX-M and/or qnr are being subjected for confirmation and further typing of antimicrobial resistance genes by using the PCR-microarray AMR05. Cefotaxim resistant E. coli have been detected in one Hungarian and one Croatian farm representing 17% of all pigs tested. In majority of the strains, the plasmid-related resistance phenotypes such as ampicillin, cefotaxim, gentamicin and tetracycline occurred in multiple combinations. In 11% of the strains the coexistence of Ctx-Nal phenotypes was detected, together with the presence of the cefotaximase gene blaCTX-M. E. coli strains with nalidixin resistance phenotype have been predominantly (70%) characterizing healthy pigs independently from the farm and country of isolation. Ciprofloxacin resistant strains occured on one farm only. The plasmid-mediated fluoroquinolone resistance gene qnrS was identified in 11% of the strains, with or without the nalidixin-ciprofloxacin resistant phenotype, while genes qnrA and qnrB were absent. Overall, our results lead to conclude that multiresistant commensal E. coli strains carrying plasmid-mediated CTX-M type cefotaximase and/or quinolone resistance genes in different combinations are widespread on some pig farms but much less on others, most likely reflecting differences in use of antimicrobials. Ama Szmolka is a holder of János Bolyai Stipend of the Hungarian Academy of Sciences

Tetracycline resistance tet(A) plasmids transferring multiresistance in enterotoxigenic E. coli strains from pigs.

Enterotoxigenic E. coli (ETEC) bacteria are frequent pathogens causing watery diarrhea in weaned pigs. Plasmids encoding different enterotoxins (estA, estB, elt) and fimbrial adhesins (f4, f18) are of prime importance in the pathogenesis of ETEC strains. Recently, the complete sequence of the ETEC pTC plasmid was described, as a first representative of hybrid ETEC plasmids carrying the tetracycline resistance gene tet(B) in combination with enterotoxin genes estA and estB. Similarly, unraveling the impact of tet(A)-plasmids in the transfer of resistance and/or virulence would also be important for the efficient therapy of ETEC infections. The main objective of the present study was to provide a comparative description of antimicrobial resistance and virulence profiles of porcine post-weaning ETEC strains representing three middle-European neighboring countries: Hungary, Austria and the Czech Republic. Furthermore, the characterization of genetic vectors for tetracycline resistance was also attempted, with special regard to plasmids responsible for the transfer of tetracycline resistance gene tet(A) and putative co-resistance/virulence genotypes. A total of 87 porcine ETEC strains isolated from post-weaning diarrhea were included in this study. Majority of them were isolated in Europe: Hungary (n=16), Austria (n=34) and the Czech Republic (n=17), and 20 ETEC strains were derived from the USA. Antimicrobial resistance phenotype of the strains was determined for 17 antimicrobial compounds with clinical relevance. Strains resistant to tetracycline were subjected to further studies, including the PCR subtyping of the tet genes, and the detection and characterization of plasmids mediating tetracycline resistance and ETEC-specific virulence in two tet(A)-positive monoplasmidic derivatives of F18+ ETEC strains from Hungary and from the Czech Republic (2172/11 and 11732/71 respectively). Regardless of the geographical origin, majority of the ETEC strains shared a common MDR pattern of sulfamethoxazole (91%), tetracycline (84%) and streptomycin (80%) resistance. The tetracyclin resistant phenotype was most frequently covered by the presence of the tet(B) gene (38%), when tet(A) was identified in 26% of the isolates. The virulence gene profile included enterotoxin genes (elt, estA and/or estB), as well as adhesin genes (f4, f18) without geographic differences. Characterization of these tet(A) plasmids revealed, that the IncI1 plasmid of the Hungarian strain mediated the co-transfer of tet(A), aadA1 (streptomycin/spectinomycin) and strA (streptomycin) genes, when the IncF plasmid of the Czech strain carried tet(A) in association with catA1 for chloramphenicol resistance. Furthermore, the tet(A) plasmid of the Hungarian strain carried a class 1 integron with an unusual variable region of estX-aadA1 responsible to streptothricin-spectinomycin/streptomycin resistance phenotype. In conclusion, the presence of IncI1 and IncF type plasmids, responsible for the co-transfer of the tet(A) gene and additional resistance determinants were demonstrated for the first time in F18+ ETEC strains. In addition to the pTC hybrid plasmid of tet(B)-type, the first description of tet(A) plasmids was provided as a vector of multidrug resistance in porcine ETEC strains