A 3-year epidemiological surveillance of Escherichia coli O157:H7 in dogs and cats in Japan.

For three years investigations from 1996 to 1998, we tried to isolate Escherichia coli O157:H7 from fecal samples collected from dogs and cats. In results, E. coli O157:H7 was isolated from 1 out of 614 samples (0.16%). This isolate produced Stx1 and Stx2 and was isolated from a dog kept by a human patient infected with E. coli O157:H7. Excluding in this case, dogs and cats as companion animals, therefore, may not give harbor to E. coli O157:H7.

pulsed-field gel electrophoresis profile changes resulting from spontaneous chromosomal deletions in enterohemorrhagic Escherichia coli O157:H7 during passage in cattle.

A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.

Phage type and antimicrobial susceptibility of Salmonella enterica serovar Enteritidis from food-producing animals in Japan between 1976 and 2004.

A total of 56 isolates of Salmonella enterica serovar Enteritidis, including 38 isolates from poultry, 16 from cattle and two from pigs, collected between 1976 and 2004, were subjected to bacteriophage typing and antimicrobial susceptibility testing. Phage type (PT) 8 was predominant in bovine isolates, whereas PT1 and PT4 were predominant in poultry isolates. Resistance was found for 8 of 11 antimicrobials tested, at the following rates: 46.4% for dihydrostreptomycin followed by ampicillin and oxytetracycline (both 8.9%).

Comparison of PCR-RFLP and PFGE for determining the clonality of enterohemorrhagic Escherichia coli strains.

We report here on a comparative evaluation of PCR-restriction fragment length polymorphism (PCR-RFLP) and pulsed-field gel electrophoresis (PFGE) assays, and ascertain the clonal relationship between 13 enterohemorrhagic Escherichia coli O157 : H7 strains isolated from fecal samples collected from three cows over a period of 2 months. PCR-RFLP analysis was carried out with either BglI or EcoRV digested LA-PCR amplicons, generated by targeting region V of the Stx-phage. While PCR-RFLP analysis placed these 13 strains into a single clonal type, pulsotyping analysis, as reported earlier, grouped these strains into four different PFGE subtypes of which three were closely related, while the other appeared to be different. The comparative analysis was extended further using two clonally different wild-type (3-0 and Sakai 215) strains and 17 derivative strains which had passed through an animal's gastrointestinal tract. The PCR-RFLP assay, which was not only able to differentiate the wild-type strains, but also placed the passaged derivative strains into their respective parental group, although PFGE patterns of the same set of strains resulted from different PFGE subtypes. These data indicate that PCR-RFLP is the more reliable and useful assay for a molecular epidemiological survey of enterohemorrhagic E. coli strains.

Pathology of cutaneous fowlpox with amyloidosis in layer hens inoculated with fowlpox vaccine.

Cutaneous fowlpox occurring in vaccinated layer hens was investigated pathologically and microbiologically. Anorexia, decrease of egg production, increased mortality, yellow scabs on faces, and alopecia of feathered skins with yellow scabs were observed in affected hens. Histologically, proliferative and necrotic dermatitis with eosinophilic ring-shaped cytoplasmic inclusions (Bollinger bodies) and clumps of gram-positive cocci (Staphylococcus hyicus) were noted in the affected birds. Fowlpox lesions were primarily observed in the feathered skins. Proliferation of feather follicle epidermal cells, with cytoplasmic inclusions and degeneration of the feather, and bacterial clumps in the feather follicles were noted in the affected skins. Ultrastructurally, characteristic fowlpox viral particles were observed in the cytoplasmic inclusions of hyperplastic epidermal cells. Amyloid deposition was observed in the Disse space of the liver, splenic sinus, and lamina propria of the bronchiolar, bronchial, and tracheal areas. Amyloidosis could be one factor inducing the fowlpox infection in vaccinated chickens.

Detection and characterization of variant Salmonella genomic island 1s from Salmonella Derby isolates.

The purpose of this study is to investigate the distribution and structure of Salmonella genomic island 1 (SGI1) among Salmonella enterica serovar Derby isolates from swine and their rearing environment. Three variants of SGI1s, specifically SGI1-A, C, and I, were identified by PCR mapping. The results of macro-restriction analysis and DNA sequencing of SGI1 flanking regions revealed that there are at least two genomic lineages of Derby strains bearing SGI1s.

[Drug sensitivity of vero toxin-producing Escherichia coli O157:H7 (VTEC O157:H7) isolated from human diarrhea and healthy cows].

