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Background: The incidence of Clostridioides difficile infection (CDI) has increased over the past 2 decades and is considered an urgent threat by the Centers for Disease Control and Prevention. Hypervirulent strains such as ribotype 027, which possess genes for the additional toxin C. difficile binary toxin (CDT), are contributing to increased morbidity and mortality., 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 completed quantitative PCR (PCR) DNA extracted from patient stool to detect cdtB gene. Lastly, 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 by ELISA, were more likely to have severe disease with higher 90-day mortality. 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 testing for C. difficile toxins A and B may not reveal the entire picture when diagnosing CDI; detection of CDT-expressing strains is valuable in identifying patients at risk of more severe disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Clostridioides difficile is a dangerous human pathogen because it can grow to high numbers in the intestine, cause colitis with its potent toxins, and persist as spores. C. difficile infection (CDI) is the primary hospital-acquired infection in North America and Europe, and it now is a global disease. Even with newer laboratory tests, there still is confusion on accurately diagnosing this disease. Three guidelines from three different healthcare-affiliated societies have recently been published. Consensus consolidated recommendations from these guidelines should be recognized by healthcare professionals, who need to understand why this disease continues to be difficult to diagnose and need a clear understanding of the advantages and limitations of current tests. Hopefully, these combined efforts will lead to an improvement in the recognition of this pathogen and a reduction in the suffering and economic loss caused by CDI.

The global emergence of epidemic Clostridium difficile PCR ribotype (RT) 027 prompted enhanced surveillance of emerging strains. Recently, there have been reports of severe C. difficile infection in Australia caused by an unusual strain of C. difficile not seen previously. Identified as PCR RT251, this strain produces toxins A (TcdA) and B (TcdB), as well as binary toxin (CDT), and shares a common phylogenetic lineage with RT027. In this study, C. difficile RT251 strains were sourced from various geographical locations and potential virulence factors were evaluated and compared to that of control strains, CD630, VPI10463 and R20291 invitro. C. difficile RT251 strains were motile, germinated and sporulated efficiently, despite producing significantly less TcdA and TcdB compared to all control strains. Genomic analyses revealed three multi-locus sequence types (MLSTs 188, 231 and 365) with four to five loci variants compared to RT027 (ST1) all MLST clade 2. C. difficile RT251 strains were susceptible to metronidazole, vancomycin and moxifloxacin, a fluoroquinolone antimicrobial to which RT027 strains are often resistant. Further studies using whole-genome sequencing are required to determine additional virulence factors that may contribute to the pathogenicity of C. difficile RT251 strains., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During a Phase 2b study with SYN-004 (ribaxamase) for prevention of Clostridium difficile infection (CDI) conducted in North America and Eastern Europe, 45 C. difficile isolates from subjects with laboratory-confirmed CDI and or colonized with C. difficile were collected and characterized. Several C. difficile PCR ribotypes, including 027 and 198, were identified., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The excess from fecal samples submitted to a centralized laboratory in Roanoke, Virginia for routine C. difficile testing was used for this research study. We tested all samples, including any formed samples usually not assayed in diagnostic laboratories. Our first aim was to rank ribotypes by their frequency. Between 2007 and 2013, fluoroquinolone resistant 027 (027FQR), a multi-drug resistant ribotype, was 32% of 3118 Clostridium difficile isolates and the most common of 128 ribotypes. 027FQR was in 45% of cytotoxin positive but only 17% of cytotoxin negative fecal samples (p = 0.001) and 34% of unformed but only 21% of formed stool samples (p = 0.001), strong associations with features of symptomatic infection. Conversely, 014/020 (10% of isolates, third most common ribotype) was more often in unformed than formed stools (14% versus 9%; p = 0.002) and in cytotoxin negative than cytotoxin positive samples (11% versus 8%, p = 0.01). Fecal lactoferrin levels, an indication of intestinal inflammation, were significantly higher with 027FQR than with 014/020 infections (median 308 versus 26 ng/mL, p = 0.02). 027FQR fecal bioburdens and toxin levels were significantly higher than their 014/020 equivalents (median 10 4.1 versus 10 3.2 /g feces, p = 0.01; median TcdA 58.7 versus 1.3 ng/g feces, p = 0.04; median TcdB 43.4 versus 0.3 ng/g feces, p = 0.001). Binary toxin was present in 40% of 027FQR positive samples but none of the 014/020 or non-toxigenic C. difficile positive samples. 027FQR made no more TcdA/cell than did 014/020 (p = 0.7) but did make close to significantly more TcdB/cell (p = 0.08)., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Clostridium difficile is the most common known cause of antibiotic-associated diarrhea. Upon the disturbance of gut microbiota by antibiotics, C. difficile establishes growth and releases toxins A and B, which cause tissue damage in the host. The symptoms of C. difficile infection disease range from mild diarrhea to pseudomembranous colitis and toxic megacolon. Interestingly, 10-50 % of infants are asymptomatic carriers of C. difficile. This longitudinal study of the C. difficile colonization in an infant revealed the dynamics of C. difficile presence in gut microbiota., Methods: Fifty fecal samples, collected weekly between 5.5 and 17 months of age from a female infant who was an asymptomatic carrier of C. difficile, were analyzed by 16S rRNA gene sequencing., Results: Colonization switching between toxigenic and non-toxigenic C. difficile strains as well as more than 100,000-fold fluctuations of C. difficile counts were observed. C. difficile toxins were detected during the testing period in some infant stool samples, but the infant never had diarrhea. Although fecal microbiota was stable during breast feeding, a dramatic and permanent change of microbiota composition was observed within 5 days of the transition from human milk to cow milk. A rapid decline and eventual disappearance of C. difficile coincided with weaning at 12.5 months. An increase in the relative abundance of Bacteroides spp., Blautia spp., Parabacteroides spp., Coprococcus spp., Ruminococcus spp., and Oscillospira spp. and a decrease of Bifidobacterium spp., Lactobacillus spp., Escherichia spp., and Clostridium spp. were observed during weaning. The change in microbiome composition was accompanied by a gradual increase of fecal pH from 5.5 to 7., Conclusions: The bacterial groups that are less abundant in early infancy, and that increase in relative abundance after weaning, likely are responsible for the expulsion of C. difficile.

