Spanish Isolates Research Articles

Pathogenicity of Phytophthora alni complex and P. plurivora in Alnus glutinosa seedlings

SummaryThe ecological importance of riparian forests is well known. However, these forest habitats have been disturbed by human activities over the past century as a result of the introduction of flow regulations. Mortality of the riparian alder population caused by Phytophthora has become an important issue in Europe in the past two decades. The main objective of this study was to examine the pathogenicity of the Phytophthora alni complex (P. xalni, P. uniformis and P. xmultiformis) and P. plurivora in Alnus glutinosa seedlings. Phytophthora alni complex has traditionally been identified as the main causal agent of alder decline; however, in this study, P. plurivora was found to cause as much damage in inoculated seedlings as P. alni complex. In fact, Spanish isolates of P. xalni caused mortality rates of ca. 30%, whereas P. plurivora killed ca. 50% of seedlings. Likewise, there were no differences between P. xalni ‐ and P. plurivora‐inoculated seedlings in either the length of lesion or biomass allocation. The pathogenicity of the species within P. alni complex did not differ significantly. The findings confirm that P. plurivora, which is one of the most common species of Phytophthora found in European nurseries, is highly pathogenic to alder seedlings. Urgent implementation of measures to prevent the risk of spread of the pathogen from nurseries to natural ecosystems is required. Further studies are required to clarify the role that P. plurivora is playing in alder decline both individually and in combination with P. alni complex.

Effect of temperature on GaRV6 accumulation and its fungal host, the conifer pathogen Gremmeniella abietina

SummaryGremmeniella abietina RNA virus 6 (GaRV6) was studied within the European race of G. abietina. We examined 97 isolates originating from Canada, Czech Republic, Finland, Italy, Montenegro, Serbia, Spain, Switzerland, Turkey and the United States. According to direct specific quantitative reverse transcription PCR (qRT‐PCR) screening based on the RNA‐dependent RNA polymerase (RdRp) sequence, the virus was mostly present in Spain, but it was also found at a very low concentration in three isolates from Canada, Italy and Finland. To gain insight into the three‐way interaction among temperature, GaRV6 and G. abietina, we performed an in vitro experiment with eight Spanish isolates (four infected and four uninfected) with four repetitions each. We assessed the virus expression (accumulation) based on the quantity of RdRp‐encoding RNA by qRT‐PCR and the G. abietina growth rate at 5, 15 and 20°C. The presence of the virus had a significant negative effect on the G. abietina growth rate, which was also significantly influenced by the temperature. However, the temperature appeared not to clearly determine the virus accumulation, and the virus accumulation did not have any influence on the growth rate of the infected G. abietina isolates. Taken together, these results suggest that the role of this virus as a relevant factor with respect to the more thermophilic nature of the Spanish population of G. abietina may be ruled out.

In vitro assessment of the antimicrobial susceptibility of caprine isolates of Mycoplasma mycoides subsp. capri

The minimum inhibitory concentration (MIC) and minimum mycoplasmacidal concentration (MMC) of 17 antimicrobials against 41 Spanish caprine isolates of Mycoplasma mycoides subsp. capri (Mmc) obtained from different specimens (milk, external auricular canal and semen) were determined using a liquid microdilution method. For half of the isolates, the MIC was also estimated for seven of the antimicrobials using an epsilometric test (ET), in order to compare both methods and assess the validity of ET. Mutations in genes gyrA, gyrB, parC and parE conferring fluoroquinolone resistance, which have been recently described in Mmc, were investigated using PCR. The anatomical origin of the isolate had no effect on its antimicrobial susceptibility. Moxifloxacin and doxycycline had the lowest MIC values. The rest of the fluoroquinolones studied (except norfloxacin), together with tylosin and clindamycin, also had low MIC values, although the MMC obtained for clindamycin was higher than for the other antimicrobials. For all the aminoglycosides, spiramycin and erythromycin, a notable level of resistance was observed. The ET was in close agreement with broth microdilution at low MICs, but not at intermediate or high MICs. The analysis of the genomic sequences revealed the presence of an amino acid substitution in codon 83 of the gene gyrA, which has not been described previously in Mmc.

IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF TOBACCO MILD GREEN MOSAIC VIRUS IN HUNGARY

Pepper (Capsicum annuum L.), an important crop in Hun- gary, is frequently infected by Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and, recently, also by Pepper mild mottle virus (PMMoV) and Tomato spotted wilt virus (TSWV). In the spring of 2015, mild mosaic symptoms were observed on cv. Ho F1. Symptoms on the fruits were more obvious, consisting of reduction in size, mottling and color changes, brown necrotic streaks and spots. To identify the putative pathogen(s), symptomatic fruits were collected and used for host range determination. Symptoms on mechani- cally inoculated Nicotiana tabacum cv. Samsun, N. tabacum cv. Xanthi-nc, Nicotiana benthamiana, Capsicum annuum cv. Albaregia (L+ gene), C. annuum cv. Feherozon (L1) and C. annuum cv. Brendon F1 (L3) suggested the presence of a tobamovirus belonging to pathotype 0 (Boukema, 1980). Moreover, total RNA was extracted from a symptomatic pepper fruit with the RNeasy plant mini kit (Qiagen, Ger- many) and used in RT-PCR using universal tobamovirus primers for the coat protein gene (Kalman et al., 2001). A products of the expected size (700 bp) was obtained, cloned into pGEM-T Easy Vector (Promega, USA) and sequenced (Biomi, Hungary). The sequence was deposited in GenBank under the accession No. KT374283. Blast analysis showed a 99% identity at nucleotide level with a Spanish isolate (P04/17, accession No. FN594859) of Tobacco mild green mosaic virus (TMGMV). To our knowledge, this is the first report of TMGMV on pepper in Hungary.

Turnip yellow mosaic virus in Chinese cabbage in Spain: commercial seed transmission and molecular characterisation

Seed transmission of Turnip yellow mosaic virus (TYMV, genus Tymovirus) was evaluated in the whole seeds and seedlings that emerged from three commercial Chinese cabbage (Brassica pekinensis) seed batches. Seedlings in the cotyledon stage and adult plants were assayed for TYMV by DAS-ELISA and confirmed by RT-PCR. The proportion of whole seeds infected with TYMV was at least 0.15 %. The seeds of the three seed batches were grown in Petri dishes, and surveyed in the cotyledon stage in trays that contained a peat:sand mixture grown in greenhouses or growth chambers, which were analysed in the cotyledon and adult stages. The seed-to-seedling transmission rate ranged from 2.5 % to 2.9 % in two different seed batches (lot-08 and lot-09, respectively). Spanish isolates derived from turnip (Sp-03) and Chinese cabbage (Sp-09 and Sp-13), collected in 2003, 2009 and 2013 in two different Spanish regions, were molecularly characterised by analysing the partial nucleotide sequences of three TYMV genome regions: partial RNA-dependent RNA polymerase (RdRp), methyltransferase (MTR) and coat protein (CP) genes. Phylogenetic analyses showed that the CP gene represented two different groups: TYMV-1 and TYMV-2. The first was subdivided into three subclades: European, Australian and Japanese. Spanish isolate Sp-03 clustered together with European TYMV group, whereas Sp-09 and Sp-13 grouped with the Japanese TYMV group, and all differed from group TYMV-2. The sequences of the three different genomic regions examined clustered into the same groups. The results suggested that Spanish isolates grouped according to the original hosts from which they were isolated. The inoculation of the Spanish TYMV isolates to four crucifer plants species (turnip, broccoli, Brunswick cabbage and radish) revealed that all the isolates infected turnip with typical symptoms, although differences were observed in other hosts.

Characterization of a Colletotrichum population causing anthracnose disease on Olive in northern Tunisia.

To phenotypically, physiologically and molecularly characterize the causal agent of olive anthracnose in the northern Tunisia and to study its genetic variability and pathogenicity. A total of 43 isolates were obtained from symptomatic olives collected from four regions in northern Tunisia. A range of morphological and physiological characteristics was recorded; and a phylogenetic study, based on the sequence analysis of both internal transcribed spacers and TUB2 gene regions, was performed. Of the 43 isolates, 41 were identified as Colletotrichum acutatum s.s, and only two were affiliated to Colletotrichum gloeosporioides s.s. Two more representative Spanish isolates, included for comparison, were identified as Colletotrichum godetiae. Using six inter-simple-sequence-repeat markers, homogeneity between isolates from different locations and within the same species was recorded. In pathogenicity and virulence studies, C.gloeosporioides s.s was found to be less virulent, while the Spanish C.godetiae isolate was significantly more virulent than the Tunisian C.acutatum s.s. Olive anthracnose in the North of Tunisia is mainly caused by C.acutatum s.s species. This is the first study of olive anthracnose in Tunisia, which combines both phenotypic and molecular approaches. Colletotrichum acutatum s.s group was recorded for the first time in the country as the causal agent of olive anthracnose.

