scholarly journals The Cytopathic Effect of Different Toxin Concentrations From Different Clostridioides difficile Sequence Types Strains in Vero Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Zohar Hamo ◽  
Maya Azrad ◽  
Boris Fichtman ◽  
Avi Peretz
Keyword(s):  
Disease Severity ◽  
Cytopathic Effect ◽  
Vero Cells ◽  
Bacterial Strains ◽  
Host Immune Responses ◽  
Clostridioides Difficile ◽  
Cell Counts ◽  
Healthcare Associated ◽  
Sequence Types

Clostridioides difficile is one of the leading causes of healthcare-associated diarrhea, with severity ranging from mild, self-limiting disease, to life-threatening toxic megacolon. C. difficile infection (CDI) pathogenesis is mediated by the TcdA and TcdB toxins. This work aimed to draw correlations between toxin levels, bacterial strains, and disease severity in 63 CDI patients. C. difficile typing was performed by multi-locus sequence types (MLST). Toxin concentrations were measured using the TOX A/B test. In addition, cell cytotoxicity assay was performed following Vero cell exposure to stool extracts (24 h). The most prevalent sequence types (ST) were ST2, ST4, ST6, ST13, ST37, ST42, and ST104, with highest toxin levels produced by ST42 and ST104 (302.5 and 297.1 ng/ml, respectively). These strains had a stronger cytopathic effect (CPE) on Vero cells as compared to strains with lower toxin concentrations (p < 0.001), as manifested by lower cell counts and higher percentages of cell rounding and adhesion loss. Although no association was found between ST, toxin concentrations, and disease severity, a diverse in vitro effect of different STs on the viability and activity of Vero cells was observed. These findings suggest that disease severity is affected by both host immune responses and by bacterial characteristics.

scholarly journals Molecular Characterization of Pathogenicity Locus (PaLoc) and tcdC Genetic Diversity Among tcdA+B+ Clostridioides Difficile Clinical Isolates in Tehran, Iran

2020 ◽  
Author(s):  
Mansoor Kodori ◽  
Zohreh Ghalavand ◽  
Abbas Yadegar ◽  
Gita Eslami ◽  
Masoumeh Azimirad ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Molecular Characterization ◽  
Disease Severity ◽  
Genetic Variants ◽  
Rflp Analysis ◽  
Clostridioides Difficile ◽  
Pcr Rflp ◽  
The Common ◽  
Healthcare Associated

Abstract Background: Clostridioides difficile is the main cause of healthcare-associated diarrhea worldwide. It is proposed that certain C. difficile toxinotypes with distinct pathogenicity locus (PaLoc) variants are associated with disease severity and outcomes. Additionally, few studies have described the common C. difficile toxinotypes, and also little is known about the tcdC variants in Iranian isolates. We characterized the toxinotypes and the tcdC genotypes from a collection of Iranian clinical C. difficile tcdA+B+ isolates with known ribotypes (RTs).Methods: Fifty C. difficile isolates with known RTs and carrying the tcdA and tcdB toxin genes were analyzed. Toxinotyping was carried out based on a PCR-RFLP analysis of a 19.6 kb region encompassing the PaLoc. Genetic diversity of the tcdC gene was determined by the sequencing of the gene.Results: Of the 50 C. difficile isolates investigated, five distinct toxinotypes were recognized. Toxinotypes 0 (33/50, 66%) and V (11/50, 22%) were the most frequently found. C. difficile isolates of the toxinotype 0 mostly belonged to RT 001 (12/33, 36.4%), whereas toxinotype V consisted of RT 126 (9/11, 81.8%). The tcdC sequencing showed six variants (35/50, 70%); tcdC-sc3 (24%), tcdC-A (22%), tcdC-sc9 (18%), tcdC-B (2%), tcdC-sc14 (2%), and tcdC-sc15 (2%). The remaining isolates were wild-types (15/50, 30%) in the tcdC gene.Conclusions: The present study demonstrates that the majority of clinical tcdA+B+ isolates of C. difficile frequently harbor tcdC genetic variants. We also found that the RT 001/ toxinotype 0 and the RT 126/ toxinotype V are the most common types among Iranian isolates. Further studies are needed to investigate the putative association of various tcdC genotypes with CDI severity and its recurrence.