Clostridium difficile infection (CDI) is a gastrointestinal disease caused by C. difficile, a spore-forming bacterium that in its spore form is tolerant to standard antimicrobials. Ramoplanin is a glycolipodepsipeptide antibiotic that is active against C. difficile with MICs ranging from 0.25 to 0.50 μg/ml. The activity of ramoplanin against the spores of C. difficile has not been well characterized; such activity, however, may hold promise, since posttreatment residual intraluminal spores are likely elements of disease relapse, which can impact more than 20% of patients who are successfully treated. C. difficile spores were found to be stable in deionized water for 6 days. In vitro spore counts were consistently below the level of detection for 28 days after even brief (30-min) exposure to ramoplanin at concentrations found in feces (300 μg/ml). In contrast, suppression of spore counts was not observed for metronidazole or vancomycin at human fecal concentrations during treatment (10 μg/ml and 500 μg/ml, respectively). Removal of the C. difficile exosporium resulted in an increase in spore counts after exposure to 300 μg/ml of ramoplanin. Therefore, we propose that rather than being directly sporicidal, ramoplanin adheres to the exosporium for a prolonged period, during which time it is available to attack germinating cells. This action, in conjunction with its already established bactericidal activity against vegetative C. difficile forms, supports further evaluation of ramoplanin for the prevention of relapse after C. difficile infection in patients., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Intestinal inflammation was evaluated using faecal lactoferrin and ribotype in 196 hospitalized adults with Clostridium difficile infection to determine the impact of ribotype 027 in long-term care facilities (LTCFs). LTCF residents (n=28) had greater antibiotic use (P=0.049) and more ribotype 027 infection [odds ratio (OR): 4.87; 95% confidence interval (CI): 2.02-11.74; P<0.01], compared to those admitted from home. Patients infected with ribotype 027 strains had worse six-month mortality (OR: 1.90; 95% CI: 1.08-3.34; P=0.03) and more inflammation (95.26 vs 36.08 μg/mL; P=0.006), compared to those infected with non-027 strains. This study was not designed to determine acquisition site, but, in this population, suggests that the location from which the patient has been admitted is strongly associated with ribotype 027 and more severe C. difficile disease., (Copyright © 2014 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