Identification and genotyping of Giardia spp. and Cryptosporidium spp. isolates in aquatic birds in the Salburua wetlands, Álava, Northern Spain

Aquatic birds are known to be suitable hosts for a number of avian-specific species and genotypes of the enteric protozoan parasites Giardia and Cryptosporidium. Waterbirds have also been reported as sporadic carriers of species of both pathogens from human or domestic animal origin via environmental contamination. Because aquatic birds can shed substantial amounts of infective Giardia and Cryptosporidium (oo)cysts to the environment including surface waters intended for human consumption, this situation may pose a potential risk of waterbone zoonotic disease.A total of 265 waterbird faecal samples were collected from May 2014 to June 2015 at Salburua (Álava), one of the most valued continental wetlands in northern Spain. The detection of Giardia oocyst and Cryptosporidium oocysts was carried out by direct fluorescence microscopy and molecular (PCR and sequence analysis) methods targeting the small subunit ribosomal RNA gene of the parasites. Typing of Giardia duodenalis isolates at the sub-assemblage level was based on the specific amplification and sequencing of a partial fragment of the glutamate dehydrogenase gene.Overall, Giardia cysts and Cryptosporidium oocysts were detected in 22 (8.3%) and 6 (2.3%), respectively, of the 265 faecal samples analysed. The two only Giardia isolates characterized (one novel, one known) were assigned to the sub-assemblage BIV of G. duodenalis, none of them previously reported in Spanish human isolates. This finding raises doubts about the actual origin of the infection and whether waterbirds may serve as potential source of infective Giardia cysts to humans via waterborne transmission or through direct contact. The six Cryptosporidium isolates obtained were characterized as avian genotype III (n=4), duck genotype b (n=1), and goose genotype Id (n=1), all considered avian-specific and therefore of negligible risk of zoonotic infection.

Citrus leaf blotch virus in Cuba: first report and partial molecular characterization

Citrus leaf blotch virus (CLBV) infection has been detected in citrus cultivars from several countries including the USA, Spain, Italy, Japan and New Zealand. To investigate whether CLBV is present in Cuba, a survey was conducted in seven commercial citrus fields of the country during 2007–2013. In all, 1940 trees were inspected and samples of two symptomatic and 18 asymptomatic sources were tested by biological indexing and molecular techniques for virus detection. Bark patches from the two symptomatic Nagami and Round kumquat sources induced chlorotic blotching and vein clearing in Dweet tangor, and only for the Round kumquat inoculums, stem pitting in Etrog citron. RT-PCR assays using specific primers to amplify fragments of polymerase (RdRp), movement (MP) and coat protein (CP) genes of CLBV yielded amplicons with the expected sizes using RNA from both sources as a template. Viral RNA was further detected by a non-radioactive dot-blot hybridization assay using a mixture of specific probes derived from a Spanish isolate of CLBV. Sequences from RdRp, MP, and CP amplicons showed identical nucleotide identities for the two Cuban isolates, and higher than 95.9 % with isolates from Spain, New Zealand and the USA.

Large Diversity of Porcine Yersinia enterocolitica 4/O:3 in Eight European Countries Assessed by Multiple-Locus Variable-Number Tandem-Repeat Analysis.

A total of 253 multiple-locus variable-number tandem-repeat analysis (MLVA) types among 634 isolates were discovered while studying the genetic diversity of porcine Yersinia enterocolitica 4/O:3 isolates from eight different European countries. Six variable-number tandem-repeat (VNTR) loci V2A, V4, V5, V6, V7, and V9 were used to study the isolates from 82 farms in Belgium (n = 93, 7 farms), England (n = 41, 8 farms), Estonia (n = 106, 12 farms), Finland (n = 70, 13 farms), Italy (n = 111, 20 farms), Latvia (n = 66, 3 farms), Russia (n = 60, 10 farms), and Spain (n = 87, 9 farms). Cluster analysis revealed mainly country-specific clusters, and only one MLVA type consisting of two isolates was found from two countries: Russia and Italy. Also, farm-specific clusters were discovered, but same MLVA types could also be found from different farms. Analysis of multiple isolates originating either from the same tonsils (n = 4) or from the same farm, but 6 months apart, revealed both identical and different MLVA types. MLVA showed a very good discriminatory ability with a Simpson's discriminatory index (DI) of 0.989. DIs for VNTR loci V2A, V4, V5, V6, V7, and V9 were 0.916, 0.791, 0.901, 0.877, 0.912, and 0.785, respectively, when studying all isolates together, but variation was evident between isolates originating from different countries. Locus V4 in the Spanish isolates and locus V9 in the Latvian isolates did not differentiate (DI 0.000), and locus V9 in the English isolates showed very low discriminatory power (DI 0.049). The porcine Y. enterocolitica 4/O:3 isolates were diverse, but the variation in DI demonstrates that the well discriminating loci V2A, V5, V6, and V7 should be included in MLVA protocol when maximal discriminatory power is needed.

