Binary Toxin Genes Research Articles

In vitro activity of MCB3681 against Clostridium difficile strains

One hundred fourteen Clostridium difficile strains were collected from 67 patients and analyzed for the presence of C. difficile toxin B by the cell cytotoxoicity neutralization assay, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped. The MICs of the isolates were determined against MCB3681 and nine other antimicrobial agents by the agar dilution method. All isolates were positive for toxin B as well as for toxin A and B genes. In addition, 13 isolates were positive for the binary toxin genes. Thirty-two different ribotypes were identified. No strain of ribotype 027 was found. All 114 isolates were sensitive to MCB3681 (0.008–0.5 mg/l), cadazolid (0.064–0.5 mg/l), fidaxomicin (0.008–0.125 mg/l), metronidazole (0.125–2 mg/l), vancomycin (0.125–1 mg/l) and tigecycline (0.032–0.25 mg/l). Three isolates were resistant to linezolid (8 mg/l), 12 isolates were resistant to moxifloxacin (8–32 mg/l), 87 isolates were resistant to clindamycin (8–256 mg/l) and 107 isolates were resistant to ciprofloxacin (8–256 mg/l). No association between toxins A, B and binary toxin, ribotypes and the sensitivity to MCB3681 could be found. MCB3681 has a potent in vitro activity against C. difficile.

Clostridium difficile and Clostridium perfringens from wild carnivore species in Brazil

Despite some case reports, the importance of Clostridium perfringens and Clostridium difficile for wild carnivores remains unclear. Thus, the objective of this study was to identify C. perfringens and C. difficile strains in stool samples from wild carnivore species in Brazil. A total of 34 stool samples were collected and subjected to C. perfringens and C. difficile isolation. Suggestive colonies of C. perfringens were then analyzed for genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota) and the beta-2 toxin (cpb2), enterotoxin (cpe) and NetB (netb) genes. C. difficile strains were analyzed by multiplex-PCR for toxins A (tcdA) and B (tcdB) and a binary toxin gene (cdtB) and also submitted to a PCR ribotyping. Unthawed aliquots of samples positive for C. difficile isolation were subjected to the detection of A/B toxins by a cytotoxicity assay (CTA). C. perfringens was isolated from 26 samples (76.5%), all of which were genotyped as type A. The netb gene was not detected, whereas the cpb2 and cpe genes were found in nine and three C. perfringens strains, respectively. C. difficile was isolated from two (5.9%) samples. A non-toxigenic strain was recovered from a non-diarrheic maned wolf (Chrysocyon brachyurus). Conversely, a toxigenic strain was found in the sample of a diarrheic ocelot (Leopardus pardallis); an unthawed stool sample was also positive for A/B toxins by CTA, indicating a diagnosis of C. difficile-associated diarrhea in this animal. The present work suggests that wild carnivore species could carry C. difficile strains and that they could be susceptible to C. difficile infection.

Occurrence of Clostridium difficile PCR-ribotype 027 and it's closely related PCR-ribotype 176 in hospitals in Poland in 2008–2010

Since 2003, a rising incidence of Clostridium difficile infection (CDI) in North America and Europe has coincided with outbreaks of C. difficile PCR ribotype 027. This ribotype was not observed in Poland until 2008. In the period 2008–2010, outbreaks of antibiotic-associated diarrhoea occurred in three different hospitals in Poland. Of 30 C. difficile isolates available for microbiological characterisation, 17 (56%) were positive for binary toxin genes and belonged to PCR ribotype 027 (n = 7) and its closely related PCR ribotype 176 (n = 10). All 17 binary toxin–positive C. difficile strains demonstrated high-level resistance to fluoroquinolones (minimum inhibitory concentration (MIC) ≥ 32 mg/L), including ciprofloxacin, gatifloxacin, and moxifloxacin, as well as erythromycin and clindamycin (MIC ≥ 256 mg/L for both). Of 14 patients from whom clinical information was available, 50% had a severe form of CDI, defined by fever (>38.5 °C), decreased kidney function, and high leucocyte count. We conclude that outbreaks of CDI associated with hypervirulent strains belonging to PCR ribotypes 027 and 176 occurred in hospitals in Poland. Further studies evaluating the clinical impact of type 176 are urgently needed.