scholarly journals In vitro Insecticidal and Time-Kill Profile of Ethyl Acetate Extract of Marine Streptomyces sp. Isolated from Sundarbans, Bangladesh

2015 ◽  
Vol 17 (2) ◽  
pp. 151-156 ◽  
Author(s):  
Md Anwarul Haque ◽  
Ashish Kumar Sarker ◽  
Mohammad Sayful Islam ◽  
Md Ajijur Rahman ◽  
Md Akter Uzzaman Chouduri ◽  
...  
Keyword(s):  
Ethyl Acetate ◽  
Mangrove Forest ◽  
Bacterial Strains ◽  
Streptomyces Species ◽  
Streptomyces Sp ◽  
Cell Counts ◽  
Wide Range ◽  
Marine Streptomyces ◽  
Time Kill

The marine soil and sediment samples were collected from different locations of mangrove forest Sundarbans, Bangladesh the largest tidal halophytic mangrove forest in the world. A total of twenty nine Actinomycete strains (AIAH-1 to AIAH-29) were isolated by serial dilution method using isolation media. Among twenty nine strains, AIAH-10 was selected for further study due to its potent antibacterial activity against a wide range of pathogenic bacterial strains. On the basis of morphological, cultural and biochemical studies, the strain AIAH-10 was assigned to Streptomyces sp. The present study was designed to investigate the in vitro insecticidal and time-kill profile of ethyl acetate extracts of marine Streptomyces sp. A dose dependent mortality was observed against the larvae of Sitophilus oryzae. The larval mortality was recorded as 100% in the concentration of 80 mg/ml and higher concentrations, LC50 was found as 11.48 mg/ml. The minimum inhibitory concentration was recorded as 8 to 32 mg/ml against six different pathogenic bacterial strains. Average Log10 reductions in viable cell counts for the extracts ranged from 1.91 Log10 and 2.86 Log10 cfu/mL after 3 h interaction and 2.10 Log10 and 2.93 Log10 after 6 h interaction at MIC, 2 × MIC, 3 × MIC and 4 × MIC concentrations. This investigation reveals that the Streptomyces species isolated from Sundarbans, Bangladesh may be interesting source for the isolation of potent bioactive compounds. DOI: http://dx.doi.org/10.3329/bpj.v17i2.22332 Bangladesh Pharmaceutical Journal 17(2): 151-156, 2014

scholarly journals 2356. Increased Clinical Specificity with Ultrasensitive Detection of Clostridioides difficile Toxins: Reduction of Overdiagnosis Compared with Nucleic Acid Amplification Tests

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S811-S812 ◽  
Author(s):  
Johanna Sandlund ◽  
Joel Estis ◽  
Phoebe Katzenbach ◽  
Niamh Nolan ◽  
Kirstie Hinson ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Disease Severity ◽  
Single Molecule ◽  
Clinical Performance ◽  
Neutralization Assay ◽  
Nucleic Acid Amplification ◽  
Clostridioides Difficile ◽  
Percent Agreement ◽  
Clostridioides Difficile Infection ◽  
Healthcare Associated