We evaluated clinical and diagnostic indicators of severe C. difficile infection (CDI) and their association with poor clinical outcome. A total of 210 patients positive according to PCR (toxin B: tcdB) were included, with patients having a median age of 62 years and a Charlson co-morbidity index (CI) score of 5. Ninety-one percent (n = 191) were positive by toxigenic culture and 61% (n = 129) had stool toxin. Toxin-positive patients had significantly higher fecal lactoferrin (mean 316 μg/g versus 106 μg/g stool; p < 0.0001). Forty percent of patients (n = 85) were infected with ribotype 027 and significantly more of these patients had measurable stool toxin (79% vs. 50%; p < 0.0001). The mean fecal lactoferrin was significantly higher for toxin-positive 027 CDI compared with the 027 toxin-negative group (317 vs 60 μg/g; p = 0.0014). Ribotype 027 CDI with stool toxin showed a higher all-cause, 100-day mortality compared with non-027 with stool toxin (36 % vs 18%; p = 0.017). Logistic regression univariate analysis for odds ratio (OR) and p values revealed that age (OR = 1.1), intensive care unit treatment (OR = 2.7), CI (OR = 1.2), 027 CDI (OR = 2.1), white blood cell count (OR = 1.0), albumin level (OR = 0.1), and stool toxin-positive 027 CDI (OR = 2.5) were significantly associated with 100-day mortality (p < 0.05). In conclusion, CDI PCR-positive patients with 027 infection and stool toxin have increased lactoferrin and are at an increased risk of death.

We evaluated blood and fecal biomarkers as indicators of severity in symptomatic patients with confirmed Clostridium difficile infection (CDI). Recruitment included patients with CDI based on clinical symptoms and supporting laboratory findings. Disease severity was defined by physician's assessment and blood and fecal biomarkers were measured. Toxigenic culture done using spore enrichment and toxin B detected by tissue culture were done as confirmatory tests. Polymerase chain reaction (PCR) ribotyping was performed on each isolate. There were 98 patients recruited, with 85 (87%) confirmed cases of toxigenic CDI (21 severe, 57 moderate, and seven mild), of which 68 (80%) were also stool toxin-positive. Elevated lactoferrin (p = 0.01), increased white blood cell (WBC) count (p = 0.08), and low serum albumin (p = 0.03) were all associated with the more severe cases of CDI. Ribotype 027 infection accounted for 71% of severe cases (p < 0.01) and patients with stool toxin had significantly higher lactoferrin levels and WBC counts (p < 0.05). Our findings show that elevated fecal lactoferrin, along with increased WBC count and low serum albumin, were associated with more severe CDI. In addition, patients infected with ribotype 027 and those with stool toxin had significantly higher fecal lactoferrin and WBC counts.

We report the selection of Clostridium difficile resistant to the rifamycin class of antibiotics in a patient within 32 h of receiving rifaximin for the treatment of recurrent C. difficile diarrhea. Resistance was associated with single nucleotide substitutions within rpoB.

We evaluated Clostridium difficile prevalence rates in 2,807 clinically indicated stool specimens stratified by inpatient (IP), nursing home patient (NH), outpatient (OP), age, gender, and specimen consistency using bacterial culture, toxin detection, and polymerase chain reaction (PCR) ribotyping. Rates were determined based on the detection of toxigenic C. difficile isolates. We identified significant differences in the rates between patient populations and with age. Specimens from NH had a higher rate (46%) for toxigenic C. difficile than specimens from IP (18%) and OP (17%). There were no gender-related differences in the rates. Liquid specimens had a lower rate (15%) than partially formed and soft specimens (25%) and formed specimens (18%) for the isolation of toxigenic C. difficile. The nontoxigenic rate was lowest for NH (4%) and highest for patients<20 years of age (23%). We identified 31 different toxigenic ribotypes from a sampling of 190 isolates that showed the lowest diversity in NH. Fluoroquinolone resistance was observed in 93% of the 027 isolates, all of the 053 isolates, and in four other ribotypes. We observed different rates for toxigenic C. difficile in stratified patient populations, with the highest rate for NH, a low overall nontoxigenic rate, and fluoroquinolone resistance.

Antibodies targeting the Clostridium difficile toxin A and toxin B confer protective immunity to C. difficile associated disease in animal models and provided protection against recurrent C. difficile disease in human subjects. These antibodies are directed against the receptor binding domains (RBD) located in the carboxy-terminal portion of both toxins and inhibit binding of the toxins to their receptors. We have constructed a recombinant fusion protein containing portions of the RBD from both toxin A and toxin B and expressed it in Escherichia coli. The fusion protein induced high levels of serum antibodies to both toxins A and B capable of neutralizing toxin activity both in vitro and in vivo. In a hamster C. difficile infection model, immunization with the fusion protein reduced disease severity and conferred significant protection against a lethal dose of C. difficile spores. Our studies demonstrate the potential of the fusion protein as a vaccine that could provide protection from C. difficile disease in humans., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

gluD was highly conserved and glutamate dehydrogenase (GDH) was readily expressed in vitro by all 77 Clostridium difficile ribotypes assayed. All ribotypes, including ARL 002, ARL 027, and ARL 106, were reactive in assays that detect C. difficile GDH.

