Haemophilus influenzae invasive disease in Italy, 1997-1998.
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Adolescent↗
Biomedical subjects
M Cerquetti
Publications and source records attributed to M Cerquetti.
Molecular and genomic analysis of genes encoding surface-anchored proteins from Clostridium difficile.
The gene slpA, encoding the S-layer precursor protein in the virulent Clostridium difficile strains C253 and 79--685, was identified. The precursor protein carries a C-terminal highly conserved anchoring domain, similar to the one found in the Cwp66 adhesin (previously characterized in strain 79--685), an SLH domain, and a variable N-terminal domain mediating cell adherence. The genes encoding the S-layer precursor proteins and the Cwp66 adhesin are present in a genetic locus carrying 17 open reading frames, 11 of which encode a similar two-domain architecture, likely to include surface-anchored proteins.
Amino Acid Sequence↗
Characterization of surface layer proteins from different Clostridium difficile clinical isolates.
In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall. In the present paper six C. difficile strains isolated from cases and healthy carriers were studied. By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C. difficile strains. In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry. The S-layer proteins from the different strains were isolated and characterized. Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36-56 kDa. Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses. N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins. These data indicate that the presence of S-layer on C. difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity.
Adult↗
Characterization of non-type B Haemophilus influenzae strains isolated from patients with invasive disease. The HI Study Group.
Forty-one non-type b Haemophilus influenzae isolates from cases of invasive disease were characterized. By PCR capsular genotyping, 33 nonencapsulated strains, 4 type f isolates, and 4 b(-) strains were identified. By pulsed-field gel electrophoresis, the nonencapsulated isolates exhibited great genetic heterogenicity, whereas the type f and the b(-) strains seemed to have a clonal spread. Occurrence of the hifA gene was found by PCR in 18% of the nonencapsulated, 50% of the b(-), and all of the type f strains. Hemagglutinating fimbriae were generally expressed by nonencapsulated isolates when fimbrial gene hifA was present. Two nonencapsulated isolates not susceptible to ampicillin were detected; no strains were positive for beta-lactamase production.
Ampicillin↗
Characterization of surface layer proteins from Clostridium difficile by liquid chromatography/electrospray ionization mass spectrometry.
Surface layers (S-layers) are regularly ordered protein subunits found as the outermost cell envelope component of many bacteria. Most S-layers are composed of a single protein or glycoprotein species with a molecular weight varying between 40 and 200 kDa. Clostridium difficile is the most common cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC) in humans. Detection of the S-layer in some C. difficile strains, and preliminary characterization of two glycoproteins, P36 and P47, involved in the composition of the S-layer of one of these strains (C. difficile C253), led us to investigate the most appropriate conditions for purification and chemical characterization of these proteins. This work describes the results obtained when liquid chromatography (LC) coupled to mass spectrometry (MS) using electrospray ionization was applied to the analysis of C. difficile S-layer proteins (SLPs). In this way the molecular weights of the two SLP components, P36 and P47, were detected to be 34,258 +/- 2 and 39,545 +/- 3 Da, respectively. These data deviate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results by 1.85 and 7.5 kDa. To confirm the LC-MS results, an alternative molecular weight analysis was performed: the two S-layer proteins were isolated by semipreparative high performance liquid chromatography (HPLC), concentrated, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). The two SLP subunits were digested with protease V8, and the peptide maps were determined by LC-MS using a C18 column. Finally, preliminary results about peptide glycosylation were obtained.
Bacterial Outer Membrane Proteins↗
Immunogenicity issues in the quality control of the new acellular pertussis vaccines.
Over the last few years our laboratory has been assessing the consistency of production of different batches of acellular pertussis vaccines to be marketed in Italy. Central to this is immunogenicity assay of the lots under control compared with those of a reference vaccine with documented clinical efficacy.However, the current assays based on the assessment of antibody (Ab) response in the mouse are unrelated to mechanisms of protection in children. The absence of a clear correlation between Ab responses and protection has also been documented in recent clinical trials. On this basis, we are currently considering the possibility of adding to the established criteria of immunogenicity in mice based on Ab responses, information from studies on cell-mediated immune responses to the vaccine constituents.
Animals↗
Clostridium difficile: an update on virulence mechanisms.
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Journal Article↗
Role of Clostridium difficile in childhood diarrhea.
