Takehisa Chuma

Genotyping and Quantitation of Noroviruses in Oysters from Two Distinct Sea Areas in Japan

Norovirus (NV) is a causative agent of acute gastroenteritis in humans, and shellfishes including oysters act as major vehicles of the virus. To investigate the genetic characteristics of NVs, we collected 1,512 oysters for raw consumption between October 2002 and March 2005 from two distinct areas (area A: the Sanriku Sea area; area B: the Setouchi Sea area). We detected the capsid gene and subjected it to phylogenetic analysis. By further quantification of the copy number of the genome by using real‐time PCR, the NV capcid gene was detected in approximately 5% of the oysters, and they showed wide diversity. Two percent of the oysters from area B showed relatively large number of NVs, i.e., over 100 copies of capsid gene/oyster, whereas this was not observed in area A. Most of the detected NVs from oysters and humans were genetically related when the capsid region was compared. These results suggested that NVs obtained from humans and those obtained from oysters showed a potential relationship to each other and that some populations of Japanese oysters accumulated a relatively large number of NVs.

Characterization of the genes encoding two of the major capsid proteins of epizootic haemorrhagic disease virus indicates a close genetic relationship to bluetongue virus

The sequences of the genes of two of the major capsid proteins of epizootic haemorrhagic disease virus serotype 1 (EHDV-1, Orbivirus genus, Reoviridae) have been determined by analyses of cDNA clones representing the L2 and S7 RNA segments. The EHDV-1 S7 RNA segment, which encodes the VP7 core protein, is 1162 nucleotides in length and has the capacity to encode 349 amino acids (M(r) 38,243). The EHDV-1 L2 RNA segment, which encodes the outer capsid VP2 protein (M(r) 113,249) is 2968 nucleotides in length and has an open reading frame of 971 codons. The potential secondary structure of the EHDV-1 S7 mRNA species, in particular that of the terminal regions, is comparable to those of the corresponding segments of bluetongue virus (BTV) and African horse sickness virus (AHSV); the EHDV-1 L2 mRNA species has a secondary structure similar to that of the L2 mRNA of BTV. The EHDV-1 VP2 and VP7 proteins, as well as those of the other two major structural proteins of EHDV published previously (the inner core VP3 protein and the second outer capsid, VP5), are closely related to the corresponding proteins of BTV. The EHDV and BTV VP7 sequences are more distantly related to the sequence of the AHSV VP7 protein published recently.

Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay.

Mutation of 23S rDNA is one of the mechanisms of erythromycin resistance. PCR and line probe assay (PCR-LiPA) with ten oligonucleotide probes were developed to detect the mutations associated with macrolide resistance at positions of 2072, 2073 and 2074 in 23S rDNA of Campylobacter jejuni/coli. A2074-->G mutation was detected in 12 of 25 isolates, which were resistant to erythromycin. No other mutations in 23S rDNA were detected. The rest of the strains were susceptible to erythromycin and no mutation in 23S rDNA was detected. Six laboratory induced erythromycin resistant mutants had no mutations in 23S rDNA. PCR-LiPA is a useful and rapid method to detect mutations in 23S rDNA associated with erythromycin resistance in C. jejuni/coli.

Simultaneous detection of mutations associated with resistance to macrolides and quinolones in Campylobacter jejuni and C. coli using a PCR-line probe assay

Quinolone and macrolide resistance of Campylobacter jejuni and Campylobacter coli mainly depend on a mutation in gyrA and in 23S rDNA, respectively. In order to detect quinolone and/or macrolide resistant C. jejuni and C. coli strains, a macrolide and quinolone line probe assay (MQ-LiPA) was developed and 42 C. jejuni and C. coli strains were tested to evaluate the efficiency of the assay. Profiles of the mutations in 23S rDNA and in gyrA characterized by MQ-LiPA agreed with resistance to macrolides and quinolones. MQ-LiPA is a rapid and simple method for simultaneous detection of quinolone and macrolide resistance of C. jejuni and C. coli. We could also discriminate between C. jejuni and C. coli using probes for detection of gyrA mutations in MQ-LiPA.

THE NON-STRUCTURAL PROTEINS OF BLUETONGUE VIRUS ARE A DOMINANT SOURCE OF CYTOTOXIC T CELL PEPTIDE DETERMINANTS

Virus-specific, CD8+ cytotoxic T lymphocytes (CTLs) were generated in two strains of mice (BALB/c, CBA/Ca) against bluetongue virus serotype 10 (BTV-10). Recombinant vaccinia viruses (VV) expressing the individual structural and non-structural proteins of BTV were used to infect syngeneic target cells. We found that in both BALB/c (H-2d) and CBA/Ca (H-2k) mice, polyclonal CTL populations recognized target cells expressing the non-structural proteins better than those expressing the structural proteins. CTLs generated against other BTV serotypes also predominantly recognized the non-structural proteins. However, the extent of cross-reactivity was dependent on the H-2 background of the animals immunized. No CTLs cross-reactive to the BTV-10 heterotype were demonstrated with the panel of molecularly cloned recombinants in the H-2d haplotype. The outer capsid proteins VP2 and VP5 which vary considerably between serotypes were not recognized by heterotypic CTLs. Using this murine model we have determined which BTV proteins are the major targets of the CTL response. The implications for the design and development of subunit vaccines are discussed.