Abstract Background Clostridioides difficile infection (CDI) is one of the most common healthcare-associated infections, resulting in significant morbidity, mortality, and economic burden. Diagnosis of CDI relies on the assessment of clinical presentation and laboratory tests. We have evaluated the clinical performance of ultrasensitive Single Molecule Counting technology for detection of C. difficile toxins A and B. Methods Stool specimens from 298 patients with suspected CDI were tested with nucleic acid amplification test (NAAT; BD MAX™ Cdiff assay or Xpert® C. difficile assay) and Singulex Clarity® C. difficile toxins A/B assay. Specimens with discordant results were tested with cell cytotoxicity neutralization assay (CCNA), and results were correlated with disease severity and outcome. Results There were 64 NAAT-positive and 234 NAAT-negative samples. Of the 32 NAAT+/Clarity− and 4 NAAT-/Clarity+ samples, there were 26 CCNA− and 4 CCNA- samples, respectively. CDI relapse or overall death was more common in NAAT+/toxin+ patients than in NAAT+/toxin− and NAAT−/toxin− patients, and NAAT+/toxin+ patients were 3.7 times more likely to experience relapse or death (Figure 1). The clinical specificity of Clarity and NAAT was 97.4% and 89.0%, respectively, and overdiagnosis was over three times more common in NAAT+/toxin− than in NAAT+/toxin+ patients (Figure 2). Negative percent agreement between NAAT and Clarity was 98.3%, and positive percent agreement increased from 50.0% to effective 84.2% and 94.1% after CCNA testing and clinical assessment. Conclusion The Clarity assay was superior to NAATs in diagnosis of CDI, by reducing overdiagnosis and thereby increasing clinical specificity, and presence of toxins was associated with disease severity and outcome. Disclosures All authors: No reported disclosures.

Composts containing fluorescent pseudomonads suppress fusarium root and stem rot development on greenhouse cucumber

2010 ◽  
Vol 56 (11) ◽  
pp. 896-905 ◽  
Author(s):  
Geoffrey G. Bradley ◽  
Zamir K. Punja
Keyword(s):  
Disease Severity ◽  
Total Population ◽  
Fusarium Species ◽  
Severity Index ◽  
Stem Rot ◽  
Bacterial Strains ◽  
Growth Room ◽  
Greenhouse Cucumber ◽  
Layer Chromatography

Three composts (Ball, dairy, and greenhouse) were tested for the ability to suppress the development of Fusarium root and stem rot (caused by Fusarium oxysporum f. sp. radicis-cucumerinum) on greenhouse cucumber. Dairy and greenhouse composts significantly reduced disease severity (P = 0.05), while Ball compost had no effect. Assessment of total culturable microbes in the composts showed a positive relationship between disease suppressive ability and total population levels of pseudomonads. In vitro antagonism assays between compost-isolated bacterial strains and the pathogen showed that strains of Pseudomonas aeruginosa exhibited the greatest antagonism. In growth room trials, strains of P. aeruginosa and nonantagonistic Pseudomonas maculicola , plus 2 biocontrol strains of Pseudomonas fluorescens , were tested for their ability to reduce (i) survival of F. oxysporum, (ii) colonization of plants by the pathogen, and (iii) disease severity. Cucumber seedlings grown in compost receiving P. aeruginosa and P. fluorescens had reduced disease severity index scores after 8 weeks compared with control plants without bacteria. Internal stem colonization by F. oxysporum was significantly reduced by P. aeruginosa. The bacteria colonized plant roots at 1.9 × 106 ± 0.73 × 106CFU·(g root tissue)–1and survival was >107 CFU·(g compost)–1after 6 weeks. The locus for 2,4-diacetylphloroglucinol production was detected by Southern blot analysis and confirmed by PCR. The production of the antibiotic 2,4-diacetylphloroglucinol in liquid culture by P. aeruginosa was confirmed by thin layer chromatography. These results demonstrate that composts containing antibiotic-producing P. aeruginosa have the potential to suppress diseases caused by Fusarium species.

scholarly journals Persistence of SARS-CoV-2 infection on personal protective equipment (PPE)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Elizabeth Córdoba-Lanús ◽  
Omar García-Pérez ◽  
Sara Cazorla-Rivero ◽  
Francisco Rodríguez-Esparragón ◽  
José-Enrique Piñero ◽  
...  
Keyword(s):  
Personal Protective Equipment ◽  
Post Infection ◽  
Cytopathic Effect ◽  
Clinical Sample ◽  
Rna Stability ◽  
Vero Cells ◽  
In Vitro Assays ◽  
Protective Equipment ◽  
Viral Loads

