Characterization of multi-antibiotic-resistant Escherichia coli Isolated from beef cattle in Japan.

The emergence of multiple-antibiotic-resistance bacteria is increasing, which is a particular concern on livestock farms. We previously isolated 1,347 antimicrobial-resistant (AMR) Escherichia coli strains from the feces of beef cattle on 14 Japanese farms. In the present study, the genetic backgrounds and phylogenetic relationships of 45 AMR isolates were characterized by the chromosome phylotype, AMR phenotype, AMR genotype, and plasmid type. These isolates were classified into five chromosome phylotypes, which were closely linked to the farms from which they were isolated, suggesting that each farm had its own E. coli phylotype. AMR phenotype and plasmid type analyses yielded 8 and 14 types, all of which were associated with the chromosomal phylotype and, thus, to the original farms. AMR genotype analysis revealed more variety, with 16 types, indicating both inter- and intra-farm diversity. Different phylotype isolates from the same farm shared highly similar plasmid types, which indicated that plasmids with AMR genes could be transferred between phylotypes, thereby generating multi-antibiotic-resistant microorganisms. This ecological study demonstrated that the chromosome phylotype was strongly correlated with the farm from which they were isolated, while the AMR phenotype, genotype, and plasmid type were generally correlated with the chromosome phylotype and farm source.

Prevalence and characteristics of Listeria monocytogenes in feces of black beef cattle reared in three geographically distant areas in Japan.

This study was conducted to determine the prevalence and characteristics of Listeria monocytogenes in the feces of black beef cattle reared in geographically distant areas in Japan. We surveyed 130 farms in the following three areas: northern (Hokkaido prefecture), central (Gifu and Mie prefectures), and southern (Oita, Miyazaki, and Kagoshima prefectures) areas and collected 1738 fecal samples. Our data showed the following isolation rate for each area: northern, 11.4% of 651; central, 2.8% of 572; and southern, 2.9% of 515, indicating that the isolation rate in the northern area was significantly higher than that in the central or southern areas (p<0.01). Moreover, serotyping of 996 isolates identified 1/2b as the most prevalent serotype (40.5%), followed by 1/2a (36.9%), 4b (21.6%), and 4ab (1.0%). In the northern area, multiple serotypes were isolated from 60% of L. monocytogenes-positive farms. In addition, multiple serotypes were isolated from individual fecal samples from 18 cattle. Pulsed-field gel electrophoresis (PFGE) characterization of 239 isolates detected 48 different PFGE types. We found that isolates from northern farms were genetically diverse compared to those from central and southern farms. Five isolates from human clinical cases and three isolates from animal clinical cases were identical to isolates from black beef cattle. Furthermore, the isolates from northern and central farms were characterized to possess epidemic clone II or III markers. We next showed that the isolates were susceptible to penicillin, ampicillin, amoxicillin, gentamicin, kanamycin, streptomycin, erythromycin, vancomycin, tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim/sulfamethoxazole. Taken together, our survey provides crucial data regarding the prevalence and characteristics of L. monocytogenes in black beef cattle farms throughout Japan.

Prevalence and molecular epidemiological characterization of antimicrobial-resistant Escherichia coli isolates from Japanese black beef cattle.

We investigated the prevalence of antimicrobial-resistant Escherichia coli in Japanese black beef cattle from the three major production regions of Japan. We collected and examined 291 fecal samples from Japanese black beef cattle in Hokkaido, Chubu, and Kyushu. Of the 3,147 E. coli isolates, 1,397 (44.4%) were resistant to one or more antibiotics; these included 553 (39.8%) of 1,388 isolates from Hokkaido, 352 (54.4%) of 647 isolates from Chubu, and 492 (44.2%) of 1,112 isolates from Kyushu. The difference in resistance rates between the three regions was significant. The antibiotics with the highest rates of resistance were oxytetracycline and dihydrostreptomycin (35.8% each), followed by ampicillin (21.4%). Further, E. coli isolates from calves had higher resistance rates than those from growing cattle and mature cattle, and the calf isolates showed high rates of resistance to gentamicin (20.2%), enrofloxacin (9.4%), and ceftiofur (4.2%). In addition, the high degrees of similarity in the genotypes of the isolates and in the resistance patterns on each farm suggest that resistance bacteria and resistance genes were horizontally transferred. Most isolates, in each of the three regions, harbored resistance genes such as blaTEM, strA, strB, aphA1, aphAI-IAB, and catI. In contrast to the isolates from Kyushu, most of which harbored aacC2, tetB, and dfrA12, the isolates from Hokkaido and Chubu harbored a variety of resistance genes. Furthermore, the prevalence of genes for resistance to dihydrostreptomycin, gentamicin, chloramphenicol, and trimethoprim differed significantly between the regions. This is the first large-scale study describing and comparing antimicrobial-resistant bacteria from different regions in Japan. The results will contribute to improving food safety and promoting careful usage of antimicrobial agents.

Prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan.

This study was conducted to determine the prevalence and characteristics of Listeria monocytogenes in bovine colostrum in Japan. We collected bovine colostrum samples from 210 dams from 21 dairy farms in Hokkaido prefecture (Japan) between March and June 2009. L. monocytogenes was detected in samples from 6 (28.6%) of the 21 farms. Of the 210 samples, 16 (7.6%) were positive for L. monocytogenes. We recovered 80 L. monocytogenes isolates; 44 (55%) isolates were classified as serotype 1/2b and 36 (45%) were classified as serotype 4b. The isolates were susceptible to penicillin, ampicillin, amoxicillin, gentamicin, kanamycin, streptomycin, erythromycin, vancomycin, tetracycline, chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole. Pulsed-field gel electrophoresis (PFGE) characterization of the 80 isolates revealed six PFGE types. Two PFGE types corresponded to human listeriosis cases. Most L. monocytogenes isolates possessed virulence-associated genes (actA, hly, iap, inlA, inlC, mpl, plcA, plcB, opuCA, prfA, and clpC). One PFGE type isolate possessed an epidemic clone II marker. Our findings suggest that isolates from bovine colostrum have the potential to cause human and animal listeriosis. This is the first study on the prevalence and characteristics of L. monocytogenes isolated from bovine colostrum obtained from dairy farms. Our results have important implications for improving public health and elucidating the epidemiology of L. monocytogenes in bovine colostrum.

Prevalence of Salmonella isolates and antimicrobial resistance patterns in chicken meat throughout Japan.

We investigated the prevalence of Salmonella in chicken meat from northern, central, and southern Japan. Between 2006 and 2008, 821 samples from these three regions were collected and examined. Salmonella isolates were detected in 164 (20.0%) of these samples, with 15 (10.0%) of 150, 113 (27.5%) of 411, and 36 (13.8%) of 260 recovered from the northern, central, and southern regions, respectively. We recovered 452 Salmonella isolates. From the isolates, 27 serovars were identified; the predominant serovars isolated were Salmonella Infantis (n=81), Salmonella Kalamu (n=56), and Salmonella Schwarzengrund (n=43). Of the 452 isolates, 443 (98.0%) were resistant to one or more antibiotics, and 221 (48.9%) showed multiple-antibiotic resistance, thereby implying that multiple-antibiotic resistant Salmonella organisms are widespread in chicken meat in Japan. Resistance to oxytetracycline was most common (72.6%), followed by dihydrostreptomycin (69.2%) and bicozamycin (49.1%). This study, the first to report Salmonella prevalence in chicken meat throughout Japan, could provide valuable data for monitoring and controlling Salmonella infection in the future.

Nationwide survey of salmonella prevalence in environmental dust from layer farms in Japan.

A nationwide survey was conducted to determine Salmonella prevalence in airborne dust from layer farms. Of the 4,090 layer farms in Japan, 203 were surveyed and 48 (23.6%) of these were positive for Salmonella. Salmonella isolation rates were higher in the eastern (24.3%), central (25.6%), western (23.9%), and southern (27.5%) prefectures than they were in the northern (13.3%) prefecture. We recovered 380 Salmonella isolates and identified 34 different Salmonella serovars. Salmonella Infantis was the most prevalent serovar (42 [11.1%] of 380), followed by Salmonella Agona (39 [10.3%] of 380), Salmonella Mbandaka (37 [9.7%] of 380), Salmonella Cerro (32 [8.4%] of 380), Salmonella Thompson (29 [7.6%] of 380), and Salmonella Braenderup (27 [7.1%] of 380). Of the 380 isolates, 273 (71.8%) were resistant to more than one antibiotic. Salmonella Infantis (41 [97.6%] of 42), Salmonella Agona (38 [97.4%] of 39), and Salmonella Mbandaka (34 [91.9%] of 37) showed the highest resistance rates. We found 18 different resistance patterns and the most common (179 [47.1%] of 273) was resistant to dihydrostreptomycin. One of the 13 Salmonella Hadar isolates was resistant to eight antibiotics. To investigate characteristics of Salmonella Agona, Salmonella Infantis, and Salmonella Mbandaka isolates across different prefectures, we performed pulsed-field gel electrophoresis by using XbaI and BlnI. The Salmonella Agona and Salmonella Mbandaka dendrograms were grouped into seven clusters, with 80 and 70% similarity, respectively. Because the Salmonella Infantis dendrogram showed low similarity, there is a possibility of genetic diffusion of this serovar across Japan. This report is the first to describe Salmonella contamination in airborne dust from layer farms in Japan. Our findings should be useful for future Salmonella infection monitoring and control.