First Complete Coding Sequence of a Spanish Isolate of Swine Vesicular Disease Virus.

Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the Enterovirus genus within the Picornaviridae family. The SVDV genome is composed of a single-stranded RNA molecule of positive polarity. Here, we report the first complete sequence of the coding region of a Spanish SVDV isolate (SPA/1/'93).

Phylogeny, resistome and mobile genetic elements of emergent OXA-48 and OXA-245 Klebsiella pneumoniae clones circulating in Spain.

The global emergence of OXA-48-producing Klebsiella pneumoniae clones is a significant threat to public health. We used WGS and phylogenetic analysis of Spanish isolates to investigate the population structure of blaOXA-48-like-expressing K. pneumoniae ST11 and ST405 and to determine the distribution of resistance genes and plasmids encoding blaOXA-48-like carbapenemases. SNPs identified in whole-genome sequences were used to reconstruct phylogenetic trees, identify resistance determinants and de novo assemble the genomes of 105 blaOXA-48-like-expressing K. pneumoniae isolates. Genome variation was generally lower in outbreak-associated isolates compared with those associated with sporadic infections. The relatively limited variation observed within the outbreak-associated isolates was on average 7-10 SNPs per outbreak. Of 24 isolates from suspected sporadic infections, 7 were very closely related to isolates causing hospital outbreaks and 17 were more diverse and therefore probably true sporadic cases. On average, 14 resistance genes were identified per isolate. The 17 ST405 isolates from sporadic cases of infection had four distinct resistance gene profiles, while the resistance gene profile differed in all ST11 isolates from sporadic cases. Sequence analysis of 94 IncL/M plasmids carrying blaOXA-48-like genes revealed an average of two SNP differences, indicating a conserved plasmid clade. Whole-genome sequence analysis enabled the discrimination of outbreak and sporadic isolates. Significant inter-regional spread within Spain of highly related isolates was evident for both ST11 and ST405 K. pneumoniae. IncL/M plasmids carrying blaOXA-48-like carbapenemase genes were highly conserved geographically and across the outbreaks, sporadic cases and clones.

Sequence variation of block III segment identifies three distinct lineages within Eggplant mottled dwarf virus isolates from Italy, Spain and Greece.

Partial polymerase (L) gene sequences of 919 nts, including the conserved segments pre-motif Aand motif Aof block III, of 20 Eggplant mottled dwarf virus (EMDV) isolates were generated, and trimmed sequences of 889 nts, based on the length of available sequences of other isolates, were used to determine phylogenetic relationships. Phylogenetic reconstructions revealed two divergent lineages, designated as genetic group A(Italian isolates) and group B, with the latter further divided into subgroups BI (Greek isolates) and BII (Spanish isolates). No evidence of recombination signals among sequences was detected, whereas analysis of the nonsynonymous/synonymous ratio indicated strong purifying selection, with codons under negative selection uniformly distributed along the sequences. An RT-PCR-RFLP method able to discriminate EMDV isolates of the two main genetic groups was proposed.

Complete Genome Sequence of a Chinese Isolate of Melon Necrotic Spot Virus and its Phylogenetic Relationship

The full-length nucleotide sequence and genomic organization of a melon necrotic spot virus isolate from Haimen, China (MNSV-HM), were determined. The MNSV-HM genome consists of a positive sense single-stranded RNA, 4267 nt in length, with at least five open reading frames (ORFs) encoding p29, p89, p42, and two small 7-kDa proteins (p7A and p7B). p89 shares a common start codon with p29 and continues through the amber stop codon of p29 to produce an 89-kDa protein. The p7A ORF terminates in an amber codon whose read-through could generate a 14-kDa protein. Phylogenetic analyses based on the p42 amino acid sequence and complete genomic sequence placed MNSV-HM and Spanish isolates of the virus in a major cluster, indicating a close genetic relationship. In conclusion, we report the full-length sequence of MNSV-HM and its translation strategy. The obtained genomic organization and phylogenetic trees indicate that MNSV-HM belongs to the MNSV genus Carmovirus. To the best of our knowledge, this is the first demonstration of the complete nucleotide sequence of an MNSV isolate from China.