As a part of studies on the source of infection of Vero toxin-producing Escherichia coli (VTEC), O157:H7 strains isolated from human infectious enteritis between 1986 and 1995 and O157:H7 strains isolated from feces of milk cows between 2001 and 2003 were subjected to drug sensitivity test with drugs widely used as therapeutic drugs for various infectious diseases in humans and animals, and the following results were obtained. 1) Drug sensitivity tests with 20 drugs were performed in 52 strains derived human from diarrhea and 100 strains derived from milk cows, and resistance was noted in 115 strains (75.7%): 36 of the 52 human diarrhea-derived strains (69.2%) and 79 of the 100 milk cow-derived strains (79.0%). 2) The human diarrhea-derived strains and milk cow-derived strains were compared with regard to MIC90 of each drug. The antibacterial activity of the drugs was generally higher against the human diarrhea-derived strains than against the milk cow-derived strains. 3) In the 115 strains exhibiting resistance, the most frequent pattern of drug resistance was single drug resistance noted in 80 strains (68.4%), and multidrug resistance was noted in 35 strains (30.4%) consisting of 17 strains with resistance to 3 drugs, 14 strains with resistance to 2 drugs, and 2 strains each with resistance to 4 drugs and 5 drugs. More strains were multidrug-resistant in the milk cow-derived strains.

[Biological properties and drug susceptibility of verotoxin-producing Escherichia coli (VTEC) isolated from healthy goats in Okinawa Prefecture].

Fecal samples from 116 healthy goats out of 25 randomly selected farms were examined for verotoxin-producing Escherichia coli (VTEC) during 1996 and 1998 in Okinawa Prefecture. VTECs were detected 204 (15.0%) from 1,361 E. coli strains, 36 (31.0%) goats out of 13 (52.0%) farms. Randomly selected 88 strains were further characterized according to VT types, serotypes, virulence markers, biochemical properties and drug susceptibility. VT types were classified as VT1 (46.6%), VT2 (6.8%), and VT1/VT2 (46.6%) by means of reversed latex agglutination test. The VTEC belonged to 18 different O serogroups: O1, O6, O22, O27, O48, O75, O76, O77, O78, O82, O91, O103, O111, O123, O125, O128, O146, and O158. Serotypes O91:H- (13 strains), O27:H- (10 strains), O22:H19 (6 strains) are considered to be predominant, whereas O serotypes O157 and O26 were not isolated. eaeA gene was detected only in 5 strains (5.7%):O103:H2 and O111:H-, in contrast, hlyA gene was found frequent in 45 strains (51.1%) belong to various O serogroups, except for O146 (8 strains). On the basis of 20 biochemical features in all isolates, characteristic patterns were divided into 14 distinct types:47 strains (57.3%) were classified as one type. The VTECs examined were resistant to streptomycin (26.7%), ampicillin (12.2%), kanamycin (8.9%), oxytetracyline (8.9%), and oxolinic acid (3.3%), respectively. The current results indicate that goats harbored VTEC at high frequencies and may be a potential reservoir of human VTEC infection.

[Isolation and serotyping of Vero toxin-producing Escherichia coli (VTEC) from pig].

As a part of basic studies to elucidate the source of infection of Verotoxin-producing Escherichia coli (VTEC) infectious disease, fresh feces were collected from pigs raised in Kanto District (A and B Prefectures) and Kyushu District (C and D Prefectures) between April and October in 2000, and isolation, serotyping, toxin typing, and drug sensitivity test of VTEC were performed. 1) Of 411 fecal samples tested, VTEC was isolated from 44 samples (10.7%), consisting of 12 of 112 samples (10.7%) from A Prefecture, nine of 100 samples (9.0%) from B Prefecture, 18 of 99 samples (18.2%) from C Prefecture, and five of 100 samples (5.0%) from D Prefecture. 2) Forty-five isolates were serotyped. Four isolates (8.9%) were typed as type 3, but the remaining 41 isolates (91.1%) could not be typed. The four typed isolates consisted of two O112ac:H- isolates and one each of O126:H- and O157:H7. 3) Toxin was typed in 45 isolates. Twenty-seven (60.0%) and 17 isolates (37.8%) produced VT 2 and VT1, respectively, and one isolate (2.2%) produced both VT1 and VT2. 4) Drug sensitivity tests of 45 isolates were performed. All 45 isolates (100%) were multidrug-resistant that were resistant to multiple drugs. Nineteen, nine, four, four, seven, one, and one isolates were resistant to five, six, two, three, four eight, and nine drugs, respectively. The above findings confirmed contamination in all districts, although the VTEC isolation rate varied among the sampling districts. Serotyping clarified the presence of O157:H7 and O112ac:H- that are detected in human VTEC infectious disease. The drug sensitivity tests clarified the presence of many multidrug-resistant strains.

Antimicrobial resistance of Salmonella enterica serovar Typhimurium isolated from cattle in Japan.