Various pathogenic clostridia produce binary protein toxins associated with enteric diseases of humans and animals. Separate binding/translocation (B) components bind to a protein receptor on the cell surface, assemble with enzymatic (A) component(s), and mediate endocytosis of the toxin complex. Ultimately there is translocation of A component(s) from acidified endosomes into the cytosol, leading to destruction of the actin cytoskeleton. Our results revealed that CD44, a multifunctional surface protein of mammalian cells, facilitates intoxication by the iota family of clostridial binary toxins. Specific antibody against CD44 inhibited cytotoxicity of the prototypical Clostridium perfringens iota toxin. Versus CD44(+) melanoma cells, those lacking CD44 bound less toxin and were dose-dependently resistant to C. perfringens iota, as well as Clostridium difficile and Clostridium spiroforme iota-like, toxins. Purified CD44 specifically interacted in vitro with iota and iota-like, but not related Clostridium botulinum C2, toxins. Furthermore, CD44 knockout mice were resistant to iota toxin lethality. Collective data reveal an important role for CD44 during intoxication by a family of clostridial binary toxins.

Clostridium difficile is a major enteropathogen of humans. It produces two main virulence factors, toxins A and B. A third, less well known toxin, C. difficile toxin (CDT), is a binary toxin composed of distinct enzymatic (CdtA) and cell binding/translocation (CdtB) proteins. We used a novel enzyme linked immunoassay (EIA) to detect CdtB protein in feces and culture fluids. Additionally, PCR was used to assay C. difficile isolates from fecal samples for the CDT locus (CdtLoc). Although the results from 80 isolates suggest no relationship between toxin concentrations in situ and in vitro, there is a good correlation between PCR detection of the cdtB gene and EIA detection of CdtB protein in vitro. Possible implications of the detection of CDT in patients are discussed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

To characterize the extent and diversity of moxifloxacin resistance among Clostridium difficile isolates recovered during a predominantly Anaerobe Reference Unit (ARU) ribotype 027-associated nosocomial outbreak of antibiotic associated diarrhea we measured the susceptibility of 34 field isolates and 6 laboratory strains of C. difficile to moxifloxacin. We ribotyped the isolates as well as assaying them by PCR for the metabolic gene, gdh, and the virulence genes, tcdA, tcdB, tcdC, cdtA and cdtB. All the laboratory isolates, including the historical ARU 027 isolate Cd196, were susceptible to moxifloxacin (or=16 microg/mL (high resistance). We sequenced the quinolone resistance determining regions of gyrA (position 71-460) and gyrB (position 1059-1448) from two susceptible laboratory strains, all five isolates with moderate resistance and two highly resistant isolates. Two highly resistant isolates (Pitt 40, ribotype ARU 027 and Pitt 33, ribotype ARU 001) had the same C245T (Thr(82)Delta Ile) mutation. No other changes were seen. Amplification with primer pairs specific for the C245T mutant gyrA and for the wild type gene respectively confirmed all 16 highly resistant ARU 027 isolates, as well as the highly resistant isolates from other ribotypes, had the C245T mutation and that the mutation was absent from all other isolates. Among the five isolates with moderate resistance we found combinations of mutations within gyrA (T128A, Val(43)Delta Asp and G349T, Ala(117)Delta Ser) and gyrB (G1276A, Arg(426)Delta Asn). The G1396A (Glu(466)Delta Lys) mutation was not associated with increased resistance.

A ciprofloxacin-resistant mutant of Clostridium perfringens, strain VPI-C, which had stable mutations in the topoisomerase genes, accumulated less norfloxacin and ethidium bromide than the wild type, strain VPI. Efflux pump inhibitors both increased the accumulation of ethidium bromide by cells of the mutant and enhanced their sensitivity to this toxic dye. Cloning a gene, which codes for a putative ABC transporter protein (NP&#95;562422) of 527 amino acids, from the mutant strain VPI-C into the wild-type strain VPI not only reduced the accumulation of ethidium bromide by the recombinant strain but also reduced its sensitivity to norfloxacin and ciprofloxacin. Efflux pump inhibitors decreased the rate at which ethidium bromide was removed from the cells of the recombinant strain. It appears that the putative ABC transporter protein (NP&#95;562422) may contribute to extrusion of drugs from C. perfringens.