Emergence of a Ribotype 244 Strain of Clostridium difficile Associated With Severe Disease and Related to the Epidemic Ribotype 027 Strain

We identified 12 patients with Clostridium difficile infection between July 2011 and March 2012 from whom an unusual C. difficile strain was isolated. This strain had a single-nucleotide deletion of the tcdC gene at position 117 and binary toxin genes, which are characteristic of the hypervirulent ribotype (RT) 027 strain. A retrospective cohort study of 12 patients infected with C. difficile RT244 and 24 patients infected with non-RT244/non-RT027 strains matched for place of diagnosis and time of collection of specimen was performed. We performed whole-genome sequencing to understand the relationship of the RT244 strain to other C. difficile strains and further understand its virulence potential. Clostridium difficile RT244 was associated with more severe disease and a higher mortality rate. Phylogenomic analysis using core genome single-nucleotide polymorphisms showed that RT244 is in the same genetic clade (clade 2) as RT027 but is distinct from all RT027 strains. The pathogenicity locus of the RT244 strain encodes a variant toxin B, and this was confirmed by demonstration of Clostridium sordellii-like cytopathic effect on Vero cells. Toxin B production in culture supernatants was lower than that seen with a RT027 strain. Our findings demonstrate the pathogenic potential of this RT244 C. difficile strain and emphasize the importance of ongoing surveillance for emergent strains.

Investigation of potentially pathogenic Clostridium difficile contamination in household environs

As Clostridium difficile spores are resistant to many household cleaning products, the potential for community household contamination is high. The purpose of this study was to assess the prevalence of toxigenic C. difficile from environmental sources from a large urban area. Three to 5 household items or environmental dust was collected from 30 houses in Houston, Texas. A total of 127 environmental samples were collected from shoe bottoms (n = 63), bathroom surfaces (n = 15), house floor dusts (n = 12), or other household surfaces (n = 37). Forty one of 127 samples (32.3%) grew C. difficile. All 41 isolates were positive for toxin A and B genes and no isolate was positive for binary toxin genes. Shoe bottom swab samples had the highest percent of positive samples (25/63; 39.7%) followed by bathroom/toilet surfaces (5/15; 33.3%), house floor dust (4/12; 33.3%), and other surface swabs (7/37; 18.9%). Strains were grouped into 25 different ribotypes, the most prevalent type was 001 (5 strains). In conclusion, a high rate of environmental contamination of C. difficile was observed from community households from a large urban area.

Regulation of toxin production in the pathogenic clostridia

The genus Clostridium comprises a large, heterogeneous group of obligate anaerobic, Gram-positive spore forming bacilli. Members of this genus are ubiquitous in the environment and although most species are considered saprophytic, several are pathogenic to both humans and animals. These bacteria cause a variety of diseases including neuroparalysis, gas gangrene, necrotic enteritis, food poisoning, toxic shock syndrome and pseudomembraneous colitis, which in most cases arise as a consequence of the production of potent exotoxins. Treatment options are often limited, underscoring the need for new treatment strategies and novel therapeutics. Understanding the fundamental mechanisms and signals that control toxin production in the pathogenic clostridia may lead to the identification of novel therapeutic targets that can be exploited in the development of new antimicrobial agents.

Spread of epidemic Clostridium difficile NAP1/027 in Latin America: case reports in Panama

The rate and severity of Clostridium difficile infection (CDI) have been linked to the emergence and spread of the hypervirulent toxigenic strain NAP1/027. This strain has been responsible for large outbreaks in healthcare facilities in North America and Europe and most recently in Latin America. This is the first report of the NAP1 strain in Panama. It suggests that the spread of C. difficile NAP1 throughout Latin America could be a possibility as evidenced in the following case reports. Five isolates typed as NAP1 had tcdA, tcdB, binary toxin gene cdtB and tcdC deletion. All isolates were resistant to clindamycin, fluoroquinolones and rifampicin. Under this scenario, surveillance programmes for CDI should be implemented in public health facilities in Latin America and diagnosis of CDI should be considered, especially in patients with predisposing factors.

Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype 027, and all 59 possessed Δ 117 in tcdC. Time to results was approximately 2.5 h per specimen. The Verigene CDF test is a novel nucleic acid microarray that reliably detects both C. difficile toxins A and B in unformed stool specimens and appears to adequately identify ribotype 027 isolates.

Clostridium difficileInfection Caused by Binary Toxin–Positive Strains

To the Editor: With interest, we read the article by Bacci et al. in which they conclude that Clostridium difficile strains containing the binary toxin gene were associated with a higher case-fatality rate after 30 days, even when the analysis was stratified for PCR ribotype (1). Although this was an appealing conclusion, in our opinion the study was severely limited by selection bias and confounding by underlying diseases. First, in Danish patients with a C. difficile infection (CDI), isolates were characterized only if they were isolated during outbreaks or from patients with severe diseases or if isolates were found to be moxifloxacin resistant. Therefore, selection bias was likely to occur. Second, adjustment for concurrent conditions was not performed. This adjustment was especially warranted because outbreaks on specific hospital wards (e.g., intensive care units) could have influenced the all-cause mortality rate. Last, the selection of specific patients and strains questions the generalizability of the authors’ conclusion. In an approach to confirm the findings of Bacci et al. (1), we used data from a cohort study conducted during 2006–2009 in 13 Dutch hospitals (2). A total of 1,350 consecutive hospitalized patients with unformed feces and a positive C. difficile toxin test result were included in the study. We checked the 30-day survival for study patients in the Dutch Civil Registration System. For 626 (46%) of the patients, a C. difficile strain was available for PCR ribotyping and binary toxin gene characterization. Patient data (e.g., age, sex, hospitalization, and antimicrobial drug use in the 3 months before onset of diarrhea) were collected by review of electronic and paper patient charts and by contacting the treating physician. Underlying diseases present at hospital admission were classified into 7 disease categories (Table, footnote). In addition, during at least 6 months, the Charlson comorbidity index at admission was determined in 9 of the 13 hospitals (total of 357 CDI patients). Proportional hazards modeling was used for survival analysis. The Medical Review Ethics Committee of the Leiden University Medical Center approved this study Table Thirty-day mortality rates, stratified by binary toxin status, for persons with Clostridium difficile infection, the Netherlands, 2006–2009* During the study period, CDI was endemic in all hospitals in the cohort study (13 cases/10,000 admissions). The all-cause risk for dying within 30 days was 22% (12/55) for persons infected with binary toxin–positive 027 strains, 15% (15/100) for those infected with binary toxin–positive non-027 strains, and 11% (50/471) for those infected with binary toxin–negative strains (Table). Selection bias (e.g., by primarily characterizing isolates of patients with severe disease) was unlikely because the number of deaths among CDI patients without strain characterization (100/724 [14%]) was similar to that among patients with a characterized strain (77/626 [12%]; p = 0.41). Thirty-day mortality rates were significantly higher among patients with CDI due to type 027 strains than among patients with binary toxin–negative strains (hazard ratio [HR] 2.2); additional adjustment for age and concurrent condition(s) resulted in a relatively constant HR of 2.0–2.4. Patients with CDI due to binary toxin–positive non-027 strains did not have a substantially higher 30-day mortality rate (HR 1.5); additional adjustment for age and concurrent condition(s) lowered the HR to 1.1–1.4, depending on the method of adjustment. In accordance with findings in the Danish study, we observed a high 30-day mortality rate among persons infected with type 027 isolates. The 30-day mortality rate was lower among persons infected with non-027 binary toxin–positive isolates, especially after correction for concurrent condition(s); however, confidence intervals overlapped with those for type 027. Therefore, we cannot statistically contradict the conclusion of Bacci et al. (1). Nevertheless, because mortality rates in our study among patients with non-027 type CDI strongly resembled mortality rates among patients with CDI caused by binary toxin–negative isolates and because the Danish study was prone to bias and lacked adjustment for confounding, we think that the results of Bacci et al. (1) should be interpreted with caution. Furthermore, a large clinical study from 2008 concluded that C. difficile type 078, which is the most frequently found binary toxin positive non-027 strain, was not associated with a high all-cause mortality rate (3). A more recent publication confirmed this finding (4). Therefore, in our opinion, there is currently no convincing epidemiologic proof that binary toxin is a marker for infection with virulent C. difficile.

