Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD)

This study was performed to determine profile of toxigenicity of 18 Clostridium difficile strains isolated from paeditric patients suffering from antibiotic associated diarrhea (AAD). Toxigenicity of C. difficile strains was tested for detection toxin A and toxin B by phenotypic methods and for detection of the tcdA and tcdB genes using of PCR. Changes in the repeating regions of the tcdA genes were detected with the NK9/NKV011 primer pairs. For detection of binary toxin (CDT) cdtA and cdtB genes, cdtApos/cdtArev i cdtBpos/cdtBrev two pair primers in PCR was used. Among C. difficile strains was detected three profiles of toxigenicity: C. difficile strains possesing of tcdA and tcdB genes but not possesing cdtA and cdtB genes of binary toxin (A+B+CDT-), strains possesing tcdA and tcdB and cdtA and cdtB genes (A+B+CDT+), strains with deletion of toxin A gene (A-B+CDT-). This is the first report on the occurence of binary positive C. difficile strains isolated from paediatric patients.     

Intestinal flora of patients with suspected antibiotic associated diarrhea (AAD). I. Clostridium perfringens

Stool samples of 158 patients suspected of antibiotic-associated diarrhoea (AAD) were studied. Toxin A of C. difficile and enterotoxin of C. perfringens were detected in stool samples by immunoenzymatic assays and PCR. In 35 stool samples toxin A of C. difficile was detected and in 48 cases (30%) C. difficile strains were cultured from 21 stool samples (13%). The presence of the cpe gene of C. perfringens, enabling the production of enterotoxin, could not be detected by PCR, both in stool samples and in isolated strains, using ent 1 and ent 2 primer pairs. C. difficile and C. perfringens were isolated from the same stool samples in 4 cases. From stool samples of two patients with AAD C. perfringens strains, thermoresistant spores were cultured.     

Detection of ermB gene responsible for high level resistance to clindamycin (MLS type resistance) among Clostridium difficile strains isolated from antibiotic associated diarrhea (AAD)

In 68 C. difficile strains isolated from feacal samples of patients with antibiotic associated diarrhoea (AAD) investigated presence of ermB gene transferable of high level resistance to clindamycin. The primers set 2980/2981 used for identification of ermB gene amplified a 688 bp segment. We used the Etest to assess all strains for susceptibility to clindamycin. This study demonstrates that 57% of strains isolated from faecal samples of patients with AAD were highly resistant to clindamycin (minimal inhibitory concentration (MIC) of clindamycin, 256 mg/L) and possessed the ermB gene.     

Detection of viral agents in fecal specimens of monkeys with diarrhea

BACKGROUND: Diarrheal disease is a major cause of morbidity and mortality in humans and animals, including non human primates. While the diagnostics for gastrointestinal bacterial and parasitic pathogens and their etiological role in disease are well established, little is known about the epidemiology, prevalence and role of viral agents in diarrheal illness among monkeys. METHODS: We collected fecal specimens from monkeys with diarrhea that were housed in two primate colonies, the Institute of Laboratory Animal Sciences, Beijing, China and the Yerkes National Primate Research Center, Georgia, USA. We screened these fecal specimens for rotaviruses and enteric adenoviruses 40/41 by using commercial EIA kits (Rotaclone and Adenoclone), enteroviruses by RT-PCR and Southern blot hybridization, and picobirnaviruses by polyacrylamide gel electrophoresis and silver staining. Some of the specimens were examined by EM for coronaviruses and noroviruses. RESULTS: Of the 92 specimens from China, we found 63 (68%) positive for viruses, including enteroviruses (52%), enteric adenoviruses (21%), rotaviruses (20%), and picobirnaviruses (2%). Coronaviruses were detected in some specimens. Mixed infection of two or more viral agents was seen in 23 (25%) specimens. In the US collection, we detected enteroviruses and enteric adenoviruses in 76% (45/59) and 14% (7/50) of the specimens, respectively. Electron microscopy showed norovirus-like particles in some specimens from both colonies. CONCLUSIONS: Our findings indicate endemic infections with enteric viruses in monkeys of both colonies. The availability of new simian rotaviruses, enteric adenoviruses, enteroviruses, and coronaviruses and the discovery of noroviruses and picobirnaviruses may allow us to develop better diagnostics for these agents and determine which of these agents are clearly associated with gastroenteritis in monkeys.     