The antimicrobial susceptibility of 144 Salmonella enterica serovar Typhimurium isolates collected from all over Japan between 1973 and 1998 were investigated. All the isolates exhibited resistance to four or more antimicrobials and 22 resistance patterns were observed. Isolates showing resistance patterns to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfonamides (Su) and tetracycline (T), which are typical resistance patterns for S. Typhimurium DT104 (DT104), were predominant. Thirty-six of the 68 isolates that exhibited resistance to five or more antimicrobials (ACSSuT+) were identified as DT104 by phage typing. Another 103 S. Typhimurium strains gathered from cattle between 1977 and 1999 in a limited area of Japan were analyzed for molecular epidemiological studies. Results using fluorescent amplified-fragment length polymorphism and pulsed-field gel electrophoresis suggest that clonal exchange of S. Typhimurium among cattle in Japan has occurred since 1992, and that contemporary strains show a remarkable degree of homogeneity with DT104 at a molecular level. The clonal replacement by DT104 affected the antimicrobial resistance pattern of S. Typhimurium from cattle in Japan.

Changes in antimicrobial susceptibility in a population of Salmonella enterica serovar Dublin isolated from cattle in Japan from 1976 to 2005.

OBJECTIVES: We investigated the antimicrobial susceptibilities and resistance mechanisms of cattle-adapted Salmonella enterica serovar Dublin isolated in Japan in the past 30 years. This study is an example of evaluation of the impact of introduction of antimicrobials in veterinary medical practice on the selection of resistance in S. enterica. METHODS: The antimicrobial susceptibilities and prevalence of R-plasmids in Salmonella Dublin isolated in Japan from 1976 to 2005 were investigated. To evaluate the importance of gyrA mutation and active efflux, we derived the gyrA revertants and acrAB deletion mutants, and then compared with their parental strains the MICs of quinolone antimicrobials such as nalidixic acid, enrofloxacin, ofloxacin and ciprofloxacin. RESULTS: Salmonella Dublin isolates with R-plasmids and resistance to more than three antimicrobials were predominant between 1981 and 1995. From the latter half of the 1990s to the present, Salmonella Dublin isolates without R-plasmids became dominant. The introduction of nalidixic acid into the veterinary field in the mid-1980s was followed by the emergence of nalidixic acid-resistant isolates, which are now predominant. We found only a single gyrA mutation (Asp-87-->Tyr) among the nalidixic acid-resistant isolates. Although the reduced susceptibilities to the fluoroquinolones were observed among the nalidixic acid-resistant isolates, none of the isolates was resistant to the fluoroquinolones used in this study. The MIC data for the fluoroquinolones differed up to 4-fold. Results of the susceptibility test using gyrA revertants and acrAB mutants suggest that the isolates with the gyrA mutation were selected by the use of nalidixic acid, and the AcrAB-TolC system accounts for the decreased fluoroquinolone susceptibilities. CONCLUSIONS: These data suggest that the introduction of nalidixic acid in veterinary medicine seemed to affect the susceptibilities of Salmonella Dublin among the cattle population in Japan, whereas the introduction of enrofloxacin has not caused any additional effect. The prudent use of antimicrobials in the veterinary field should be continuously stressed.

The artAB genes encode a putative ADP-ribosyltransferase toxin homologue associated with Salmonella enterica serovar Typhimurium DT104.

Many bacterial pathogens encode ADP-ribosyltransferase toxins. The authors identified an ADP-ribosyltransferase toxin homologue (ArtA, ArtB) in Salmonella enterica serovar Typhimurium (S. typhimurium) DT104. ArtA is most homologous to a putative pertussis-like toxin subunit present in Salmonella typhi (STY1890) and Salmonella paratyphi A (SPA1609), while ArtB shows homology to a hypothetical periplasmic protein of S. typhi (STY1364) and S. paratyphi A (SPA1188), and a putative pertussis-like toxin subunit in S. typhi (STY1891) and S. paratyphi A (SPA1610). The artA gene was detected from the phage particle fraction upon mitomycin C induction, and the flanking region of artAB contains a prophage-like sequence, suggesting that these putative toxin genes reside within a prophage. Southern blotting analysis revealed that artA is conserved in 12 confirmed DT104 strains and in four related strains which are not phage-typed but are classified into the same group as DT104 by both amplified-fragment length polymorphism and pulsed-field gel electrophoresis. Except for one strain, NCTC 73, all 13 S. typhimurium strains which were classified into different groups from that of DT104 lacked the artA locus. The results suggest that phage-mediated recombination has resulted in the acquisition of art genes in S. typhimurium DT104 strains.

Molecular characterization of a prophage of Salmonella enterica serotype Typhimurium DT104.

Isolates of the Salmonella enterica serotype Typhimurium definitive phage type (DT104) were found to contain the same prophage (designated phage ST104). The complete sequence of the DNA genome of prophage ST104 was determined. The entire DNA sequence consisted of 41,391 bp, including 64 open reading frames, and exhibited high similarity to P22 and to phage type conversion phage ST64T.