Kazumichi Tamura

Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning

Enterohemorrhagic Escherichia coli O157 is one of the leading worldwide public health concerns, causing large outbreaks of hemorrhagic colitis as well as numerous small outbreaks and sporadic cases. The variability of restriction enzyme-digestion patterns of O157 genomes, which is widely used to distinguish strains in the molecular epidemiology of O157 infections, suggests the presence of some genomic diversity among the strains. Based on the complete genome sequence of O157 Sakai, we analyzed the whole genome structures of eight O157 strains displaying diverse XbaI-digestion patterns by a systematic PCR analysis that we have named whole genome PCR scanning. This analysis identified not only the O157-specific sequences that are highly conserved among the strains, but also revealed an unexpectedly high degree of genomic diversity. In particular, prophages, including Shiga toxin-transducing phages, exhibited extensive structural and positional diversity, implying that variation of bacteriophages is a major factor in generating genomic diversity among the O157 lineage.

Ciprofloxacin and loperamide in the treatment of bacillary dysentery.

Antimotility drugs are not recommended in the treatment of dysentery. In guinea pigs, the motility of the small intestine is an important defense mechanism. Eight of 10 starved guinea pigs that were experimentally infected with Shigella and given opium died, whereas 10 similarly infected guinea pigs not given opium survived [1]. In a study of diphenoxylate-atropine (Lomotil, Searle and Company, Chicago, Illinois) used alone or in combination with oxolinic acid to treat experimental shigellosis in 25 volunteers [2], the authors concluded that drugs that reduced intestinal motility should not be used to treat invasive diarrhea. The report acknowledged that the results were inconclusive and suggested that larger trials should be done in naturally occurring illness to document the frequency and severity of prolongation of fever and pathogen carriage when patients are treated with Lomotil or paregoric [2]. Loperamide, a synthetic antidiarrheal agent, has not been studied in large numbers of patients with dysentery. Although it does not contain atropine (as does Lomotil), its primary action is to slow intestinal motility. In studies designed to look at nondysenteric diarrhea, loperamide has recently been used, either alone or in combination with antibiotics (cotrimazole or ciprofloxacin), to treat more than 25 adults with Shigella infections [3-7]. Although loperamide did not deliver a definable therapeutic advantage, the drug appeared to be safe in treating patients infected with Shigella. Because loperamide is frequently used in the treatment of diarrhea, which is often due to Shigella, a double-blind, placebo-controlled, randomized clinical trial of loperamide in the treatment of bacillary dysentery (primarily due to Shigella) was done. Methods Study Design Between 28 November 1990 and 29 February 1992, we studied patients with dysentery at Bamrasnaradura Hospital, Nonthaburi, Thailand. Dysentery was defined as three or more loose stools containing blood or mucus associated with at least one of the following symptoms: abdominal cramps, nausea, vomiting, or temperature greater than 38 C. Patients entered in the study had symptoms for fewer than 60 hours. Patients were excluded if they had received antimotility drugs or antibiotics in the previous 7 days, were pregnant, were immunosuppressed, or could not be followed for the next 10 days. Adults admitted to the Bamrasnaradura Hospital who met the study criteria and gave consent were treated with 500 mg of ciprofloxacin (Cipro, Miles Pharmaceuticals, West Haven, Connecticut) twice daily for 3 days. Patients were sequentially assigned a study number and an associated vial containing 16 caplets of loperamide (Imodium, McNeil Consumer Products Company, Fort Washington, Pennsylvania) or identical placebo. Numbered vials were randomized before the beginning of the study using a computer-generated random number table. Nurses, physicians, investigators, and patients were blinded to treatment regimen throughout the study. Patients received 4 mg (two caplets) of loperamide as an initial dose under the observation of a nurse. Patients were instructed to take one caplet after every loose stool (as many as eight caplets per day). Age, duration of diarrhea in hours, number of previous unformed stools, previous medication, and history of symptoms were recorded. Patients were examined by a physician, and stool specimens were collected at study entry. Patients were interviewed daily for 3 days (or until diarrheal symptoms had resolved) and again 10 days later. Symptoms, the time of the last unformed stool, and the number of unformed stools occurring during the previous 24 hours were recorded. Stools (or rectal swabs) were collected at the time of each interview. Laboratory Studies Stools were examined microscopically for fecal leukocytes and erythrocytes after staining with methylene blue [8] and were cultured promptly on MacConkey and Hektoen agar before and after inoculation in Selenite F broth (Difco, Detroit, Michigan). Bacterial, viral, and intestinal protozoa were identified as previously described [9, 10]. Up to 10 lactose-fermenting and up to 10 nonlactose-fermenting Escherichia coli, as identified on a MacConkey plate, were saved on Dorset egg yolk media slants. Escherichia coli isolates were tested within 1 month of isolation for heat-labile and heat-stable toxin