Karoku Okamoto

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.

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.

Chronological Change of Resistance to β-Lactams in Salmonella enterica serovar Infantis Isolated from Broilers in Japan

Epidemiologic surveillance study was conducted in southern Japan to determine the antimicrobial resistance phenotypes and characterize the β-lactamase genes and the plasmids harboring these genes in Salmonella enterica serovar Infantis (S. Infantis) isolates from broilers. Between January, 2007 and December, 2008, a total of 1,472 fecal samples were collected and examined at the Laboratory of Veterinary Public Health, Kagoshima University, Japan. In 93 (6.3%) isolates recovered, 33 (35.5%) isolates showed resistance to cefotaxime, an extended-spectrum cephalosporin (ESC), conferred by TEM-20, TEM-52 and CTX-M-25 extended-spectrum β-lactamases (ESBLs). In addition to ESC-resistance, eight (8.6%) isolates exhibited resistance to cefoxitin mediated by CMY-2 AmpC β-lactamase. Plasmid analysis and polymerase chain reaction replicon typing revealed the blaTEM-20 and blaCMY-2 genes were associated with IncP plasmids, blaTEM-52 was linked with a non-typable plasmid and blaCTX-M-25 was carried by an IncA/C plasmid. Non-β-lactam resistance to streptomycin, sulfamethoxazole, and oxytetracycline encoded by the aadA1, sul1, and tet(A) genes, respectively, was found in 86 (92.5%) isolates. Resistance to kanamycin and ofloxacin was exhibited in 12 (12.9%) and 11 (11.8%) isolates, respectively, the former was mediated by aphA1-Iab. These data indicate that S. Infantis isolates producing ESBLs and AmpC β-lactamase have spread among broiler farms in Japan. These data demonstrated that the incidence of ESC-resistant S. Infantis carrying blaTEM-52 remarkably increased and S. Infantis strains harboring blaCMY-2, blaTEM-20, or blaCTX-M-25 genes emerged from broilers in Japan for the first time in 2007 and 2008.

Re-emergence of multi-drug resistant Salmonella enterica serovar Stanley from cattle

During 2009, Salmonella enterica subspecies enterica serovar Stanley isolates were recovered from cattle diagnostic specimens in southern Japan, and the isolates were examined to characterize the genetic determinants involved in this new pathogenicity that associated with mortality in cattle. All the isolates were multi-drug resistance exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, oxytetracycline, and kanamycin (ACSSuT-Km) encoded by blaTEM, catA, aadA1, sul1, tet(A), and aphA1 genes, respectively. Class 1 integrons of 1.5-kb size were detected in all MDR isolates. The isolates harboured easily transferable plasmids of ca. 210-kb with the potential of transmitting resistance phenotype and genotype detected in the donor isolates. XbaI-digested PFGE patterns generated two related clusters implicated in the dissemination of multi-drug resistance amongst Salmonella Stanley isolates. An emergence of multi-drug resistant Salmonella Stanley amongst food-producing animals, including cattle is a threat to human health, as resistant isolates may be transmitted to humans through the food chain.

Hematological, Osmotic, and Scanning Electron Microscopic Study of Erythrocytes of Dogs Given β-acetylphenylhydrazine

Hematologic examinations, osmotic fragility tests, and scanning electron microscopy of erythrocytes were done on blood of dogs given 5 mg/kg of β-acetylphenylhydrazine for 5 weeks. Reticulocytes, Heinz bodies, and serum total bilirubin values increased in the 1st week. Reticulocyte numbers peaked in the 2nd week, and reticulocytosis persisted through the 5th week. Erythrocyte, packed cell volume, and hemoglobin values decreased markedly and became lowest in the 2nd week. Mean corpuscular volume increased in the 1st week and remained increased for the duration of treatment. Erythrocyte osmotic fragility was increased after 1 week of treatment. Echinocytes were increased with a peak level of 47.6% at week 1 of treatment. Increased numbers of acanthocytes and schizocytes also were detected.

Erratum to “Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay”: International Journal of Antimicrobial Agents 2001;18:359–364

Erratum to ‘‘Rapid detection of mutations associated with resistance to erythromycin in Campylobacter jejuni/coli by PCR and line probe assay’’ International Journal of Antimicrobial Agents 2001;18:359 /364 H. Niwa , T. Chuma , K. Okamoto , K. Itoh * a Laboratory of Veterinary Public Health, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. b Laboratory of Veterinary Public Health, Department of Veterinary Medicine, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. International Journal of Antimicrobial Agents 22 (2003) 461 /463