Ikuo Uchida

Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Isolates from Cattle in Hokkaido, Japan: Evidence of Clonal Replacement and Characterization of the Disseminated Clone

ABSTRACT The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla TEM-1 gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla TEM-1-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.

Molecular typing of Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates from cattle by multiple-locus variable-number tandem-repeats analysis.

To evaluate the usefulness of multiple-locus variable-number tandem-repeats analysis (MLVA) as a tool for the epidemiological analysis of bovine Salmonellosis, Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates (544 and 18, respectively) obtained from cattle in Hokkaido, Japan, between 1977 and 2009, were characterised by MLVA. MLVA identified 184 profiles versus 121 profiles identified by pulsed-field gel electrophoresis (PFGE). Cluster analysis of the MLVA profiles demonstrated 3 major clusters (A, B, and C) and 3 minor clusters (D, E, and F). Cluster A was associated with PFGE cluster I, which included isolates of definitive phage type 104 (DT104), while cluster C was associated with PFGE cluster VII, which has been disseminating among cattle since 2002. An isolate of serotype Typhimurium belonging to MLVA cluster F, in which 10 serotype 4,5,12:i:- isolates were included, was found to have an MLVA profile closely related to those of serotype 4,5,12:i:- isolates, suggesting that such a strain may be an ancestral candidate for serotype 4,5,12:i:-. Overall, the discriminatory power of MLVA was higher than that of PFGE, and MLVA differentiated between the isolates of the DT104 family, which appeared to be clonal by PFGE. However, this depended on PFGE clusters because PFGE allowed greater discrimination between isolates within PFGE cluster IV and VI than MLVA. The combination of PFGE and MLVA data allowed for improved subtype discrimination and enabled the identification of recently disseminated clones. Hence, MLVA can be used in combination with PFGE to effectively accelerate the molecular epidemiologic investigation of Salmonella.

A Fluoroquinolone-Resistant Escherichia coli Clinical Isolate without Quinolone Resistance-Determining Region Mutations Found in Japan

Fluoroquinolones are broad-spectrum and highly bactericidal antimicrobials agents that are used to treat various bacterial infections. Escherichia coli infections, especially in the urinary tract, are frequently treated with fluoroquinolones, and fluoroquinolone resistance has increased in the

Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD

Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB - encoded by a prophage in S. Typhimurium DT104 - are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [(32)P]NAD and ArtA, and the label was released by HgCl(2), which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA(6Arg-Ala) and ArtA(115Glu-Ala), in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.

Molecular cloning of bovine (Bos taurus) cDNA encoding a 94-kDa glucose-regulated protein and developmental changes in its mRNA and protein content in the mammary gland

We isolated and sequenced cDNA clones encoding a 94-kDa glucose-regulated protein (GRP94) from a cDNA library constructed using bovine (Bos taurus) mammary gland poly(A)(+) RNA. The coding nucleotide sequence and the deduced amino acid sequence of bovine GRP94 shared 94.2-88.4% and 98.1-96.5% identity with those of other mammalian species, respectively. The primary structure contained a carboxyl-terminal signal sequence for retention in the endoplasmic reticulum, six potential sites for N-linked glycosylation and two potential adenosine 5-triphosphate binding sites, similar to other mammalian and avian GRP94 homologues. In Northern blot hybridization using a cDNA probe from the bovine GRP94 cDNA sequence, a transcript 3.0 kb in size was detected. We measured the amounts of GRP94 and its mRNA in mammary glands from cows at various developmental stages of hormonally induced lactation. The highest level of GRP94 mRNA, determined by dot blot analysis, was detected in the developing stage. In contrast to the mRNA level, the amount of protein, determined by immunoblot analysis using rabbit antiserum raised against GRP94 purified from bovine brain, was higher in lactating stages than in others. The increased level of GRP94 mRNA during the developing stage and the maintenance of GRP94 protein during lactation suggest that the synthesis of GRP94 is regulated during mammary development and differentiation, and also that the protein is involved in a function related to lactation.

Antigenic variation among recent Japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups

Bovine coronaviruses (BCoVs) isolated in Japan consist of four genetic groups, as determined by phylogenetic analysis using the polymorphic region (aa 456–592) of the S glycoprotein gene. Japanese field isolates of BCoV, reference Kakegawa strain, and vaccine strain 66/H were analyzed for their antigenic properties by indirect immunofluorescence and neutralization testing. There were no significant differences observed among these BCoVs in direct immunofluorescence tests. However, antigenic differences were observed between BCoVs in the neutralization tests, although there was no clear indication of a distinct serotype. A monoclonal antibody, 4H4, against the Kakegawa strain belonging to group 1 lacked significant neutralizing activity for viruses of groups 2, 3, and 4. Therefore, we speculate that the genetic differences between these groups may have altered their antigenicity. Analysis of mutant viruses resistant to neutralization by 4H4 revealed that the antigenic site of the Kakegawa strain maps to amino acid position 284 of the S glycoprotein. This site is not homologous to a known antigenic site (aa 528) of the Quebec strain belonging to group 1, and it is not located in the conformational domain comprising domain I (aa 351–403) and domain II (aa 517–621). This amino acid constitutes a neutralization epitope of BCoV, which is distinct from aa 528 of the Quebec strain. These results indicate antigenic evolution of BCoV between the genetic groups circulating in Japan.