production in the Y-1 adrenal cell [11] and suckling mouse [12] assays. Escherichia coli isolates were also fixed on Whatman 541 filters (Millipore Corporation, Bedford, Massachusetts) as described by Maas [13] and were tested for genes coding for Shiga-like toxin I and II as well as those with the 17-kilobase EcoRI fragment of pWR100 [14]. Isolates that hybridized with the 17-kilobase EcoRI probe were speciated and tested using the Sereny test [15]. Shigella, enteroinvasive E. coli, and enterotoxigenic E. coli as well as Aeromonas, Plesiomonas, and Vibrio species were tested for susceptibility to ampicillin, chloramphenicol, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, and tetracycline by disc diffusion [16] using CM471 agar (Oxoid Ltd., Basingstoke, United Kingdom). Enteroinvasive and enterotoxigenic E. coli were serotyped as described by Orskov and Orskov [17]. Intestinal parasites were identified by direct microscopy of saline suspension of stools after formalin-ether concentration, polyvinyl alcohol fixation of stools, and trichrome staining [18]. Cryptosporidium was identified microscopically with a modified dimethyl-sulfoxide method [19]. Rotavirus was identified using a monoclonal enzyme-linked immunosorbent assay (Pathfinder, Kallsted Laboratories, Austin, Texas) [20]. Data Analysis All investigators, nurses, and laboratory personnel were blinded to treatment group until all end points were determined at the end of the study. Data were entered from data collection forms and laboratory records into a computer database. Patients not followed until the resolution of diarrhea and those found not to have met the enrollment criteria were excluded from the analysis before unblinding. Infections with enteroinvasive E. coli or Shigella were evaluated together because of the similar pathogenicity of these organisms [21]. Statistical comparisons were made using Epi Info software [22]. A P value of less than 0.05 was considered statistically significant. The sample size necessary to show a difference of 24 hours in duration of diarrhea (80% power, P < 0.05) was 66 patients with Shigella or enteroinvasive E. coli. We planned to conduct this study over a 1-year period or until 66 patients with Shigella or enteroinvasive E. coli infections were entered. Data analysis included a chi-square with Yates correction (or two-tailed Fisher exact test if an expected cell was less than five) for the detection of proportional differences between the two treatment groups; the Student t-test for the comparison of mean ages and weights; and the Mann-Whitney-Wilcoxon test for the evaluation of non-normally distributed outcome variables (duration of diarrhea and number of diarrheal stools). Confidence intervals (CIs) of 95% are given where appropriate. Results From November 1990 to February 1992, patients with dysentery were screened by a nurse each morning, Monday through Friday. Ninety-two adults met the enrollment criteria. Two patients refused to submit additional specimens, one vomited his initial medication, and one could not be located after discharge from the hospital. Our final sample included 88 analyzable patients with dysentery. Forty-two patients received loperamide and 46 received placebo. Characteristics of the evaluable patients are shown in Table 1. Patients in the loperamide and placebo groups did not differ significantly in age, weight, sex, fecal leukocytes or erythrocytes, proportion who had fever at enrollment, duration of diarrhea before enrollment, or number of diarrheal stools during the 24 hours immediately preceding enrollment. Shigella species were isolated from 44 (50%) and enteroinvasive E. coli from 3 (3%) study participants (Table 2). The patients in whom Shigella or enteroinvasive E. coli were isolated had a lower mean weight (53 compared with 58 kg) and had a longer duration of diarrhea before enrollment (26 compared with 8 hours) than did the patients in whom these organisms were not cultured. Severity of illness was greater in the Shigella-enteroinvasive E. coli group, which had a higher proportion of volunteers with fecal leukocytes (87% compared with 46%), fecal erythrocytes (94% compared with 34%), and fever at enrollment (47% compared with 22%) than did other patients. The median number of stools passed in the 24 hours before enrollment was similar in the two groups (10 and 8 stools, respectively). Table 1. Patient Characteristics at Study Entry* Table 2. Enteric Pathogens Identified in 88 Patients with Dysentery Receiving Ciprofloxacin and Loperamide or Placebo* After treatment, the number of diarrheal stools was fewer for patients with dysentery who received loperamide (median, 4.5 and 7.0 stools, respectively; P = 0.030). This difference was caused by patients infected with Shigella or enteroinvasive E. coli (P = 0.016) (Table 3). In addition, the duration of diarrhea was less in patients infected with Shigella or enteroinvasive E. coli who received loperamide (P = 0.028) (Table 3). During the observation period after the start of treatment, the percentage of patients infected with Shigella or enteroinvasive E. coli receiving loperamide compared with placebo did not differ statistically in the following parameters: abdominal cramps (21% compared with 27%, P = 0.282), nausea (16% compared with 18%, P = 0.903), vomiting (16% compared with 19%, P = 0.770), or constipation (5% compared with 0%, P = 0.447). An oral temperature greater than 38 C was not found in any participant for more than 1 day after enrollment. Shigella or enteroinvasive E. coli were isolated 1 day after begin