Abstract Background SARS-CoV-2 stability and infection persistence has been studied on different surfaces, but scarce data exist related to personal protective equipment (PPE), moreover using realist viral loads for infection. Due to the importance for adequate PPE management to avoid risk of virus infection, RNA stability was evaluated on PPE. Methods Persistence of SARS-CoV-2 infection and detection of genomic RNA in PPE (gowns and face masks) were determined by in-vitro assays and RT-qPCR, respectively. Samples were infected with a clinical sample positive for SARS-CoV-2 (Clin-Inf), and with a heat-inactivated SARS-CoV-2 strain sample (Str-Inf) as a control. Results PPE samples infected with Clin-Inf were positive for the 3 viral genes on gowns up to 5 days post-infection, whereas these overall genes were detected up to 30 days in the case of face masks. However, gowns and FFP2 masks samples contaminated with Clin-Inf showed a cytopathic effect over VERO cells up to 5–7 days post-infection. Conclusions SARS-CoV-2 RNA was detected on different PPE materials for 5 to 30 days, but PPE contaminated with the virus was infectious up to 5–7 days. These findings demonstrate the need to improve PPE management and to formulate strategies to introduce viricidal compounds in PPE fabrics.

scholarly journals Binary toxin expression by Clostridioides difficile is associated with worse disease

2021 ◽  
Author(s):  
Mary K Young ◽  
Jhansi L Leslie ◽  
Gregory R Madden ◽  
David M Lyerly ◽  
Robert J Carman ◽  
...  
Keyword(s):  
Clinical Outcomes ◽  
Disease Severity ◽  
Severe Disease ◽  
Patient Treatment ◽  
Binary Toxin ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rrna Gene ◽  
Clostridioides Difficile ◽  
Cell Counts ◽  
Significant Difference

Background. The incidence of Clostridioides difficile infection (CDI) has increased over the past two decades and is considered an urgent threat by the Centers for Disease Control. Hypervirulent strains such as ribotype 027, that possess genes for an additional toxin C. difficile binary toxin (CDT) are contributing to increased morbidity and mortality. In the mouse model, CDT activates Toll-like receptor 2 resulting in suppression of a protective type 2 innate immune response mediated by eosinophils. Methods. We retrospectively tested stool from 215 CDI patients for CDT by enzyme-linked immunosorbent assay (ELISA). Stratifying patients by CDT status, we assessed if disease severity and clinical outcomes correlated with CDT positivity. Additionally, we performed 16 S rRNA gene sequencing to examine if CDT positive samples had an altered fecal microbiota. Results. We found that patients with CdtB, the pore forming component of CDT, detected in their stool were more likely to have severe disease and had higher 90-day mortality compared to CDT negative patients. CDT positive patients also had higher C. difficile bacterial burden and white blood cell counts. There was no significant difference in gut microbiome diversity between CDT positive and negative patients. Conclusions. Patients with fecal samples that were positive for CDT had increased disease severity and worse clinical outcomes. Utilization of PCR and C. difficile Toxins A and B testing may not reveal the entire picture when diagnosing CDI and the detection of CDT-expressing strains may be valuable during patient treatment.

scholarly journals Effect of 1-(phenyl)-N’-(4-methoxybenzylidene)-9H-pyrido[3,4-b] indole-3-carbohydrazide on in vitro poliovirus replication

2015 ◽  
Vol 65 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Patricia Regina Santos ◽  
Anelise S. Nazari Formagio ◽  
Benedito Prado Dias Filho ◽  
Celso Vataru Nakamura ◽  
Maria Helena Sarragiotto ◽  
...  
Keyword(s):  
Host Cell ◽  
Mode Of Action ◽  
Cytopathic Effect ◽  
Vero Cells ◽  
Replication Cycle ◽  
Clinical Use ◽  
Future Studies ◽  
Post Exposure