Genetic variability and evolutionary analysis of parietaria mottle virus: role of selection and genetic exchange.

The genetic variability and evolution of parietaria mottle virus (PMoV) of the genus Ilarvirus was studied by analyzing nucleotide sequences of 2b and CP genes from isolates collected in different countries. Phylogenetic analysis showed that PMoV isolates clustered in different clades: one (clade I) composed of only Italian isolates and three clades (clades II-IV) including the Spanish isolates. The Greek isolate GrT-1 used in this study was in clade IV for the CP phylogenetic tree whereas it formed a separate branch in the 2b phylogenetic tree. The nucleotide sequence diversity of both the 2b and CP genes was low (0.062 ± 0.006 and 0.063 ± 0.006 for 2b and CP, respectively) but higher than those of other ilarviruses. Distribution of synonymous and nonsynonymous substitutions revealed that 2b and CP proteins are under purifying selection, with some positions under diversifying selection. Genetic exchange among Spanish isolates was also detected.

Characterization of a new Spanish Potato virus X strain inducing symptoms in tomato

Potato virus X (PVX), one of the most common plant viruses causing disease to solanaceous crops around the world, is usually asymptomatic in tomato. After mechanical inoculation, a PVX isolate from Northeastern Spain (Sp2013) induced leaf yellowing and light curling in tomato plants, while no symptoms were observed with strain CP4 (GenBank accession No. AF172259.1). Both PVX strains were detected by ELISA with the same antiserum. In order to determine PVX population variability and structure, the full-length coat protein (CP) gene of this Spanish isolate (accession No. KJ631111 corresponding to SPCP1 strain in GenBank) was cloned, sequenced and compared with 90 PVX CP gene sequences available in the database. The global PVX population is divided in three clusters comprising: (i) mainly of South American isolates, including strain CP4; (ii) a mix of isolates from different continents, including isolate Sp2013; (iii) Europan isolates only. The low variability and lack of recombination events in the CP gene, except for isolate Sp2013, which has 77% nucleotide sequence identity with strain CP4, point to a homogeneous structure. Marked genetic differences between the CP gene sequence of PVX strains Sp2013 and CP4 could explain the differential symptomatology on tomato.

Complete sequence of three different biotypes of tomato spotted wilt virus (wild type, tomato Sw-5 resistance-breaking and pepper Tsw resistance-breaking) from Spain.

Tomato spotted wilt virus (TSWV) occurs worldwide and causes production losses in many important horticultural crops such as tomato and pepper. Breeding resistant cultivars has been the most successful method so far for TSWV disease control, but only two genes have been found to confer resistance against a wide spectrum of TSWV isolates: Sw-5 in tomato and Tsw in pepper. However, TSWV resistance-breaking isolates have emerged in different countries a few years after using resistant cultivars. In this paper, we report the first complete nucleotide sequences of three Spanish TSWV isolates with different biotypes according to their abilities to overcome resistance: LL-N.05 (wild type, WT), Pujol1TL3 (Sw-5 resistance breaking, SBR) and PVR (Tsw resistance-breaking, TBR). The genome of these TSWV isolates consisted of three segments: L (8913-8914 nt), M (4752-4825 nt) and (S 2924-2961 nt). Variations in nucleotide sequences and genomic RNA lengths among the different virus biotypes are reported here. Phylogenetic analysis of the five TSWV open reading frames showed evidence of reassortment between genomic segments of LL-N.05 and Pujol1TL3, which was supported by analysis with different recombination-detecting algorithms.

DETECTION OF ALFALFA MOSAIC VIRUS IN LAURUSTINUS IN APULIA

Laurustinus (Viburnum tinus L.), an evergreen shrub of the Mediterranean scrub, is widely used in Italy as an ornamental in private and public gardens. In the spring of 2014, conspicuous interveinal yellow spots and mottling were observed on the foliage of a V. tinus plant cv. Eve Price growing in the Campus of the University of Bari (Italy). Since Alfalfa mosaic virus (AMV) has been reported from V. tinus and related species (V. opulus and V. lucidum) in several countries, and in northern Italy (De Sanctis and Quacquarelli, 1985; Parrella et al., 2011), the symptomatic plant was tested by RT-PCR for the presence of this virus. The specific primer set CPAMV1/CPAMV2, designed in the viral RNA3 sequence (Finetti-Sialer et al., 1997) amplified the expected 754 bp fragment, spanning the whole coat protein gene and the 3’-UTR. There was no amplification from symptomless plants. The amplified fragment was cloned into pSC-A-amp/kan, custom-sequenced (Macrogen Europe, The Netherlands) and the sequence was deposited in GenBank as accession No. KP233749. The cloned sequences were BLAST compared with those of a Chilean strain of AMV from V. tinus and a Spanish isolate from V. lucidum, with which an identity at the nucleotide level of 99% and 94%, respectively, was found. These results demonstrate the presence of an AMV strain in laurustinus in southern Italy.