DNA Sequence Analysis of DNA Gyrase and DNA Topoisomerase IV Quinolone Resistance-Determining Regions of Salmonella enterica Serovar Typhi and Serovar Paratyphi A

ABSTRACT The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.

Selective Amplification of tyv (rfbE), prt (rfbS), viaB, and fliC Genes by Multiplex PCR for Identification of Salmonella enterica Serovars Typhi and Paratyphi A

ABSTRACT The PCR primers for O, H, and Vi antigen genes, tyv (rfbE), prt (rfbS), fliC-d, fliC-a, and viaB, were designed and used for the rapid identification of Salmonella enterica serovars Typhi and Paratyphi A with multiplex PCR. The results showed that all the clinical isolates examined of Salmonella serovars Typhi and Paratyphi A were accurately identified by this assay.

Leclercia adecarboxylata Gen. Nov., Comb. Nov., formerly known asEscherichia adecarboxylata.

The nameLeclercia adecarboxylata is proposed for a group of the family Enterobacteriacae previously known asEscherichia adecarboxylata. Leclercia adecarboxylata can be phenotypically differentiated from all other species of Enterobacteriaceae. The members of this species are positive for motility, indole production, methyl red, growth in the presence of KCN, malonate, beta-galactosidase, beta-xylosidase, esculin hydrolysis, gas production fromd-glucose, and acid production fromd-cellobiose,d-lactose, melibiose,l-rhamnose, adonitol,d-arabitol, dulcitol, and salicin; the strains were negative for Voges-Proskauer, citrate (Simmons), H2S (Kligler), lysine and ornithine decarboxylases, arginine dihydrolase, phenylalanine deaminase, gelatinase, DNase, Tween-80 hydrolysis, and acid production from myoinositol and alpha-methyl-d-glucoside. Fermentation ofd-raffinose,d-sucrose, andd-sorbitol is variable with strains. DNA relatedness of 11 strains ofL. adecarboxylata to three strains including the type strain of this species averaged 80% in reactions at 65°C. DNA relatedness to other species in Enterobacteriaceae was 2%–32%, indicating that this species was placed in a new genusLeclercia gen. nov. The type strain ofL. adecarboxylata is ATCC 23216.