To investigate the etiologic role of Clostridium difficile in childhood acute diarrhea, stool specimens from 618 children with diarrhea and 135 controls without enteric symptoms were examined by cell culture assay for the presence of free toxin B. This toxin was found in 4.2% of the fecal specimens examined without finding a significant difference between cases and controls, suggesting no causal relationship between diarrhea and the presence of free C. difficile toxin B. C. difficile strains isolated from toxin B-positive specimens were characterized by cytotoxin and enterotoxin production and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of EDTA-extracted proteins. All but two isolates produced toxin B and toxin A and the remaining were negative for both toxins. Polyacrylamide gel electrophoresis analysis showed eight electrophoretic types, none of them was clearly related with the cases of diarrhea. The majority of isolates from children with diarrhea did not belong to types previously observed in adults with pseudomembranous colitis or antibiotic-associated diarrhea. This study provides additional evidence that C. difficile is not involved in the etiology of childhood diarrhea.
Bacterial Toxins↗
Detection of intestinal and extra-intestinal strains of enterotoxigenic Bacteroides fragilis by the HT-29 cytotoxicity assay.
Bacteroides fragilis strains with enterotoxic activity can be isolated from the faeces of newborn farm animals with diarrhoea and are called enterotoxigenic B. fragilis (ETBF). These strains can now be detected in an in-vitro cytotoxicity assay with HT-29 cells. In this study, 146 B. fragilis strains (95 faecal and 40 extra-intestinal isolates) and 64 Bacteroides isolates belonging to species other than B. fragilis were tested for their ability to produce enterotoxin. Sixteen strains of ETBF were identified; all belonged to the fragilis species and represented 11% of all B. fragilis examined. The prevalence was similar among extraintestinal and faecal strains, 11.5% and 10%, respectively. The production of enterotoxin in clinical isolates appeared to be associated with infections where tissue destruction was more prominent. Enterotoxigenicity was not associated with the presence of a plasmid and the plasmid profiles of ETBF strains that harboured plasmids were different. These results show that enterotoxin production by human isolates of B. fragilis is not uncommon and could represent a new virulence factor of B. fragilis.
Adult↗
Purification and characterization of an immunodominant 36 kDa antigen present on the cell surface of Clostridium difficile.
The 36 kDa antigen represents the major EDTA-extracted protein of Clostridium difficile strains belonging to electrophoretic group 2. Antibodies to this antigen are found in sera of patients with C. difficile-associated diarrhoea. The 36 kDa antigen was extracted from C. difficile C253 by EDTA and purified by gel filtration (Sephacryl S300) and ion exchange chromatography (DEAE-Trisacryl M). The molecular weight of the purified protein was 36 kDa as determined by SDS-PAGE, also in non-reducing conditions. By gel filtration, the molecular size appeared to be 72 kDa and was not modified by the presence of EDTA or SDS in the column buffer. Since IEF showed a single isoelectric form of pI 4.6, the protein could be a homodimeric molecule. Immunofluorescence demonstrated that the 36 kDa antigen was located on the surface of the C. difficile cell. Monospecific antiserum raised in rabbits reacted positively with the 36 kDa protein of most group 2 C. difficile strains isolated from antibiotic-associated diarrhoea (AAD) outbreaks in Italy and other European countries.
Antigens, Surface↗
Capsule-like structures in Clostridium difficile strains.
Fourteen strains of Clostridium difficile, previously characterized by SDS-PAGE, were examined for the presence of surface structures. None of the strains were fimbriated but, when grown in the presence of glucose, all exhibited a thin ruthenium red-positive layer. Two strains, belonging to different electrophoretic groups, were also observed after treatment with homologous and heterologous antisera. The strain belonging to the electrophoretic group 2, usually associated with the disease, showed a 30-80nm thick capsule in ultrathin sections. The strains belonging to group 5, mainly observed in isolates from healthy children, exhibited a thinner polysaccharide layer (10-20 nm). No stabilization was observed when these strains were treated with heterologous antisera. This capsule-like material did not seem to be associated with adhesive properties of C. difficile strains.
Cell Wall↗
Immunoblot analysis of serum immunoglobulin G response to surface proteins of Clostridium difficile in patients with antibiotic-associated diarrhea.