Prevalence of enterotoxigenic Bacteroides fragilis strains (ETBF) in the gut of children with clinical diagnosis of antibiotic associated diarrhoea (AAD)

Prevalence of enterotoxin producing Bacteroides fragilis (ETBF) strains in faecal samples of children with clinical diagnosis antibiotic associated diarrhoea (AAD) was investigated. Out of faecal samples collected from sixty children, thirty C. difficile strains were isolated. Enterotoxigenic B. fragilis (ETBF) strains were cultured from four children what made 6.7% of investigated faecal samples. Out of two samples toxinogenic C. difficile strains [tox A(+) tox B(+)] were cultured together with enterotoxinogenic B. fragilis. From sample of one child C. difficile A negative/B positive strains [tox A(-) tox B(+)] was found together with B. fragilis (ETBF). From faecal sample of one child enterotoxinogenic B. fragilis only was isolated. It was shown that in the gut of children with clinical diagnosis of (AAD) enterotoxinogenic B. fragilis (ETBF) can be present. B. fragilis (ETBF) can be observed in concomitance with toxinogenic C. difficile.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.     

Egg counts utilizing trichrome-stained smears from polyvinyl alcohol (PVA)-preserved fecal specimens.

A simple method of making egg counts on fecal specimens is described. It utilizes PVA-preserved stool, yields a permanent stained slide, and is as accurate and reproducible as other methods in common use.     

610例浅部致病真菌分析

目的了解本地区浅部真菌病的致病菌菌种的分布情况。方法用沙堡琼脂培养基分离培养浅部真菌病的致病菌,并分析各年份菌种分离结果。结果共分离出6种610株致病菌,其中红色毛癣菌居首位,念珠菌居第2位,红色毛癣菌和念珠菌的分离率随年份有明显变化。结论红色毛癣菌居患者浅部真菌病致病菌首位,念珠菌也是重要病原菌,且病原菌分离率随年份有明显变化。     

Intestinal lactoflora in Estonian and Norwegian patients with antibiotic associated diarrhea

The disruption of intestinal microbiota is an important risk factor for the development of Clostridium difficile caused antibiotic associated diarrhea (AAD). The role of intestinal lactoflora in protection against C. difficile is unclear. Fecal samples (n = 74) from AAD patients were investigated for C. difficile and lactobacilli by culture and real-time PCR. Lactobacilli were identified by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and sequencing of 16S rRNA. In C. difficile negative cases we found somewhat higher counts of intestinal Lactobacilli (5.02 vs. 2.15 CFU log₁₀/g; p = 0.053) by culture and more frequently Lactobacillus plantarum (33.3% vs. 9.4%; p = 0.03) as compared with positive ones. Results of total counts of lactobacilli comparing Estonian and Norwegian samples were conflicting by culture and PCR. We found higher colonization of Norwegian AAD patients with L. plantarum (21% vs. 5%, p = 0.053) and Estonians with Lactobacillus gasseri (19% vs. 2%, p = 0.023). Particular lactobacilli (e.g. L. plantarum) may have a role in protection against C. difficile, whereas the meaning of total counts of lactobacilli remains questionable. In different persons and nations, different lactobacilli species may have a protective role against C. difficile.     

Clostridium difficile strains not producing Toxin A,but producing Toxin B [toxA(-)tox B(+)]--etiologic agent of antibiotic associated diarrhoea (AAD) in Poland

Previously, toxin A-negative/toxin B-positive Clostridium difficile strains were not thought to be associated with clinically significant diseases. In our study among 159 tested C. difficile strains isolated from feacal samples from 413 patients with antibiotic associated diarrhoea (AAD) 17 strains (11%) were negative in the "Culturette Brand Toxin" CD (Becton-Dickinson) for detection toxin A and positive in the TOX A/B test, designed for detection of both toxins. The conserved regions of both toxin genes were detectable in all of isolates studied by the PCR. Nine of these C. difficile strains had a deletion in the A gene and remaining 8 strains, revealed an amplicon with the expected size of approximately 2500 bp. In this paper we described the first time the toxin A-negative/toxin B-positive C. difficile strains with deletion in toxin A gene, isolated from the faecal samples of patient with AAD in Poland.     

Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis)

Thermotolerant fecal indicator organisms carried by migratory waterfowl may serve as reservoirs of antibiotic resistance. To determine the extent to which such antibiotic resistance markers were present in migratory Canada geese (Branta canadensis) on the Maryland Eastern Shore, we isolated Enterococcus spp. and Escherichia coli from fresh feces and examined the antibiotic resistance profiles of these bacteria. Samples were obtained in October 2002, January 2003, and March 2003. Thermotolerant E. coli counts ranged from 0 to 1.0x10(7) colony forming units (CFU)/0.1g (g-1) wet weight of feces, whereas Enterococcus spp. counts ranged from 1.0x10(2)-1.0x10(7) CFU g-1 wet weight of feces. Primary isolates of each indicator organism were tested against a panel of 10 antibiotics. Greater than 95% of E. coli isolates were resistant to penicillin G, ampicillin, cephalothin, and sulfathiazole; no E. coli were resistant to ciprofloxacin. Enterococcal isolates showed highest resistance to cephalothin, streptomycin, and sulfathiazole; no enterococci were resistant to chloramphenicol. The tetracyclines, streptomycin, and gentamycin provided the greatest discrimination among E. coli isolates; chlortetracycline, cephalothin, and gentamycin resistance patterns provided the greatest discrimination between enterococcal strains. Multiple antibiotic resistance (MAR) profiles were calculated: fall (E. coli=0.499; enterococci=0.234), winter (E. coli=0.487; enterococci=0.389), and spring (E. coli=0.489; enterococci=0.348). E. faecalis and E. faecium, which are recognized human nosocomial pathogens, were cultured from winter (44 and 56%, respectively) and spring (13 and 31%, respectively) fecal samples.     

Selective-enrichment procedure for isolation of Listeria monocytogenes from fecal and biologic specimens

A selective-enrichment procedure (SEP) was developed to isolate Listeria monocytogenes from fecal and biologic specimens. This procedure was compared with direct plating with McBride listeria agar and 2-, 4-, and 8-week cold-enrichment procedures in recovering L. monocytogenes from mouse fecal, liver, and brain specimens. Although the SEP occasionally did not isolate the organism from specimens proved positive by the other procedures, the SEP isolated L. monocytogenes from about two and five times as many specimens as the cold-enrichment and direct-plating procedures, respectively.     

Clonal lineages, antibiotic resistance and virulence factors in vancomycin-resistant enterococci isolated from fecal samples of red foxes (Vulpes vulpes)

Fourteen vanA-containing enterococcal isolates were detected in seven of 52 fecal samples (13.5%) from free-ranging red foxes in Portugal. Nine of the vanA-containing isolates were Enterococcus faecium and five were E. durans. Both sequence types, ST262 and ST273, were identified among E. faecium isolates.     

Isolation of enteric pathogens from patients with travelers' diarrhea using fecal transport media

Abstract We investigated the effectiveness of a two-vial fecal transport system: glycerol-buffered saline (GBS) and modified Cary-Blair with antibiotics (CBA) to transport stool samples to an enteric disease reference laboratory. In a blind study we compared the results of culturing 41 specimens promptly in the field laboratory to subculturing GBS and CBA in the reference laboratory. A pathogen was isolated from 72% of the cases in each laboratory. In addition, a pathogen was isolated from 32 of 45 samples inoculated into GBS and CBA and transported to the reference laboratory for subculture. These results demonstrate that GBS and CBA are effective means of transporting specimens to a reference laboratory when studying the etiology of diarrhea in remote parts of the world.     