Molecular characterization of mosquitocidal Bacillus sphaericus isolated from Tamil Nadu, India

Forty-two Bacillus sphaericus strains were isolated from soil around Tamil Nadu, India. The phylogenetic relationship among the B. sphaericus isolates was analysed by REP-PCR and multiplex PCR was performed for the detection of mosquito larvicidal genes binA, binB, mtx1, mtx2 and mtx3 in B. sphaericus isolates. According to the REP-PCR band pattern, B. sphaericus isolates were divided into group A comprising I–XI clusters and group B comprising cluster XII. Three of the isolates BSTN01, 23 and 24 were gathered under cluster XII showed a high level of larvicidal activity against Culex quinquefasciatus and Anopheles stephensi, the other 39 isolates grouped under I–XI clusters were non-toxic or weak or moderately toxic to mosquito larvae. Even though BSTN23 and 24 were isolated from the same location and both contained all the five mosquito larvicidal genes, their intraspecies difference was clearly elucidated by REP-PCR analysis. Among high toxic isolates, BSTN23 and 24 were observed to contain all the five toxin genes and BSTN01 showed the presence of binary toxin and Mtx1 toxin genes. The isolates BSTN02, 03, 07, 14, 16, 19, 20, 21, 25, 31, 36 and 39 were found to contain mtx1 gene with combination of mtx2 and/or mtx3 showed moderate or low toxicity against mosquito larvae. binA, binB and mtx1 genes were not present in non-toxic isolates. The present study revealed the genetic heterogeneity between both toxic and non-toxic isolates and indicates that there is a good correlation between the presence of toxin genes and toxicity of the strains. These techniques could be developed in screening of novel highly toxic B. sphaericus strains from environment without bioassay on mosquito larvae.

Screening of vip1/vip2 binary toxin gene and its isolation and cloning from local Bacillus thuringiensis isolates

Efforts were carried out to isolate vegetative insecticidal protein genes from local isolates of Bacillus thuringiensis. The study focused on vip1/vip2 binary toxin, considering its insecticidal potential against coleopteran and hemipteran pests. Thirty nine B. thuringiensis local strains and one standard reference strain (HD 1) were screened for the presence of vip1/vip2 gene by using a PCR approach. Among 39 isolates only four isolates (PDKV-08, PDKV-27, PDKV- 28 and NCIM-5112) showed the presence of the desired gene. SDS-PAGE screened profiling of the isolates showed the presence of 95 kDa and 50 kDa protein which confirmed our PCR study. For further characterization, the vip1/vip2 gene was cloned from the PDKV-08 isolate by using the pJET1 cloning vector. Sequence homologous analysis confirms the presence of the vip1/vip2 gene. A further BLAST analysis also revealed that the isolated vip1/vip2 gene is highly conserved and showed a maximum of 88% sequence homology with existing vip1/vip2 genes. Insect toxicological potential was also elucidated by performing bioassays of PDKV-08 supernatant proteins against the coleopteran store grain pest, Sitophilus zeamais. The results from a bioassay revealed 60% mortality.

Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil

The objective of this study was to detect C. difficile A/B toxins and to isolate strains of C. perfringens and C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficile A/B toxins were detected by ELISA, and C. perfringens and C. difficile were identified by multiplex PCR. C. difficile A/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+ and two were A−B−. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtB was found in one strain, which was A+B+ and was derived from a non-diarrheic dog. C. perfringens strains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringens type A and there was an association between the detection of the cpe gene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2 gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficile and C. perfringens in dogs and to better our understanding of C. difficile as a zoonotic agent. This is the first study to report the binary toxin gene in C. difficile strains isolated from dogs in Brazil.