A Novel Binary Mixture of Helicoverpa armigera Single Nucleopolyhedrovirus Genotypic Variants Has Improved Insecticidal Characteristics for Control of Cotton Bollworms.

The genotypic diversity of two Spanish isolates of Helicoverpa armigera single nucleopolyhedrovirus (HearSNPV) was evaluated with the aim of identifying mixtures of genotypes with improved insecticidal characteristics for control of the cotton bollworm. Two genotypic variants, HearSP1A and HearSP1B, were cloned in vitro from the most pathogenic wild-type isolate of the Iberian Peninsula, HearSNPV-SP1 (HearSP1-wt). Similarly, six genotypic variants (HearLB1 to -6) were obtained by endpoint dilution from larvae collected from cotton crops in southern Spain that died from virus disease during laboratory rearing. Variants differed significantly in their insecticidal properties, pathogenicity, speed of kill, and occlusion body (OB) production (OBs/larva). HearSP1B was ∼3-fold more pathogenic than HearSP1-wt and the other variants. HearLB1, HearLB2, HeaLB5, and HearLB6 were the fastest-killing variants. Moreover, although highly virulent, HearLB1, HearLB4, and HearLB5 produced more OBs/larva than did the other variants. The co-occluded HearSP1B:LB6 mixture at a 1:1 proportion was 1.7- to 2.8-fold more pathogenic than any single variant and other mixtures tested and also killed larvae as fast as the most virulent genotypes. Serial passage resulted in modified proportions of the component variants of the HearSP1B:LB6 co-occluded mixture, suggesting that transmissibility could be further improved by this process. We conclude that the improved insecticidal phenotype of the HearSP1B:LB6 co-occluded mixture underlines the utility of the genotypic variant dissection and reassociation approach for the development of effective virus-based insecticides.

Molecular Characterization, Antibiotic Resistance, and Virulence Factors of Methicillin-Resistant Staphylococcus aureus Strains Isolated from Imported and Domestic Meat in Korea.

During a nationwide surveillance in Korea, 13 methicillin-resistant Staphylococcus aureus (MRSA) strains were isolated from imported and domestic meat between 2009 and 2011. The predominant MRSA genotype was SCCmec type V, and only two agr types (I and II) were found. Unexpectedly, sequence type ST72 comprised more than 50% of the isolates; this is the first instance of type ST72 in food from Canada. Two Spanish pork isolates were ST398, which caused human disease in Europe, and they carried leukotoxin genes, lukS, lukF, and lukE-lukD. Furthermore, P71 and P6 harbored all of the known leukocidin genes, lukS-lukF-lukE-lukD-lukM. Our collected MRSA strains were multidrug resistant with various antimicrobial and heavy-metal resistance genes. Toxin genes that are commonly found in clinical MRSA also were detected in our meat strains. One MRSA strain exhibited an uncommon type of enterotoxin, sec-see-seg-sei-sel-sem-sen-seo-sep. Plasmids (1.5-15.0 kb) were found in 12 of the 13 MRSA isolates. Repetitive sequence-based polymerase chain reaction of the genomic DNA showed 3 clusters with 95% similarity. The presence of multidrug-resistant and toxigenic MRSA in meat products suggests that comprehensive surveillance should be continued for imported meats in Korea.

Full genome analysis of a European-type genotype 3 hepatitis E virus variant obtained from a Japanese patient with autochthonous acute hepatitis E.

A unique European-type HEV strain (HE-JA12-0725) classifiable into subgenotype 3f was recovered from a 66-year-old Japanese female with autochthonous acute hepatitis E, and its entire genomic sequence was determined and characterized. The HE-JA12-0725 strain shared the highest identity of 92.7% with a Spanish swine isolate (EU723514) over the entire genome and possessed a long hypervariable region sequence of 111 amino acids, identical to the 3f strains of European origin. The patient had consumed pork liver obtained via home delivery in Japan approximately two months before the disease onset. These results suggest the circulation of rare 3f HEV strains in Japan.