Mycorrhizal fungi associated with Monotropastrum humile (Ericaceae) in central Japan

We explored the diversity of mycorrhizal fungi associated with Monotropastrum humile in the central part of Japan's main island. We collected 103 M. humile individuals from 12 sites with various forest types. We analyzed the DNA sequences of the internal transcribed spacer region from fungal and plant nuclear ribosomal DNAs to assess the genetic diversity of the fungi associated with M. humile roots and to position the plant with respect to known Monotropoideae groups, respectively. The plants formed a monophyletic clade with other members of M. humile but were separated from M. humile var. glaberrimum and other monotropes (97% bootstrap support). Of the 50 fungal phylotypes, 49 had best matches with the Russulales, and the other had highest similarity with the Thelephoraceae. Our phylogenetic analysis suggests that M. humile roots have a highly specialized association with fungal partners in the Russulaceae. Moreover, a few fungal phylotypes from the M. humile roots had positions neighboring those from Monotropa uniflora roots. These results indicated that the genetic diversity of mycorrhizal fungi of M. humile was highly specific to the Russulaceae, but with high diversity within that family, and that the fungi associated with M. humile differ from those associated with M. uniflora.     

Factors associated with fecal glucocorticoids in Alaskan brown bears (Ursus arctos horribilis)

The aims of this study were to validate a radioimmunoassay (RIA) for quantifying glucocorticoid metabolite concentrations in the feces of Alaskan brown bears (Ursus arctos horribilis) and to investigate whether any of the following factors are associated with those concentrations: the presence of humans or other bears, fishing difficulty, sex-age class, diet, and season. We tested an established corticosterone RIA for assay sensitivity, similarity, precision, and sample matrix effects of brown bear feces, and it proved satisfactory. We collected fecal samples from brown bears along salmon-spawning streams and assessed fecal glucocorticoid (FG) concentrations. We observed that the factors explaining the most variation in measured concentrations were date and diet type and that there was a significant interaction between the two. We did not observe a significant effect of human and bear activities or sex-age class on FG concentrations. This study demonstrates that although FG concentrations may be assessed in brown bears, complex dietary patterns and seasonal variations must be taken into consideration in the study design in order to make inferences regarding stress.     

The presence of bacteria and yeast like fungi in specimens from surgical patients

The aim of this study was to determine the incidence of Candida spp. strains in specimens obtained from surgically treated patients as well as to analyze the accompanying bacterial flora, both aerobic and anaerobic. The material came from two groups of patients. In the first group consisting of patients operated for colon and rectum carcinoma, the samples included peritoneal fluid, colon or rectum bioptates, pus, blood, and wound swabs. In the other group, biopsy material and smears from post operation wounds were taken from patients who underwent a surgical treatment of larynx carcinoma. Altogether, 282 various clinical specimens from 165 patients were analysed, and 41 Candida spp. strains were isolated: 39 strains of C. albicans and 2 strains of C. tropicalis. In 20 out of 41 specimens infected with Candida spp. (48.8%) the co-infection with bacterial aerobic flora was found. In 10 cases (24.4%), the fungi were isolated together with aerobic and anaerobic bacterial flora, whereas in 2 specimens (4.9%) the anaerobes and Candida albicans were diagnosed. The remaining 9 samples showed only the presence of Candida spp. (21.9%). From among aerobic bacterial flora Enterococcus spp. strains (n = 17) and Gram negative rods from Enterobacteriaceae family (n = 13) were the most frequently isolated. The bacterial strains of Streptococcus spp. (n = 5), Pseudomonas spp. (n = 3), Staphylococcus spp. and Corynebacterium spp. (2 strains, both) were identified more rarely. Bacteroides spp. were the most frequent members of bacterial anaerobic flora (n = 10). Other isolated anaerobic bacteria were classified as Fusobacterium spp. or Peptostreptococcus spp. (1 strain each). E. coli and Enterococcus spp. strains of aerobic bacterial flora were more frequently isolated together with Candida spp. CONCLUSIONS: (i) Mixed bacterial flora was found to predominate in the clinical material from the patients after surgery. (ii) Candida spp. were most frequently found together with aerobic bacterial flora.