Characteristics of patients withClostridium difficileinfection in Taiwan

The medical records of 84 patients with stool cultures positive for Clostridium difficile during the period August 2007 to June 2009 were retrospectively reviewed. A case of confirmed (toxigenic)C. difficile infection (CDI) was defined by the presence of symptoms (fever, diarrhoea, abdominal discomfort or distension, ileus) and the presence of toxigenic C. difficile. Patients with compatible clinical symptoms and stool cultures positive for non-toxigenic C. difficile isolates were defined as probable (non-toxigenic) CDI cases. Of these 84 patients, 50 (59.5%) were diagnosed as confirmed CDI and 34 (40.5%) as probable CDI. Thirteen (15.5%) of the 84 patients died during their hospital stay. Usage of proton pump inhibitors was a significant independent risk factor for CDI (OR 3.21, P=0.014). Of the 50 isolates associated with confirmed CDI, seven (8.3%) carried binary toxin genes (cdtAB), and six (7.1%) had a deletion in the tcdC gene. The mortality rate in confirmed CDI patients with isolates exhibiting deletion in the tcdC gene (2/6, 33.3%), those with isolates harbouring binary toxin genes (2/7, 28.6%), and those with isolates containing mutations in gyrA (2/7, 28.6%) and gyrB (1/2, 50%) was higher than the overall mortality rate (10/50, 20%) in patients with confirmed CDI.

Characterizations of Clinical Isolates of Clostridium difficile by Toxin Genotypes and by Susceptibility to 12 Antimicrobial Agents, Including Fidaxomicin (OPT-80) and Rifaximin: a Multicenter Study in Taiwan

A total of 403 nonduplicate isolates of Clostridium difficile were collected at three major teaching hospitals representing northern, central, and southern Taiwan from January 2005 to December 2010. Of these 403 isolates, 170 (42.2%) were presumed to be nontoxigenic due to the absence of genes for toxins A or B or binary toxin. The remaining 233 (57.8%) isolates carried toxin A and B genes, and 39 (16.7%) of these also had binary toxin genes. The MIC(90) of all isolates for fidaxomicin and rifaximin was 0.5 μg/ml (range, ≤ 0.015 to 0.5 μg/ml) and >128 μg/ml (range, ≤ 0.015 to >128 μg/ml), respectively. All isolates were susceptible to metronidazole (MIC(90) of 0.5 μg/ml; range, ≤ 0.03 to 4 μg/ml). Two isolates had reduced susceptibility to vancomycin (MICs, 4 μg/ml). Only 13.6% of isolates were susceptible to clindamycin (MIC of ≤ 2 μg/ml). Nonsusceptibility to moxifloxacin (n = 81, 20.1%) was accompanied by single or multiple mutations in gyrA and gyrB genes in all but eight moxifloxacin-nonsusceptible isolates. Two previously unreported gyrB mutations might independently confer resistance (MIC, 16 μg/ml), Ser416 to Ala and Glu466 to Lys. Moxifloxacin-resistant isolates were cross-resistant to ciprofloxacin and levofloxacin, but some moxifloxacin-nonsusceptible isolates remained susceptible to gemifloxacin or nemonoxacin at 0.5 μg/ml. This study found the diversity of toxigenic and nontoxigenic strains of C. difficile in the health care setting in Taiwan. All isolates tested were susceptible to metronidazole and vancomycin. Fidaxomicin exhibited potent in vitro activity against all isolates tested, while the more than 10% of Taiwanese isolates with rifaximin MICs of ≥ 128 μg/ml raises concerns.

Clostridium difficile: 18 months surveillance at the Niguarda Hospital - Milano

Clostridium difficile is currently one of the most common cause of diarrhea in hospitalized or residents in long term care institutions patients, symptoms of infection ranging from diarrhea to pseudo-membranous colitis and toxic megacolon.The disease is due to the production of enterotoxin A, cytotoxin B or binary toxin. The emergence of hypervirulent new strains showing a tcdC regulatory gene deletion (ribotype 027) has been observed in recent years. Stool or rectal swab specimens were screened using automated real-time multiplex PCR (GeneXpert, Cellbio) to detect the presence of toxins producing (B toxin, binary toxin and tcdC gene deletion) C. difficile. All positive samples were cultured in order to isolate the causing infection strains.The binary toxin and/or tcdC deletion producing strains were genotyped using Multilocus Sequence Typing (MLST). MLST compares the intragenic sequences of seven housekeeping genes and provides a unique combination of alleles; to each combination is then assigned a Sequence Typing (ST). In the period January 2010 – June 2011, 3681 samples from 2234 patients were analyzed. Four hundred seventy-three patients (21.2%) were positive for the presence of C. difficile, 34 (7.2%) of there were also positive for binary toxin and 3 were positive for the tcdC gene deletion (suspected C. difficile NAP027).All the strains having tcdC gene deletion, showes ST3 typing by MLST method.These strains were isolated during the same period. The three patiens were epidemiologically linked: the patient A was hospitalized in the same room of patient B, while patient C was managed by the same team care of patient A.The spread ofsuch C. difficile strains is at the present very low in Italy. Our data confirm the poor prevalence of ribotype NAP027 and the usefulness of a presumptive diagnosis to implement immediately appropriate control measures.