Detection and characterization of extended-spectrum β-lactamase (TEM-52)-producing Salmonella serotype infantis from broilers in Japan.

During 2004 and 2006, multidrug-resistant Salmonella enterica subspecies enterica serovar Infantis (Salmonella Infantis) isolates (n = 120) were recovered from broiler cecal samples collected from a meat-processing plant, and the isolates were examined. The study was conducted to detect and characterize extended-spectrum beta-lactamase (ESBL)-producing Salmonella Infantis isolates recovered from broiler chickens and determine the mechanisms of transfer of the resistance traits. Extended-spectrum cephalosporins-resistant Salmonella Infantis isolates producing ESBL TEM-52 were detected. The mutant bla(TEM-52) gene and the wild-type bla(TEM-1) gene that mediated resistance to ampicillin (an extended-spectrum penicillin) and cephalothin (a narrow-spectrum cephalosporin) were located on approximately 50-kb conjugative plasmids among beta-lactam-resistant (n = 29) isolates. The bla(TEM) genes did not cotransfer with aadA1, sul1 (both associated with class 1 integrons), tetA, and dfrA5, signifying a chromosomal location of these non-beta-lactam resistance-encoding genes. This is the first report describing TEM-52-producing S. enterica from food-producing animals in Japan. An emergence of TEM-type ESBL is an important concern to public health because this readily transferable resistance mechanism threatens the value of the third-generation cephalosporins and may reduce the clinical utility of this class of antibiotics against pathogenic Gram-negative bacteria.

Genetic analysis of multidrug-resistant Salmonella enterica serovars Stanley and Typhimurium from cattle

During 2005-2008, a longitudinal study was conducted in southern Japan to detect and characterize multidrug-resistant Salmonella enterica serovars recovered from cattle diagnostic specimens. Determination of antimicrobial resistance phenotypes and genotypes, identification of Salmonella genomic island 1 (SGI1), detection of virulence genes, plasmid analysis, conjugal transfer experiments, and sequencing of class 1 integrons were conducted. Multidrug-resistant Salmonella detected were serovars Stanley, Typhimurium, and O4:d. Salmonella Stanley isolates exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, trimethoprim, and kanamycin (ACSSuT+) encoded by bla(TEM), catA, aadA2, tetA, sul1, dfrA12, and aphA1 genes, respectively. Sequencing analysis revealed that aadA2 and dfrA12 were integrated as gene cassettes within the class 1 integrons of 1.5kb size. Importantly, the isolates harboured easily transferable plasmids of ca. 210kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. Moreover, Salmonella Typhimurium DT104 isolates with typical SGI1 were detected and presented ACSSuT+ resistance phenotype encoded by bla(PSE-1) and bla(TEM); floR; aadA1; sul1; and tetA and tetG, respectively. Salmonella Typhimurium isolates carried plasmids of variable sizes ranging from 3.5 to 100 kb with DT104 isolates harbouring plasmids of ca. 90 kb. Salmonella serovar O4:d had ACSSuT+ resistance phenotype mediated by bla(TEM), catA, aadA1, sul1, tetA, and aphA1 genes. A virulence gene invA was found in all multidrug-resistant Salmonella Typhimurium, Stanley and O4:d clinical isolates. In conclusion, this is the first report describing the occurrence of multidrug-resistant Salmonella Stanley from bovine species. The emergence of Salmonella Stanley isolates exhibiting plasmid-encoded high-level multidrug resistance is an important health concern because this new pathogenecity was associated with mortality in cattle.

Genetic analysis of multi-drug resistance and the clonal dissemination of β-lactam resistance in Salmonella Infantis isolated from broilers