Abstract The effect of the alkaloid 1-(phenyl)-N’-(4-methoxybenzylidene)- 9H-pyrido[3,4-b]indole-3-carbohydrazide (PMC) on the poliovirus (PV) replication cycle in Vero cells was assayed by inhibition of the cytopathic effect (CPE) and inhibition of plaque forming units (PFU). Both methodologies suggested that the mode of action was avoidance of infection progression in the host cell. The compound was able to prevent CPE and PFU formation when the cells were pretreated with PMC for 24 h prior to PV infection. In addition, when the alkaloid was continuously maintained in the infected cultures, the spread of PV to adjacent cells was impaired. The pre-exposure and post-exposure prophylactic applications are possible situations in which an anti-PV drug might be used. Future studies are needed to elucidate the PMC mode of action and verify the feasibility of PMC use in vivo. No antipicornavirus agent is currently approved for clinical use

279 IN VITRO DEVELOPMENT IN THREE CULTURE SYSTEMS OF BOVINE OOCYTES FERTILIZED WITH SEX-SORTED SPERM

2011 ◽  
Vol 23 (1) ◽  
pp. 237
Author(s):  
B. Trigal ◽  
E. Gomez ◽  
M. Muñoz ◽  
J.N. Caamaño ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Culture System ◽  
Final Analysis ◽  
Vero Cells ◽  
Bovine Embryos ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Culture Systems ◽  
Cell Counts ◽  
Vitro Development

Following in vitro fertilization and culture, the timing of both embryonic cleavage and appearance of blastocysts has been reported to be altered when using sorted spermatozoa (Lu et al. 1999 Theriogenology 52, 1393–1405). To optimize an in vitro culture system for bovine embryos produced with sexed sperm, sorted and unsorted (control) semen samples of 5 bulls (semen samples provided by XY Inc., Fort Collins, CO, USA) were separated using a Percoll gradient followed by a washing step with Fert-TALP medium (Trigal et al. 2010 Reprod. Domest. Anim. 45, 83). The obtained pellet was used for fertilizing in vitro matured cumulus–oocyte complexes from slaughterhouse ovaries. Zygotes were cultured in 1) Vero cells monolayer + B2 + 10% FCS; 2) SOFaaci + 10% FCS; or 3) SOFaaci + 6 g L–1 BSA. A total of 1980 oocytes were in vitro matured and fertilized. Development was recorded on Days 3 and Days 6 to 8, and analysed by ANOVA and Duncan test. Bull, replicate, and culture effects were nonsignificant, and consequently were not considered in the final analysis. Data are percentages of development related to the zygotes cultured, and are expressed as LSM ± SE (see Table 1). The use of sexed sperm significantly reduced development rates compared with unsorted sperm. Thus, sorted sperm showed reduced 5- to 8-cell, Day-6 morulae, and Day-8 blastocyst rates over unsorted sperm within the 3 culture systems analysed. Culture in SOF + BSA was the least efficient embryo culture system in terms of Day-7 blastocyst rates, both with unsorted and sorted sperm. Day-7 blastocyst rates produced with unsorted sperm after culture in Vero cells or SOF + FCS were significantly higher than those obtained after culture in SOF + BSA. Day-7 blastocyst rates obtained with sorted sperm and cultured in Vero cells or SOF + FCS were comparable to those obtained with unsorted sperm in SOF + BSA. No differences were detected between male- and female-sorted sperm (data not shown). Sorted sperm is an effective tool for producing sex-known embryos. Quality assessment (differential cell counts and cryosurvival) of embryos produced with sorted sperm under different conditions are being undertaken. Table 1.In vitro development of bovine embryos fertilized with either untreated or sorted sperm and cultured in Vero cells, SOF + FCS, or SOF + BSA Supported by INIA RTA2008.#0082. M. Muñoz, B. Trigal and D. Martin are sponsored by RYC08-03454, Cajastur and PTA2007-#0268-I. We acknowledge Sexing Technologies for collaboration.