We examined by immunoblot analysis the serum immunoglobulin G antibody response to EDTA-extracted surface proteins of Clostridium difficile in 16 patients with antibiotic-associated diarrhea. For each patient, paired serum samples were tested against proteins of the infecting strain and of a collection strain (C253) known to belong to the electrophoretic group 2 pattern. Eight patients, all harboring group 2 C. difficile strains, exhibited responses to the proteins of the infecting strain; six patients showed increases in the level of antibodies between acute-phase and convalescent-phase sera. A great variability in the antigens recognized was found; however, seven patients possessed antibodies directed against an antigen of about 35 kilodaltons, corresponding to the major protein of group 2 strains. The sera of these seven patients cross-reacted also with the 35-kilodalton and other proteins of strain C253. Our data show that C. difficile proteins other than toxins can elicit an immune response in patients with C. difficile-associated disease; in this group of patients, the major surface protein of the group 2 strains was the antigen most often recognized.
Adolescent↗
[Hospital epidemic of Clostridium difficile diarrhea: demonstration of cross-infection using a typing technic].
Two hospital outbreaks of Clostridium difficile diarrhoea in two general surgery units of different hospitals are described. Moreover, the results of a study on the circulation of C. difficile in a neurosurgery unit following two cases of colitis are reported. C. difficile strains isolated from patients and environment have been typed using antimicrobial susceptibility testing and electrophoretic profiles of EDTA-extracted proteins. The majority of strains isolated in each hospital shared the same protein profile and the same pattern of antibiotic susceptibility. This provides strong evidence of cross-infection and hospital acquisition of C. difficile.
Bacterial Proteins↗
Evidence for cross-infection in an outbreak of Clostridium difficile-associated diarrhoea in a surgical unit.
Environmental studies were performed in a hospital outbreak of Clostridium difficile-associated diarrhoea. Transmission was associated with the sluice room and the storage room where medical equipment was found to be contaminated with C.difficile. Typing of isolates by antibiotic-susceptibility patterns and profiles of EDTA-extracted proteins showed the presence of an "epidemic" strain common to the majority of patients and environmental sites. Control of the outbreak was achieved by improvement of environmental hygiene and use of disposable equipment.
Adult↗
Electrophoretic characterization of Clostridium difficile strains isolated from antibiotic-associated colitis and other conditions.
Clostridium difficile has been recognized as the cause of antibiotic-associated pseudomembranous colitis and of less severe diarrheal diseases associated with the use of antimicrobial agents. However, healthy carriers of this microorganism have been found, particularly healthy neonates and small children. Various typing systems have been used to clarify the epidemiology of C. difficile. We used the electrophoretic patterns of EDTA-extracted proteins to characterize C. difficile strains from various sources. Altogether, 110 strains were studied, including 2 reference strains, and 21 different protein profiles were obtained. However, two patterns were the most common: the group 2 pattern, characterized by a major 35-kilodalton polypeptide band, and the group 5 pattern, identified by principal bands of 37 and 56 kilodaltons. The group 2 pattern was characteristic of strains isolated during hospital outbreaks and from sporadic cases of pseudomembranous colitis and antibiotic-associated diarrhea. The group 5 pattern was obtained only from isolates from healthy neonates and children. A correlation between electrophoretic characteristics and virulence can be hypothesized, namely that group 2 strains are more prone to induce diseases and cause outbreaks. It is noteworthy that strains isolated from children with diarrhea of unknown etiology, not related to antibiotic use, belong to the "virulent" group 2; strains from leukemic patients showed a variety of different patterns, and only two belong to group 2. This characterization can be used to aid studies on the virulence and clinical significance of C. difficile.
Adult↗
Synthesis and pharmacological evaluation of a new class of 2-(2-aminothiazol-4-yl)-2-hydrazonoacetamido cephalosporins.
A series of 2-(2-aminothiazol-4-yl)-2-hydrazonoacetamido cephalosporins 1a-h was prepared. Whenever possible, E and Z isomers were isolated, and their relative stabilities and their interconversions were tested. The antibacterial activity was tested against Gram-positive and Gram-negative bacteria. For compound 1c, whose Z and E forms do not interconvert rapidly, the Z form was the more active one. Among the other compounds, for which the E form is the only stable one for practical purposes, compound 1a was the most active. When compared with cefuroxime and cefotaxime, compound 1a showed slightly lower antibacterial activity but good serum level and half-life values.
Acetamides↗