Complete Nucleotide Sequences of Virulence-Resistance Plasmids Carried by Emerging Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated from Cattle in Hokkaido, Japan

In the present study, we have shown that virulence-resistance plasmids from emerging multidrug-resistant isolates of Salmonella enterica serovar Typhimurium were derived from a virulence-associated plasmid, essential for systematic invasiveness of S. Typhimurium in mice (pSLT), through acquisition of a large insert containing a resistance island flanked by IS1294 elements. A bla CMY-2-carrying plasmid from a cefotaxime-resistant isolate comprised a segment of Escherichia coli plasmid pAR060302 and the replication region (IncFIB) of a virulence-resistance plasmid. These results provide insights into the evolution of drug resistance in emerging clones of S. Typhimurium.

Molecular typing of Salmonella enterica serovar 4,[5],12:i:- isolates from humans, animals and river water in Japan by multilocus variable-number tandem repeat analysis and pulsed-field gel electrophoresis

Fifty-one Salmonella enterica serovar 4,[5],12:i:- (S. 4, [5],12:i:-) isolates (14 human strains, 34 animal strains and 3 river water strains) which are assumed to be monophasic variants of S. Typhimurium were analyzed using pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem repeat analysis (MLVA) in order to investigate their genetic diversities and relationships. PFGE, MLVA and combination of them identified 28, 27 and 34 profiles (Simpson’s diversity indices [DI]=0.94, 0.96 and 0.97), respectively. No correlations were detected between MLVA clustering and PFGE clustering or phage typing. These results suggested that S. 4,[5],12:i:- originated from multiple S. Typhimurium ancestors. Two cattle and one pig isolates showing identical phage types as well as PFGE and MLVA profiles to human isolates S. 4,[5],12:i:- suggested the existence of the links between human infections and animal reservoirs.

Molecular typing of Salmonella enterica serovar Enteritidis isolates from food-producing animals in Japan by multilocus variable-number tandem repeat analysis: evidence of clonal dissemination and replacement

BackgroundSalmonella enterica serovar Enteritidis is a zoonotic pathogen. Human infections are associated with contaminated eggs and egg products. In Japan, since 1989, the incidence of food-borne disease caused by S. Enteritidis has increased and a pandemic has occurred; however, little is known about changes that occurred before and after this pandemic event in the dominant lineage of isolates from food-producing animals. This study aimed to determine the S. Enteritidis lineages in Japan over the last few decades by using multilocus variable-number tandem repeat analysis (MLVA).FindingsMLVA was used to analyse 79u2009S. Enteritidis isolates collected from chickens (nu2009=u200963), cattle (nu2009=u200912), pigs (nu2009=u20092), and goats (nu2009=u20092) during 1975–2009. The S. Enteritidis isolates showed 14 different MLVA allele combinations, which were classified into two major clusters (A and C) and a minor cluster (B). All the 62 isolates in cluster A were isolated after 1988, whereas 13 of the 17 isolates belonging to cluster B and C were isolated before 1989.ConclusionsThe MLVA results showed that cluster C was predominant before 1989, and isolates in cluster A disseminated since 1989 and replaced the previous dominant clone, suggesting that isolates of cluster A originated from imported S. Enteritidis infection.

Rapid detection of Salmonella enterica serovar Typhimurium DT104 strains by the polymerase chain reaction

BackgroundInfection with Salmonella enterica is a major public health concern in developed countries, and multidrug-resistant strains have become increasingly prevalent. S. enterica serovar Typhimurium DT104 (DT104) strains are prevalent in livestock in Japan and include numerous strains of multidrug-resistant S. enterica. Epidemiological analysis of these strains is critical for both agriculture and public health; however, diagnostic tests for these strains have yielded inconsistent results.ResultsWe developed a rapid, simple, and inexpensive polymerase chain reaction test to detect multi-drug resistant DT104 strains. We designed primers specific to the prophage ST104 sequence encoded by DT104 strains and assessed the specificity of these primers by assaying a panel of 50 S.enterica isolates. Amplification products of the expected size were generated from the genomes of each of the DT104 strains; however, the ST104 primers failed to amplify products from non-DT104 strains of S.enterica serovar Typhimurium or other S. enterica serovars. Furthermore, a probe generated using the ST104 primers detected a restriction fragment encoding the ST104 region of DT104 by Southern hybridization.ConclusionsThe ST104 primers exhibit specificity to DT104 strains and are suitable for epidemiological applications.