scholarly journals Natural Clostridioides difficile Toxin Immunization in Colonized Infants

2019 ◽  
Vol 70 (10) ◽  
pp. 2095-2102 ◽  
Author(s):  
Larry K Kociolek ◽  
Robyn O Espinosa ◽  
Dale N Gerding ◽  
Alan R Hauser ◽  
Egon A Ozer ◽  
...  
Keyword(s):  
Cord Blood ◽  
Neutralizing Antibody ◽  
Toxin B ◽  
Humoral Immune ◽  
Clostridioides Difficile ◽  
Healthy Infants ◽  
Unrelated Cord Blood ◽  
Duration Of Protection ◽  
Sequence Types

Abstract Background Clostridioides (Clostridium) difficile colonization is common among infants. Serological sequelae of infant C. difficile colonization are poorly understood. Methods In this prospective cohort study of healthy infants, stools serially collected between ages 1-2 and 9-12 months were tested for non-toxigenic and toxigenic C. difficile (TCD). Cultured isolates underwent whole-genome sequencing. Serum collected at 9–12 months underwent measurement of IgA, IgG, and IgM against TCD toxins A and B and neutralizing antibody (NAb) titers against toxin B. For comparison, antitoxin IgG and NAb were measured in cord blood from 50 mothers unrelated to study infants. Results Among 32 infants, 16 (50%) were colonized with TCD; 12 were first colonized >1 month before serology measurements. A variety of sequence types were identified, and there was evidence of putative in-home (enrolled siblings) and outpatient clinic transmission. Infants first colonized with TCD >1 month prior had significantly greater serum antitoxin IgA and IgG against toxins A (P = .02 for both) and B (P = .009 and .008, respectively) compared with non–TCD-colonized infants, and greater IgG compared with unrelated cord blood (P = .005). Five of 12 (42%) colonized infants had detectable NAb titers compared with zero non–TCD-colonized infants (P = .02). Breastfeeding was not associated with differences in serological measurements. Conclusions TCD colonization is associated with a humoral immune response against toxins A and B, with evidence of toxin B neutralization in vitro. The extent and duration of protection against CDI later in life afforded by natural C. difficile immunization events require further investigation.

scholarly journals Virulence parameters in the characterization of strains of Entamoeba histolytica

1997 ◽  
Vol 39 (2) ◽  
pp. 65-70 ◽  
Author(s):  
Maria A GOMES ◽  
Maria N. MELO ◽  
Gil P.M. PENA ◽  
Edward F. SILVA
Keyword(s):  
Entamoeba Histolytica ◽  
Cytopathic Effect ◽  
Axenic Culture ◽  
Vero Cells ◽  
In Vitro Assays ◽  
Biological Assays ◽  
Restriction Fragment Pattern

Differences in virulence of strains of Entamoeba histolytica have long been detected by various experimental assays, both in vivo and in vitro. Discrepancies in the strains characterization have been arisen when different biological assays are compared. In order to evaluate different parameters of virulence in the strains characterization, five strains of E. histolytica, kept under axenic culture, were characterized in respect to their, capability to induce hamster liver abscess, erythrophagocytosis rate and cytopathic effect upon VERO cells. It was found significant correlation between in vitro biological assays, but not between in vivo and in vitro assays. Good correlation was found between cytopathic effect and the mean number of uptaken erythrocytes, but not with percentage of phagocytic amoebae, showing that great variability can be observed in the same assay, according to the variable chosen. It was not possible to correlate isoenzyme and restriction fragment pattern with virulence indexes since all studied strains presented pathogenic patterns. The discordant results observed in different virulence assays suggests that virulence itself may not the directly assessed. What is in fact assessed are different biological characteristics or functions of the parasite more than virulence itself. These characteristics or functions may be related or not with pathogenic mechanisms occurring in the development of invasive amoebic disease

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