Staphylococcus aureus is a facultative, Gram-positive coccus well known for its disease-causing capabilities. In particular, methicillin and vancomycin resistant strains of S. aureus (MRSA and VRSA, respectively) isolated globally represent daunting medical challenges for the 21(st) Century. This bacterium causes numerous illnesses in humans such as food poisoning, skin infections, osteomyelitis, endocarditis, pneumonia, enterocolitis, toxic shock, and autoimmune disorders. A few of the many virulence factors attributed to S. aureus include antibiotic resistance, capsule, coagulase, lipase, hyaluronidase, protein A, fibronectin-binding protein, and multiple toxins with diverse activities. One family of protein toxins is the staphylococcal enterotoxins (SEs) and related toxic shock syndrome toxin-1 (TSST-1) that act as superantigens. There are more than twenty different SEs described to date with varying amino acid sequences, common conformations, and similar biological effects. By definition, very low (picomolar) concentrations of these superantigenic toxins activate specific T-cell subsets after binding to major histocompatibility complex class II. Activated T-cells vigorously proliferate and release proinflammatory cytokines plus chemokines that can elicit fever, hypotension, and other ailments which include a potentially lethal shock. In vitro and in vivo models are available for studying the SEs and TSST-1, thus providing important tools for understanding modes of action and subsequently countering these toxins via experimental vaccines or therapeutics. This review succinctly presents the pathogenic ways of S. aureus, with a toxic twist. There will be a particular focus upon the biological and biochemical properties of, plus current neutralization strategies targeting, staphylcoccocal superantigens like the SEs and TSST-1.

Clostridium difficile is an emerging nosocomial pathogen and one of the major causes of antibiotic-associated diarrhoea. Cases of Clostridium difficile-associated disease (CDAD) are likely initiated by the ingestion of dormant C. difficile spores, which then germinate, outgrow and rapidly proliferate to cause gastrointestinal (GI) infections. To understand the initial stages of CDAD pathogenesis, we have characterized the germination of spores from a collection of C. difficile strains, including some clinical isolates obtained from a CDAD outbreak (CDAD isolates). Spores of one laboratory strain and five CDAD isolates did not germinate with amino acids, but did germinate on a nutrient-rich medium. However, bile salts had little effect on spore germination, either alone or in a nutrient-rich medium. These spores also germinated with KCl, as well as the non-nutrient germinants dodecylamine and a 1 : 1 chelate of Ca(2+) and dipicolinic acid. An unexpected finding was that spores of most of the C. difficile strains also germinated with inorganic phosphate (P(i)) with a pH optimum of 6. The in vitro germination of spores of CDAD strains with KCl and P(i), two molecules present at significant levels in the GI tract, suggests that C. difficile spores germinate in the human body by sensing P(i) in the early segments of the duodenum and KCl in the colon.

We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.

Amongst 25 extra-intestinal clinical isolates of Clostridium difficile, A(+)B(+) (72%) and A(-)B(+) (4%) toxigenic phenotypes, as well as the non-toxigenic phenotype (A(-)B(-)) (24%), were identified. The A(-)B(-) isolates did not express toxin, yet carried part of the tcdA and tcdB gene and are of a previously unreported toxinotype. Six A(+)B(+) isolates also carried binary toxin genes. Resistance to erythromycin (20%), clindamycin (48%), tetracycline (16%), moxifloxacin (16%) and imipenem (11%) occurred but with no apparent correlation to phenotype. None of the strains was resistant to vancomycin or metronidazole. Imipenem-resistance decreased by EDTA, but susceptibility to meropenem suggests the presence of an imipenem specific metalloenzyme.

Binary toxin CDT or its genes have been identified in some strains of Clostridium difficile that also produce the large clostridial toxins, toxins A and B (A+B+CDT+), including a newly recognized epidemic strain in the United States and Canada. To study the effects of binary toxin alone, we characterized 4 binary toxin CDT-positive only (A-B-CDT+) C. difficile strains. Unlike other clostridial binary toxins, binary toxin CDT required exogenous trypsin for activation. Supernatants from all A-B-CDT+ strains caused marked fluid accumulation in the rabbit ileal loop assay after concentration and trypsinization. In addition, the ileal loop response was neutralized by antisera raised against other binary toxin-producing clostridia. Challenge of clindamycin-treated hamsters with these strains resulted in colonization but not diarrhea or death. Binary toxin CDT may play an adjunctive role to toxins A and B in the pathogenesis of C. difficile-associated disease but by itself may not be sufficient to cause disease.