Identification of mosquito larvicidal bacterial strains isolated from north Sinai in Egypt.

In the present study, two of the most toxic bacterial strains of Bacillus sphaericus against mosquito were identified with the most recent genetic techniques. The PCR product profiles indicated the presence of genes encoding Bin A, Bin B and Mtx1 in all analyzed strains; they are consistent with protein profiles. The preliminary bioinformatics analysis of the binary toxin genes sequence revealed that the open reading frames had high similarities when matched with nucleotides sequence in the database of other B. sphaericus strains. The biological activity of B. sphaericus strains varied according to growing medium, and cultivation time. The highest yield of viable counts, spores and larvicidal protein were attained after 5 days. Poly (P) medium achieved the highest yield of growth, sporulation, protein and larvicidal activity for all tested strains compared to the other tested media. The larvicidal protein produced by local strains (B. sphaericus EMCC 1931 and EMCC 1932) in P medium was more lethal against the 3rd instar larvae of Culex pipiens than that of reference strains (B. sphaericus 1593 and B. sphaericus 2297). The obtained results revealed that P medium was the most effective medium and will be used in future work in order to optimize large scale production of biocide by the locally isolated Bacillus sphaericus strains.

Rapid detection of vip1-type genes from Bacillus cereus and characterization of a novel vip binary toxin gene

A PCR-restriction fragment length polymorphism (PCR-RFLP) method for identifying vegetative insecticidal protein (vip) 1-type genes from Bacillus cereus was developed by designing specific primers based on the conserved regions of the genes to amplify vip1-type gene fragments. PCR products were digested with endonuclease AciI, and four known vip1-type genes were identified. Vip1Ac and vip1Aa-type genes appeared in 17 of 26 B. cereus strains. A novel vip1-type gene, vip1Ac1, was identified from B. cereus strain HL12. The vip1Ac1 and vip2Ae3 genes were co-expressed in Escherichia coli strain BL21 by vector pCOLADuet-1. The binary toxin showed activity only against Aphis gossypii (Homoptera), but not for Coleptera (Tenebrio molitor, Holotrichia oblita), Lepidoptera (Spodoptera exigua, Helicoverpa armigera, and Chilo suppressalis), Diptera (Culex quinquefasciatus). The LC(50) of this binary toxin for A. gossypii is 87.5 (34.2-145.3) ng mL(-1) . This is probably only the second report that Vip1 and Vip2 binary toxin shows toxicity against homopteran pests. The PCR-RFLP method developed could be very useful for identifying novel Vip1-Vip2-type binary toxins, and the novel binary toxins, Vip1Ac1 and Vip2Ae3, identified in this study may have applications in biological control of insects, thus avoiding potential problems of resistance.

Detection of enterotoxin A and cytotoxin B, and isolation of Clostridium difficile in piglets in Minas Gerais, Brazil

Clostridium difficile has emerged as a major cause of neonatal colitis in piglets, displacing classic bacterial pathogens. However, there is no information regarding the distribution of this microorganism in pig farms in Brazil. In the present study, the presence of toxins A/B and of C. difficile strains in stool samples from 60 diarrheic or non-diarrheic newborn piglets (one to seven days old), from 15 different farms, was studied. The presence of toxins A/B was detected by ELISA and PCR was used to identify toxin A, toxin B and binary toxin gene in each isolated strain. C. difficile A/B toxins were detected in ten samples (16.7%). Of these, seven were from diarrheic and three were from non-diarrheic piglets. C. difficile was recovered from 12 out of 60 (20%) fecal samples. Of those, three strains were non-toxigenic (A-B-) and nine were toxigenic. Of the nine toxigenic strains, four were A+B+ strains and five were A-B+ strains. The presence of binary toxin observed in the present study was much higher (50%) than in previously reported studies. All three non-toxigenic strains were isolated from otherwise healthy piglets. The results suggest the occurrence of neonatal diarrhea by C. difficile in farms in Brazil.

