Renáta Kugler

Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers.

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe.

Lineage diversification, homo- and heterologous reassortment and recombination shape the evolution of chicken orthoreoviruses

The near complete genome sequences of ten field avian orthoreovirus (ARV) strains collected from young chicken between 2002 and 2011 in Hungary have been determined in order to explore the genetic diversity and evolutionary mechanisms affecting ARVs in this region. Sequence analyses and phylogenetic calculations revealed similar geographic distribution and distinct evolution in case of eight studied strains that were closely related to the recently described Hungarian strain T1781. The remaining two strains showed the highest similarity with the US origin AVS-B. The topology of the phylogenetic trees of certain segments was affected by several potential homologous reassortment events between strains of Hungarian, Chinese and US origin. Analyzing the μB gene a possible heterologous reassortment event was identified in three Hungarian strains. Recombination events were detected in as much as a dozen cases implying that beside point mutations and reassorment this mechanism also plays an important role in the diversification of ARVs. All these mechanisms in concert may explain the reduced effectiveness of immunization using commercial vaccine strains.

Whole genome characterization of a chelonian orthoreovirus strain identifies significant genetic diversity and may classify reptile orthoreoviruses into distinct species

In this study we report the sequence and phylogenetic characterization of an orthoreovirus strain, CH1197/96, isolated from a spur-thighed tortoise (Testudo graeca) on chicken embryo fibroblast cells. The 23,957 bp long genome sequence was obtained by combined use of semiconductor and capillary sequencing. Although the genomic characterization showed that the virus was most similar to the bush viper reovirus strain, 47/02, and in phylogenies performed with all segments the two strains formed a monophyletic group, the nucleotide (48.4-70.3%) and amino acid (39.2-80.7%) sequence identity values were moderate between the two reptile origin reoviruses. Based on our results and existing classification criteria for the genus Orthoreovirus, the tortoise reovirus strain CH1197/96 might be the first representative of a novel reptilian origin Orthoreovirus species.

Coding-complete sequencing classifies parrot bornavirus 5 into a novel virus species

In this study, we determined the sequence of the coding region of an avian bornavirus detected in a blue-and-yellow macaw (Ara ararauna) with pathological/histopathological changes characteristic of proventricular dilatation disease. The genomic organization of the macaw bornavirus is similar to that of other bornaviruses, and its nucleotide sequence is nearly identical to the available partial parrot bornavirus 5 (PaBV-5) sequences. Phylogenetic analysis showed that these strains formed a monophyletic group distinct from other mammalian and avian bornaviruses and in calculations performed with matrix protein coding sequences, the PaBV-5 and PaBV-6 genotypes formed a common cluster, suggesting that according to the recently accepted classification system for bornaviruses, these two genotypes may belong to a new species, provisionally named Psittaciform 2 bornavirus.

Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis)

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1–L3, M2–M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.

Pathogenic potential and virulence genotypes of intestinal and faecal isolates of porcine post-weaning enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) are frequent causes of diarrhoea in infants and in young mammals by inducing attaching effacing (AE) lesions of the intestinal epithelium. EPEC bacteria have also been implicated in cases of porcine post-weaning diarrhoea but their pathogenicity for conventional weaned pigs remains less elucidated. This present study investigates differences in pathogenic potential and virulence genotypes of intestinal and faecal isolates of EPEC from newly-weaned pigs. For this we inoculated ligated ileal loops of four weeks old weaned pigs to assess EPEC adherence to enterocytes by histology and immunohistology. Virulence gene patterns were identified by using a PCR-microarray. Intestinal EPEC isolates of sero-/intimin types O45:H11:eae-β, O49:NM:eae-β, O84:H7:eae-γ, and O123:H11:eae-β formed adherent microcolonies of EPEC with AE lesions on ileal villi more frequently than faecal isolates of O28:H28:eae-NT, O108:H9:eae-β, O145:H28:eae-γ and O157:H2:eae-β (p≤0.05). The PCR-array analysis of both groups detected all together 25 virulence genes of LEE (Locus of Enterocyte Effacement), and of non-LEE pathogenicity islands, of plasmids and phages characteristic to EPEC. Intestinal isolates carried significantly more virulence genes than faecal isolates (p≤0.05). Intestinal isolates possessed efa1, lpfA, and tsh genes most likely contributing to enterocyte adhesion while faecal isolates did not carry these genes (p≤0.05). Overall, the ileal loop model in weaned pigs combined with virulence genotyping PCR-array indicated a greater pathogenic potential of intestinal isolates over faecal isolates of porcine post-weaning EPEC. Differing virulence genotypes of the intestinal and faecal isolates as demonstrated here suggests dynamic evolutionary events within the population of porcine EPEC.

Phylogenetic analysis of a novel reassortant orthoreovirus strain detected in partridge (Perdix perdix)

Avian orthoreoviruses cause various diseases in wild birds and domesticated poultry. In this study we report the detection and genomic characterization of a partridge (Perdix perdix) origin reovirus strain, D1007/2008. The virus was isolated on cell culture from acute pneumonia and infra-orbital sinusitis. The 23,497 nucleotide long genome sequence was obtained by combined use of semiconductor and capillary sequencing. Sequence and phylogenetic analyses showed that the partridge reovirus strain was related to orthoreoviruses of gallinaceous birds. In fact, five (λB, λC, μB, σC, σNS) and one (σB) out of 10 genes clustered definitely with turkey or chicken origin orthoreoviruses, respectively, whereas in the λA, μA, μNS and σA phylogenies a more distant genetic relationship was observed. Our data indicate that the identified reovirus strain is composed of a mixture of chicken and turkey orthoreovirus related alleles. This finding implies that partridges may serve as natural reservoirs for orthoreoviruses of domesticated poultry.

Development of a consensus reverse transcription PCR assay for the specific detection of tortoise picornaviruses.

Picornaviruses (PVs) of different terrestrial tortoise species, previously designated as Virus “X,” have been frequently detected from various tissues by virus isolation in Terrapene heart cell culture as the preferred laboratory method for diagnosis. Here, we describe the development of 2 diagnostic reverse transcription (RT)-PCR–based assays for the identification and characterization of tortoise PVs belonging to the tentative genus Topivirus. To test the novel diagnostic systems, PVs were isolated from swab and tissue samples collected in Germany, Italy, and Hungary between 2000 and 2013. All 25 tested isolates gave positive results with both novel consensus primer sets. Sequencing of the amplified products confirmed that all studied viruses were members of the new proposed genus Topivirus. Phylogenetic analyses clearly distinguished 2 lineages within the genus. Based on sequence analysis, no association was observed between the geographic distribution and genetic relatedness. Furthermore, no strict host specificity was indicated. The PCR-based diagnosis may provide a time-saving and sensitive method to detect tortoise PVs, and evaluation of PV presence in these animals may help control virus spread.