Importance of thymidine phosphorylase, dihydropyrimidine dehydrogenase and thymidylate synthase expression at the invasive front of T3 rectal cancer as prognostic factors.

BACKGROUND/AIMS: We evaluated a relationship between postoperative recurrence and thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) expression at the invasive front of T3 rectal cancer. METHODOLOGY: This study was conducted on 61 patients with T3 rectal cancer who underwent surgically curative resection between 1998 and 2002. Paraffin-embedded sections of these patients were immunostained for TP, DPD and TS. Relationship between expression level of the three factors and postoperative recurrence were evaluated. RESULTS: There was no relationship between expression of DPD or TS in the tumor cells and recurrences. Although no relationship was present between expression of TP in the stromal cells around the invasive front of the tumor and postoperative recurrences, there was a strong correlation between expression of TP in the invasive front of the tumor and postoperative recurrence. Moreover, by multivariate logistic regression analysis, TP expression in the tumor cells was the only independent contributory factor for postoperative recurrences (p = 0.021) with an odds ratio of 8.27. CONCLUSIONS: TP expression at the invasive front of the tumor may be an important prognostic factor for T3 rectal cancer, and patients with such a condition may benefit from intensive chemotherapy.

Mesorectum amputation technique for laparoscopic anterior resection.

The objective of this study was to assess our original procedure, the cutting off method (COM), where the rectum and mesorectum are cut off with an autosuture device simultaneously in laparoscopic anterior resection. From 2001 to 2003, laparoscopic anterior resection was performed in 21 patients at our institution using the COM (n = 14) and trimming method (TM; n = 7). We reviewed the medical records for operative duration, intraoperative blood loss, and postoperative complications. The operation lasted 165 minutes (range, 140-215 minutes) in the TM group, but only 120 minutes (range, 95-135 minutes) in the COM group (P < 0.01). Intraoperative blood loss was 90 ml (range, 60-140 ml) in the TM group and 30 ml (range, 5-90 ml) in the COM group (P < 0.01). There were no postoperative complications in the COM group, although one complication of minor leakage was observed in the TM group.

Sliding functional end-to-end anastomosis for colorectal surgery.

Conventional functional end-to-end anastomosis (FEEA) is not indicated for left hemicolectomy, sigmoidectomy, and anterior resection. However, our original anastomosis with stapling devices (SFEEA) can be performed at any site in the intestine. We report our novel surgical technique compared with the double stapling technique (DST). Between January 2001 and August 2003, anterior resection with stapling devices was performed in 74 patients (DST, 54; SFEEA, 20). The SEEEA group was greater than the DST group in operation time and significant intraoperative blood loss. In the DST group, two postoperative complications (3.7%) occurred (leakage and stenosis). On the other hand, no complications were noted in the SFEEA group. Our novel technique for colorectal anastomosis, SFEEA, allows safe, wide, physiological, and clean anastomosis compared with FEEA.

Importance of thymidine phosphorylase expression at the invasive front of T3 rectal cancer as a prognostic factor.

PURPOSE: Vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP) are angiogenic factors. We evaluated the relationship between the degree of expression of binary angiogenic factors and the malignant potential of colorectal cancer. METHODS: This study was conducted in 61 patients with T3 rectal cancer who underwent surgical curative resection between 1998 and 2002. Paraffin-embedded sections of their surgical specimens were immunostained for VEGF and TP. The relationship between the expression level of binary angiogenic factors and lymph node metastasis was evaluated. RESULTS: Expression of VEGF was present only in the tumor cells of all patients, and expression was absent in the stromal cells around the invasive front of the tumor. Expression of TP was mainly present in the stromal cells around the cancer nests or around the invasive margin of the cancer. There was no relationship between expression of TP in the stromal cells around the invasive front of the tumor and lymph node metastasis. However, there was a significant relationship between expression of TP at the invasive front of the tumor and lymph node metastasis (p = 0.014). Moreover, TP expression in the tumor cells was the only independent factor that correlated with the postoperative recurrence (p = 0.024) with an odds ratio of 11.973 by multivariate logistic regression analysis. CONCLUSION: Expression of TP at the invasive front of the tumor may be a useful indicator of the malignant potential of advanced rectal cancer.