An epidemiologic study was conducted to investigate the incidence and characterize the antimicrobial resistance determinants, analyzing plasmid profiles, and establishing the genetic relationship among beta-lactam-resistant isolates of Salmonella Infantis from broilers in Southern Japan. A total of 120 isolates were recovered from 56 flocks belonging to 44 holdings during 2004-2006. The percentages of resistance were as follows: ampicillin (24%), cephalothin (23%), cefoxitin (0%), ceftazidime (11%), cefotaxime (11%), chloramphenicol (0%), kanamycin (7.5%), ofloxacin (20%), oxytetracycline, streptomycin and sulfamethoxazole (100%) and trimethoprim (75%). The incidence of bla(TEM)-encoded beta-lactam resistance in 2004-2006 was significantly higher than in 1998-2003 (P<0.001). BlnI-digested PFGE patterns generated two related clusters implicated in the dissemination of beta-lactam resistance. Two types of plasmid profiles were observed and two plasmids of ca. 50 and 180-kb size were carried by beta-lactam-resistant isolates. Streptomycin resistance was conferred by aadA1 (n=116), aadA1-aadA2 (n=1), and aadA1-strA-strB (n=3). Resistances to kanamycin, oxytetracycline, sulfamethoxazole and trimethoprim were conferred by aphA1 (n=9, 100%), tetA (n=120, 100%) sul1 (n=120, 100%) and dfrA5 (n=90, 100%), respectively. Two types of class 1 integrons were detected: 1.0 kb (n=120) and, 1.0/1.5 kb (n=3). Integrons of 1.0/1.5 kb were found in isolates with the aadA1-strA-strB gene combination. For the first time, all S. Infantis isolates showed resistance to at least three classes of antimicrobial agents; and the intestinal tract of healthy poultry was a reservoir of the extended-spectrum cephalosporin-resistant isolates of serovar Infantis.

Temporal Distribution and Genetic Fingerprinting of Salmonella in Broiler Flocks from Southern Japan

During the 1998 to 2003 period, cecal contents of 4,024 broiler chickens from 252 flocks raised in 63 holdings were examined for Salmonella. The aims were to establish the actual status of the infection, its temporal distribution, prevalent serotype, and common genotype among broiler flocks brought at the slaughterhouse. Collected samples were preenriched in Hajna tetrathionate broth, and after 24 h of incubation, 10 microL of the broth was streaked on selective Rambach agar plate. Suspected scarlet color colonies of Salmonella were cloned on nutrient agar, confirmed through biochemical tests and sero-typed using O and H antigens. Pulsed field gel electrophoresis technique generated DNA fragments banding patterns and established their clonal relatedness. Salmonella was isolated from 563 (14%) samples in 179 (71%) flocks. The flock situation varied from Salmonella-negative holdings (n = 9), positive-flocks from persistently infected holdings (n = 21), and holdings (n = 19) that showed fluctuations with alternating negative and positive flocks for variable time periods. Fourteen holdings (negative, n = 5 and positive, n = 9) were sampled once throughout the study period. Seasonality component was not observed, and salmonellae were found colonizing broiler ceca in warm and cold months. Predominant serovar was Salmonella Infantis (93.3%; n = 525). Macrorestriction fingerprints of Salmonella Infantis using XbaI presumed the isolates to be derived from a common parent. Enhanced discrimination by BlnI digestion produced 3 banding patterns that were closely related genetically and hence epidemiologically related. Such epidemiological information may enable producers to formulate effective control action plan tailored for individual holdings with special emphasis on biosecurity, hygiene, and pest control.

Distribution of extended-spectrum cephalosporin resistance determinants in Salmonella enterica and Escherichia coli isolated from broilers in southern Japan

This study was conducted to investigate the distribution and diversity of extended-spectrum cephalosporin (ESC) resistance determinants in Salmonella enterica and Escherichia coli obtained from the same cecal samples and to provide evidence of transmission of the resistance determinants among these bacteria in broiler farms in southern Japan. Salmonella enterica and E. coli were characterized by serotyping and multilocus sequence typing, respectively. An antimicrobial susceptibility test, plasmid analysis, and identification and localization of resistance genes were performed to determine the relatedness of ESC resistance determinants among the isolates. Of 48 flocks examined, 14 had S. enterica. In total, 57 S. enterica isolates were obtained, 45 of which showed ESC resistance. Extended-spectrum cephalosporin-resistant E. coli were also obtained from all of these ESC-resistant Salmonella-positive samples. β-Lactamase genes, blaTEM-52 (38 isolates), blaCTX-M-14 (1 isolate), and blaCMY-2 (6 isolates), were carried by conjugative untypable or IncP plasmids detected in the S. enterica serovars Infantis and Manhattan. The β-lactamase genes blaCTX-M-14 (3 isolates), blaCTX-M-15 (3 isolates), blaSHV-2 (1 isolate), blaSHV-12 (2 isolates), and blaCMY-2 (32 isolates) associated with IncI1-Iγ, IncFIB, IncFIC, IncK, IncB/O, and IncY plasmids were detected in E. coli co-isolates. Restriction mapping revealed similar plasmids in Salmonella Infantis and Salmonella Manhattan and in different sequence types of E. coli. Intraspecies transmission of plasmids was suggested within S. enterica and E. coli populations, whereas interspecies transmission was not observed. This study highlights the importance of plasmids as carriers of ESC resistance determinants.