A chemostat model of the healthy human large bowel ecosystem was used to establish no effect levels for tetracycline, neomycin, and erythromycin. For each compound, the equivalent to four oral doses (0, 1.5, 15, and 150 mg/60 kg person/d) was studied. Concentrations of the test compounds in the chemostat medium were intended to simulate fecal levels that might be expected following consumption of food containing antibiotic residue and were based on published oral doses and fecal levels. We monitored the following parameters: short chain fatty acids, bile acids, sulfate reduction, azoreductase and nitroreductase activities, beta-glucosidase and beta-glucuronidase activities, a range of bacterial counts and, lastly, the susceptibility among sentinel bacteria to each test compound. Neomycin and erythromycin reduced bile acid metabolism. Neomycin elevated propionate levels and caused a marginal diminution in azoreductase activity. Based on our results, the no observed effect level (NOEL) of both tetracycline and erythromycin was 15 mg/60 kg person/d. The NOEL for neomycin was 1.5 mg/60 kg person/d.

Introduction: The binary toxin genes cdt and cdtB have been detected in approximately 5% of Clostridium difficile strains. Severe C. difficile disease (CDD) may be associated with strains that carry the binary toxin genes., Methods: From April 2001 through March 2002, 8 severe and 41 nonsevere cases of nosocomial CDD were studied. Severe cases of CDD were defined by the presence of >or=2 of the following criteria: (1) abdominal pain, (2) a white blood cell count of >20,000 or <1500 cells/mm(3), and (3) ileus or bowel wall thickening with ascites. Underlying disease was assessed by 2 methods: a modified Horn score and the presence of comorbid conditions. The presence of cdtA, cdtB, and the toxin A and toxin B genes was determined, and molecular subtyping was performed., Results: All strains were positive for the toxin A and B genes, and 65.3% of the strains carried the cdtA and cdtB genes. Strains that carried the binary toxin genes accounted for 87.5% of the cases of severe CDD and 61.0% of the nonsevere cases (P=.23). Severity of CDD was not associated with either severe underlying disease or comorbid conditions. The strains that caused severe CDD belonged to 4 protein profile groups and >or=3 restriction endonuclease analysis (REA) groups. All (i.e., 5 of 5) strains in REA group BI, compared with none (i.e., 0 of 7) of the strains in REA group J carried the binary toxin genes (P=.001). Strains that belonged to REA groups BK and BR also carried the binary toxin genes., Conclusions: The binary toxin genes were present in nearly two-thirds of the C. difficile strains, and they were correlated with the REA group. Severity of CDD was not closely associated with a specific clone or underlying disease, but it may be associated with the presence of the binary toxin genes. Larger studies are needed to discern whether a true association exists and whether the binary toxin alters the pathogenicity of the C. difficile strain.

We studied the in vitro effects of a range of ciprofloxacin (CI) concentrations on the human intestinal flora's colonization resistance (CR) to Salmonella kedougou NCTC 12173. Four steady state microbial communities were established in chemostats using inocula from a single pool of human feces. Three chemostats were exposed to CI (0.1, 0.43 and 5 microg/mL, respectively); one served as a no-drug control. The CR of each community was tested by three successive daily challenges of 10(8) S. kedougou, each delivered in a 1 mL bolus. There was no colonization of the no-drug chemostat. Likewise, after exposure to only 0.1 microg/mL CI there was no loss of CR and S. kedougou did not colonize. Conversely, both the 0.43 and the 5 microg/mL-exposed floras suffered a loss of CR and these chemostats were colonized. S. kedougou overgrew faster and reached higher counts in the presence of 0.43 than it did in the presence of 5 microg/mL. One possible explanation is that CI had a dose-dependent effect on both the challenge strain and CR. Thus, at higher levels, even though CR was disrupted by CI, so too was the growth of the challenge strain. Since exposure to CI elicited a dose-dependent reduction in Escherichia coli counts [Reg. Pharmacol. Toxicol. 33 (2001) 276] our new data suggest that E. coli may contribute to the CR against salmonella. We further conclude that, even at fecal levels below those reached during therapy, CI may impact the human gut flora sufficiently to facilitate colonization by S. kedougou.

Clostridium difficile causes approximately 25% of nosocomial antibiotic-associated diarrheas and most cases of pseudomembranous colitis. We evaluated C. DIFF CHEK, a new screening test that detects glutamate dehydrogenase of C. difficile. Our results showed that this test was comparable to PCR in sensitivity and specificity and outperformed bacterial culture.