Detection of Clostridium difficile in animals: comparison of real-time PCR assays with the culture method

Clostridium difficile has emerged as a pathogen or commensal in food animals. There is overlap between isolates from animals, retail meats and humans, suggesting that animals may be a C. difficile reservoir. For direct detection of variant C. difficile strains in faecal samples of symptomatic and asymptomatic animals, we developed and validated a new TaqMan real-time PCR (TMrtPCR) assay targeting the tcdA, tcdB and cdtB genes. We compared it with the enrichment culture method and with two real-time PCR (rtPCR) assays, BrtPCR and PCRFast, targeting tcdB and tcdA/tcdB, respectively. All ten tested C. difficile toxinotypes, except one (XIa) with PCRFast and two (X, XIa) with BrtPCR, were detected with the test assays. A total of 340 (100 %) samples were cultured and amplified with TMrtPCR. Results correlated in 75.3 % samples. Forty (11.8 %) samples were culture positive/TMrtPCR negative, possibly because of the low numbers of bacteria in the samples or because of DNA extraction failure. Forty (11.8 %) samples were TMrtPCR positive/culture negative. Among 79 samples included in the rtPCR assays/culture comparison, 50.6 % were in complete concordance. The results showed that TMrtPCR performed better than BrtPCR and PCRFast, and 67 % of the culture-positive samples were TMrtPCR positive in comparison to 40 % of the samples positive in BrtPCR and 7 % of the samples positive in PCRFast, respectively. Another advantage of TMrtPCR over BrtPCR and PCRFast is its ability to detect a binary toxin gene. Therefore, the TMrtPCR results can provide the first information about the toxin type present in the sample. According to the results of our study, TMrtPCR could be a preferred screening method for the rapid detection of C. difficile in animal faecal samples, although an enrichment culture has to be performed for the specimens with negative or inconclusive rtPCR results.

Characterization and antimicrobial susceptibility of Clostridium difficile strains isolated from adult patients with diarrhoea hospitalized in two university hospitals in Poland, 2004–2006

This study analysed 330 Clostridium difficile strains isolated from patients with C. difficile infection who were hospitalized in two university hospitals (H1 and H2) in Warsaw, Poland, over the period 2004-2006. Strains were investigated for the presence of tcdA (A), tcdB (B) and binary toxin (CDT) genes, and antimicrobial susceptibility was determined against nine agents. Among the 330 C. difficile isolates, 150 (45.4 %) were classified as A(+)B(+)CDT(-), 18 (5.5 %) as A(+)B(+)CDT(+), 144 (43.6 %) as A(-)B(+)CDT(-) and 18 (5.5 %) as A(-)B(-)CDT(-). The predominant PCR ribotype in hospitals H1 and H2 was type 017 and accounted for 48.3 and 40.0 %, respectively. Only one PCR ribotype 027 strain was found. The rates of resistance to erythromycin and clindamycin in hospitals H1 and H2 were 53.6 and 53.6 %, and 48.6 and 47.5 %, respectively, whereas resistance rates to the newer fluoroquinolones gatifloxacin and moxifloxacin were 38.5 and 38.5 % (H1) and 38.4 and 40.1 % (H2). Erythromycin resistance was frequently associated with resistance to clindamycin and newer fluoroquinolones in strains belonging to type 017. No metronidazole- and vancomycin-resistant isolates were found, although two C. difficile isolates had elevated MIC values of metronidazole (MIC range 1.0-1.5 mg l(-1)) and 15 strains revealed elevated MIC values for vancomycin (MIC range 1.5-2.0 mg l(-1)). In conclusion, an increase in non-027 CDT-producing C. difficile strains was observed in Poland, but C. difficile PCR ribotype 017 remains a major circulating type.