Usefulness of automatic triangular anastomosis for esophageal cancer surgery using a linear stapler (TA-30).

In a clinical evaluation, thirty-one patients with thoracic esophageal cancer who were treated from 1997 to 2000 were selected as subjects including those who underwent hand sewn anastomosis method, circular stapler method and triangular anastomosis method. Incidence of anastomosis failure was 27.3%, 25.0% and 8.3% for the hand sewn anastomosis method, circular stapler method and triangular anastomosis method, respectively. Anastomosis stenosis was found in 32.4%, 45.6% and 8.3% for the hand sewn anastomosis method, circular stapler method and triangular anastomosis method, respectively. In the basic examination, reduction rate of anastomosis caliber was 22.1+/-4.8%, 14.9+/-1.4% and 7.37+/-0.9%, for the hand sewn anastomosis method, circular stapler method and triangular anastomosis method. Microscopic evaluation revealed serious problems with the circular stapler method. The cause of anastomotic stenosis may include the fact that if anastomosis is performed by a circular stapler method, all the layers of gastrointestinal tract are punched out at the anastomosis portion, and mucosal conjugation is not observed and the muscular layer is exposed in the inner lumen of the gastrointestinal tract. Taking that the ulcer is formed circularly at the anastomotic portion into account, it is easily understood that this circular ulcer develops into stenosis in the healing process. The advantage of triangular anastomosis for esophago-gastric anastomosis is less suture failure, and is extremely advantageous for prevention of stenosis at the anastomotic portion when compared with other anastomosis methods. However, with regard to the healing process of eversion anastomosis in gastrointestinal tract instrumental anastomosis, detailed examination is expected hereafter.

New approach for laparoscopic surgery of the right colon.

BACKGROUND: In patients in whom the ventral aspect of the root of the mesentery was obscured by the adherent greater omentum, laparoscopic surgery is usually abandoned. To forcefully loosen the adhesions by a laparoscopic maneuver may cause inadvertent intestinal injury. We describe a procedure, named the 'retromesen teric approach', which enables a safe laparoscopic right-sided colic operation in such circumstances. PATIENTS AND METHODS: From 2000 to 2003 in our institute, a laparoscopic right-sided colic operation was performed in 21 patients in whom the ventral aspect of the root of the mesentery was obscured by the adherent greater omentum using the retromesenteric approach (RMA; n = 16) and a conventional approach (CA; n = 5). We reviewed the medical records for the operative duration, intraoperative blood loss, conversion rates and postoperative complications. RESULTS: The duration of operation in the RMA group ranged from 75 to 120 (median 95) min, which was shorter than that in the CA group (p < 0.05). Perioperative bleeding in the RMA group ranged from 0 to 115 (median 30) ml, which was smaller than that in the CA group (p < 0.05). No conversion and no postoperative complications were noted in the both groups. CONCLUSIONS: Our new technique, in which the right colon and the ileum are dissected and lifted en bloc from the retroperitoneum, is safe and useful for laparoscopic right-sided colic operation.

Identification and characterization of an immunodominant 28-kilodalton Coxiella burnetii outer membrane protein specific to isolates associated with acute disease.

Coxiella burnetii causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among C. burnetii isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol. 42:81-85, 1998). In order to clone the adaA gene, the N-terminal amino acid sequence of adaA was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an approximately 28-kDa mature protein with a typical signal sequence. The adaA (acute disease antigen A) gene was detected in acute group C. burnetii isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in adaA, and specific antibody to adaA reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that adaA is exposed on the outer surface of C. burnetii. adaA was overexpressed in pET23a as a fusion protein in Escherichia coli to develop anti-recombinant adaA (anti-radaA) specific antibody, which recognized a approximately 28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that radaA reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an adaA gene-targeted PCR assay and an radaA antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.

Implementation of a clinical pathway for laparoscopic colorectal surgery.