To study the utility of an in vitro model system for assessing the effect of low concentrations of a fluoroquinolone (FQ) drug on the ecology of the human intestinal microflora, chemostats containing human fecal flora were exposed to 0.43, 4.3, and 43microg of ciprofloxacin (CI) per milliliter. Prior to and during drug exposure, we assayed short-chain fatty acids (SCFA), bacterial populations, and the relative levels of susceptibility of these populations to CI and trovafloxacin (TV), a newer related FQ with increased activity against anaerobes. The degree to which CI affected the chemostat ecology was measured statistically by comparing observed data with the corresponding predicted "no effect" level. No changes in total SCFA were observed; only butyrate was significantly higher at the intermediate and high-dose levels. Enterococci counts and the levels of susceptibility to CI among enterococci were also unaffected. Escherichia coli counts decreased in a dose-dependent manner. Susceptibility levels in E. coli followed no interpretable pattern. Bacteroides fragilis group (BfG) counts decreased significantly following exposure to 43 and 4.3microg/mL CI. Ciprofloxacin susceptibility among the BfG in these chemostats was not determined because the BfG counts were too low (less than 30 colonies per plate) when undiluted chemostat samples were plated. However, within 2 days of exposure to 0.43microg/mL CI, the percentage of BfG resistant to 4microg/mL CI increased to over 95%. Before exposure, all BfG were susceptible to both CI (2microg/mL) and TV (0.25microg/mL). All BfG isolated during exposure were resistant to both CI (4microg/mL) and TV (2microg/mL). Resistance selection in the BfG was unexpected as the MIC(90) of CI for B. fragilis is 8microg/mL. Since the average colon flora is about 20% B. fragilis and other bacteroides, CI may impact the human gut flora even at subtherapeutic levels., (Copyright 2001 Academic Press.)

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest that C. perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal cpe gene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpe isolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive, C. perfringens fecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpe gene, but the North American food poisoning isolates all carry a chromosomal cpe gene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpe isolates cause most CPE-associated AAD cases and chromosomal cpe isolates cause most C. perfringens type A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.

The output power from a 25-mm-diameter (volume, 0.49 L) and a 40-mm-diameter (volume, 1.9 L) copper-vapor laser (nominally 20- and 65-W devices, respectively) was approximately doubled to >50 and >100 W , respectively, by addition of small partial pressures of both H(2) and HCl to the neon buffer gas. The specific output powers of these lasers are believed to be records for any copper-vapor lasers of this size.

A rate-equation analysis has been used to investigate the feasibility of exciting new UV laser transitions in Cu II (3d(9)4p-3d(9)4s) by use of a pulsed Cu-Ne discharge. The model predicts average output powers in excess of 100 mW at 10 kHz from the combined output at 201.5 and 211.2 nm.

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms.

The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.

We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.

The composition and functions of the intestinal floras from humans and other animals is compared and contrasted. Intrinsic and extrinsic factors, such as anatomy, age, and diet, help define the nature of the flora typical to each species of animal. Stable differences, such as the ability to reduce cholesterol or not, between the flora of individuals of the same species are described. The difficulties of characterizing a flora by cultural taxonomy are discussed and alternatives are suggested. These alternatives, collectively known as microflora associated characteristics (MACs), are measures of the biochemical activity of the bacterial flora. How MACs can be used to measure the effects of subtherapeutic levels of antibiotics on a flora is described.

Ten healthy ponies from a single herd were found by repeated fecal culture to be free of Salmonella species and Clostridium cadaveris. In a preliminary study, four ponies administered a single oral dose of 10 mg/kg lincomycin did not develop idiopathic colitis when the drug was administered alone. Four other ponies were administered 10 mg/kg lincomycin by stomach tube together with 0.45 L of colonic content from a horse with idiopathic colitis induced earlier by lincomycin alone. Two of the four ponies were treated with 25 g oral zinc bacitracin premix (110 g/kg active ingredient) 24 h later. Forty-two hours after inoculation the two untreated ponies had severe signs of idiopathic colitis and were euthanized. Postmortem findings were typical of idiopathic colitis. The two treated ponies had milder illness but the more severely affected was also euthanized; the other was retreated at 42 h with bacitracin pre-mix and again 12 h later. Its illness and diarrhea resolved over the next 24 h. Clostridium cadaveris was isolated in large numbers from the cecum of the euthanized ponies and their cecal content contained mouse lethal and guinea pig dermonecrotic, but not cytotoxic, activity. Enterotoxins of Clostridium perfringens and Clostridium difficile could not be demonstrated. No toxin could be demonstrated in culture supernatants of C. cadaveris or in supernatants of cecal contents treated with ethanol prior to culturing in anaerobically incubated broth. No Salmonella spp. were isolated. A further two ponies were administered 10 mg/kg lincomycin orally with 0.45 L colonic content from a horse with idiopathic colitis, as described.(ABSTRACT TRUNCATED AT 250 WORDS)