Klebsiella ornithinolytica sp. nov., formerly known as ornithine-positiveKlebsiella oxytoca

The nameKlebsiella ornithinolytica sp. nov. is proposed for a group ofKlebsiella strains referred to previously as NIH Group 12 at the National Institute of Health, Tokyo. The members of this species are Gram-negative, encapsulated, nonmotile rods with the general characteristics of the familyEnterobacteriaceae and of the genusKlebsiella. They give positive results in tests for indole production, Voges-Proskauer, citrate utilization, lysine and ornithine decarboxylases, urease, β-galactosidase, malonate utilization, growth in KCN, and esculin hydrolysis, and they produce acid and gas fromd-glucose, and acid froml-arabinose, cellobiose, lactose, melibiose, raffinose, rhamnose, sucrose, trehalose,d-xylose, adonitol,d-arabitol, myo-inositol, sorbitol, arbutin, salicin, α-methyl-d-glucoside, and mucate. They give negative drolysis, DNase, pectinase, and acid production fromd-arabinose, melezitose, and dulcitol. They can grow at 4°C and 42°C, and do not produce any pigment. DNA relatedness of eight strains ofKlebsiella ornithinolytica to three strains including the type strain of this species averaged 88% in reaction at 75°C. DNA relatedness to the already recognizedKlebsiella species inEnterobacteriaceae was 1 to 20%. Phenotypic and DNA relatedness data also indicated that a group of organisms referred to as Enteric Group 47 orKlebsiella Group 47 at the Centers for Disease Control (Atlanta, Georgia) was identical withK. ornithinolytica. The type strain ofK. ornithinolytica is NIH 90-72 (JCM 6096).

Two community outbreaks of human infection with Yersinia enterocolitica.

Two outbreaks of human infection with Yersinia enterocolitica in Shizuoka, Japan are described. This is the first report of community outbreaks of infection with this organism in Japan, and possibly in the world. All the strains isolated in each outbreak belonged to O antigen group 3, biotype 4, of the species. Despite much effort, the source and mode of spread of the infection were not established.

Extended-Spectrum β-Lactamase-Producing Shiga Toxin Gene (stx1)-Positive Escherichia coli O26:H11: a New Concern

ABSTRACT Escherichia coli strain TUM2139 was isolated from a stool sample from a 9-year-old girl on 16 June 2004. This strain was categorized as Shiga toxin-producing Escherichia coli (STEC) because the Shiga-like toxin gene stx1 was detected by immunochromatography and PCR assay. The strain was highly resistant to cefotaxime (256 μg/ml) and was also resistant to cefepime, cefpodoxime, ceftriaxone, and aztreonam. In the presence of 4 μg of clavulanic acid per ml, the MIC of cefotaxime decreased to ≤0.12 μg/ml, indicating that this strain was an extended-spectrum β-lactamase (ESBL) producer. Cefotaxime resistance was transferred to E. coli C600 by conjugation at a frequency of 3.0 × 10−6. A PCR assay was performed with primer sets specific for TEM-type and SHV-type ESBLs and for the CTX-M-2 (Toho-1), CTX-M-3, and CTX-M-9 groups of ESBLs. A specific signal was observed with the primer set specific for the CTX-M-9 group of β-lactamases. This β-lactamase was confirmed to be the ESBL CTX-M-18 by DNA sequencing. This is the first report of an ESBL-producing STEC isolate.

Characterization of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated in Japan

ABSTRACT A total of 221 isolates of multidrug-resistant Salmonella enterica serovar Typhimurium in Japan were characterized in the present study. The results revealed that clonal serovar Typhimurium definitive phage type 104 strains prevailed and that these strains had drug resistance patterns, integron types, and pulsed-field gel electrophoresis patterns similar to those predominant among isolates in Western countries.

Antimicrobial Susceptibility of Shigella sonnei Isolates in Japan and Molecular Analysis of S. sonnei Isolates with Reduced Susceptibility to Fluoroquinolones

ABSTRACT We performed susceptibility testing with Shigella sonnei isolates from imported and domestic cases of infection in Japan during 2001 and 2002. Some S. sonnei isolates were resistant to nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole. Most of the nalidixic acid-resistant strains showed reduced susceptibility to fluoroquinolones but did not show fluoroquinolone resistance.