The purpose of this study was to assess through questionnaire the validity of our original clinical pathway for laparoscopic colorectal surgery. From 2001 to 2002, laparoscopic colorectal surgery was performed in 52 patients in our institution. All patients were out of bed within 3 days after the operation. The average postoperative hospital stay was 10.9 days. The implementation rate of the clinical pathway was 100%. As to the results of the questionnaire, 92.4% of the patients felt they could be discharged within 7 days after the operation, whereas >95% of the patients wanted to go home on a Sunday/holiday or the day before. All but 2 patients (96.2%) were satisfied with our therapy. In a clinical pathway for laparoscopic colorectomy, a high implementation rate and patients' satisfaction can be achieved by taking into account the patients' viewpoints; however, better patient education is necessary to shorten postoperative stay.

Comparison of Japanese isolates of Coxiella burnetii by PCR-RFLP and sequence analysis.

The genetic variation of Japanese isolates of Coxiella burnetii, the agent of Q fever, was found for the first time. Forty-nine out of 72 isolates had the chronic pattern of the isocitrate hydrogenase gene. Sequence analysis revealed that the isolates have a specific nucleotide sequence. The putative amino acid sequence was the same as that of chronic reference strains. These results suggest the variation of C. burnetii isolates in Japan.

Use of monoclonal antibodies for analyses of Coxiella burnetii major antigens.

Monoclonal antibodies (MAbs) to major antigens of Coxiella burnetii were produced. Some of the MAbs to a 62-kDa protein antigen, peptidoglycan protein complex and lipopolysaccharide (LPS) O-chains reacted with other bacteria whereas none of the MAbs to outer membrane proteins and LPS outer-core did. The LPS outer-core and OMPs may be useful antigens for specifically detecting antibodies to C. burnetii.

In vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of Coxiella burnetii.

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.

[Home chemotherapy by concomitant UFT + Leucovorin (po.) in postoperative colorectal cancer patients assessed with Dukes D and curability C colorectal cancer].

UNLABELLED: Concomitant treatment with 5-fluorouracil (5-FU) and Leucovorin (LV) is positioned as the standard chemotherapy against colorectal cancer. We noted the action of LV to enhance the effect of biochemical modulation by 5-FU, and made an attempt at home chemotherapy with UFT + LV by oral administration, in consideration to the convenience of patients. SUBJECTS: The subjects of this study were 24 post-operative patients who had been assessed with Dukes D and curability C colorectal cancer with measurable metastatic lesions and who could tolerate chemotherapy. METHODS: 1 course of treatment consisted of 2 weeks of UFT at 300-400 mg/m2/day and LV at 15 mg/body/day followed by 2 weeks of drug withdrawal. The administration was conducted for 4 courses or more as the target. Unless serious adverse reaction occurred, dose increase of UFT was allowed. RESULTS: The efficacy rate in the 22 patients who were assessable was 22.7%. There were 11 NC patients, accounting for half (50%) of the subjects. This home chemotherapy is expected to become an alternative chemotherapy against colorectal cancer in the future, because the treatment does not require hospitalization and has less impact on the QOL of patients.

Proventricular adenocarcinoma in a Humboldt penguin (Spheniscus humboldti) and a great horned owl (Bubo virginianus); identification of origin by mucin histochemistry.

Cases of proventricular neoplasm in a Humboldt penguin (Spheniscus humboldti) and a great horned owl (Bubo virginianus) were observed. Microscopically, the neoplastic cells formed branching tubules or acini in both cases. Galactose oxidase-Schiff (GOS) staining revealed that the cytoplasm of the normal surface epithelium and surface mucosubstances of the proventriculus adjacent to the neoplasm were positive in both cases. The neoplastic cells in both cases were also classified as GOS-positive. Therefore, the two proventricular neoplasms in this report were diagnosed as proventricular adenocarcinoma that arose from the proventricular surface epithelium. This study suggests that the mucosubstances, which the neoplastic cells produced, were a useful index for identifying the origin of the neoplastic cells in the birds.

Leucocytozoon lovati infections in wild rock ptarmigan (Lagopus mutus) in Japan.

Leucocytozoon lovati infections were detected in free-flying rock ptarmigan (Lagopus mutus), an endangered species that inhabits alpine areas in Japan. Eight of nine adult birds tested positive for L. lovati infection. For comparison, two captive rock ptarmigans hatched in a breeding facility at the foot of the mountains were examined. Both were negative for L. lovati infection. This is the first report of L. lovati infection in the rock ptarmigan in Japan.