The identity of the major surface antigens of Porphyromonas gingivalis was investigated. Outer membranes of P. gingivalis strains W83, W50, 381 and NCTC 11843 were prepared following inactivation of the trypsin-like enzyme activity. Three proteins, molecular weight 115, 55 and 40 kDa, were major components of the outer membranes of strains W83 and W50 and were also present in strains 381 and NCTC 11834. Two proteins, 55 and 47 kDa, were released from the cells during the sonication step of the outer membrane preparation procedure. Immunoblots using preparations of P. gingivalis W83 and serum from a case-control study of adult periodontitis demonstrated higher mean antibody reactivity in the case population to all the major proteins except for the 115 kDa outer membrane protein, which was recognized equally well by both populations. We conclude that the 55, 47 and 40 kDa proteins are important surface antigens of P. gingivalis. Characterization of the structure and function of these components should lead to an improved understanding of the host-parasite interactions in adult periodontitis.

Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains of iota-toxigenic strains of Clostridium spiroforme. Each strain was isolated from a separate outbreak of toxic diarrhoea of rabbits. Vancomycin and bacitracin, both agents used to treat intestinal clostridioses of humans and other animals, had a relatively high MIC (8 micrograms/ml or more). Metronidazole was uniformly active against C. spiroforme. With MIC of 8 micrograms/ml or more, both lincomycin (11 strains) and erythromycin (9 strains) were relatively inactive against C. spiroforme, conversely, penicillin G was active (MIC for 8 strains was 0.5 micrograms/ml or less). Exposure to any one of these drugs has been implicated as a predisposing factor for C. spiroforme mediated diarrhoea of rabbits. The greatest variation in MIC was seen for erythromycin (8-fold), penicillin G (8-fold) and tetracycline (16-fold).

The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.

We have reviewed the recent literature on the humoral immune responses to a variety of subgingival plaque bacterial species in patients with destructive periodontal diseases. We do not feel that the information presently available on the specific antibody responses to proposed pathogens such as Bacteroides gingivalis and Actinobacillus actinomycetemcomitans allows antibody responses to be diagnostic. All control subjects without periodontal destruction have antibodies to candidate pathogens but the generally higher levels in patients are not sufficiently elevated to be diagnostic. Nor can they be used to predict the initiation of disease or the onset of new episodes of destruction where disease had previously occurred. Successful treatment of patients may lead to lower levels of antibodies to some organisms, including possible pathogens, and thus support a given species in the aetiopathogenesis of disease. It appears that unsuccessful treatment may be accompanied by continuing high antibody levels to some organisms and further studies may enable this observation to be used to monitor therapy. There is some evidence from serological studies that each destructive episode may be induced by a different bacterial species or consortium. The start of studies using single antigens and the techniques of molecular biology will provide not only antibody-based diagnostic methods but also allow us to determine which bacterial antigens are virulence factors and thus the role of the antibody responses, whether protective or damaging, in the periodontal diseases.

Washed cells and Sarkosyl-insoluble outer membrane preparations of the black-pigmented bacteroides Porphyromonas gingivalis W50 bound hemin. The amount of hemin removed from a buffered solution by both cells and outer membranes was significantly larger if bacteria had been grown in broths supplemented with 5 mg of hemin per liter rather than none. Conversely, cells grown without supplemental hemin bound relatively little. However, all preparations bound some hemin. In addition, hemin regulated the production of significantly higher levels of trypsinlike protease by P. gingivalis W50. The nonpigmented variant, W50 BE1, showed no such responses to the levels of hemin in the growth medium.

Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.

The histological evaluation of surgical biopsies from affected tissues is a standard way of assessing pathological change and determining treatment in many diseases. In most forms of periodontal disease, however, this approach finds limited application. Here, we review what uses the histopathological approach has in the study and evaluation of the periodontal diseases. Current understanding of the changes in epithelial anatomy during pocket formation, the cellular composition and dynamics of the inflammatory infiltrate and the mechanisms of bone resorption and repair are reviewed from the perspective of the information available from microscopical investigation, including the uses and potential application of modern immunocytochemical methods to these questions. The usefulness of histological study of biopsy material is reassessed in the light of advances made in immunohistochemical techniques and their application to gingival inflammatory infiltrates and epithelia. Such techniques offer immediately valuable research opportunities with potential for diagnostic applications, noteably the recognition of phases of destructive activity and their differentiation from periods of effective host defence.