A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates.

Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10,463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortened tcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability in C. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile.

We have used six independent polymerase chain reactions (A1-A3 and B1-B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5' third of tcdB (B1 fragment) and/or the 3' third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.

Influence of ceftriaxone on emergence of Clostridium difficile.

The influence of long-term ceftriaxone administration on the emergence of Clostridium difficile was studied with 80 patients receiving ceftriaxone for 14 days. In five patients (6.3%) C. difficile was cultured. The appearance of gastrointestinal disturbances during treatment with ceftriaxone was common, but the rate of emergence of C. difficile in immunocompetent patients was not high.

[Variation in phage typing and pyocin typing of Pseudomonas aeruginosa]. / Varijabilnost fagotipizacije i piocinske tipizacije Pseudomonas aeruginosa.

The most frequently used microbiological methods in detecting hospital infection reservoirs caused by bacterial species Pseudomonas aeruginosa (P. a.) are serotyping, phagotyping and pyocin typing. Isolated were 86 strains of P. a. from various material of 38 patients hospitalized in University Clinical Centre in Ljubljana during 1986-7. Efforts have been made to find out the variability of results obtained by phago- and pyocin typing of successively isolated P.a. strains from the same patient. Changes found in these samples have been significant.

Chicken-to-Human Infection with Campylobacter jejuni and Campylobacter coli : Biotype and Serotype Correlation.

Isolates of Campylobacter jejuni and Campylobacter coli isolated from two flocks of parent hens and their progeny which were followed from hatch to slaughter in 10 different farms within a 6-month period in the area of Ljubljana, Yugoslavia were bio- and serotyped. They were compared to those isolated from diarrheic patients within the same period of time. C. jejuni biotype I of Lior's biotyping scheme was found most predominant. Using 25 unabsorbed antisera raised against live C. jejunicoli cultures, 62.2% and 44.8% of the isolates from patients and chickens, respectively, could be serotyped. Penner sergroups (PG) 1, 2, 5, 7, 9, and 22 were found common to both patients and chickens. PG 2 was the most common isolate. PG 8, which was the second most frequently isolated serogroup from patients was not isolated from chickens. No Campylobacters were isolated from 71 farm family members.

Yoghurt: an unlikely source of Campylobacter jejuni/coli.

Knowledge of the relative insensitivity of Campylobacter jejuni to moderately acid environments prompted us to study its survival in different batches of yoghurt of pH range 4.2-5.3 and the role of organic or inorganic acid in the die-off of this pathogen. None of the 11 strains of C. jejuni or C. coli survived more than 25 min in yoghurt. Suspecting that this rapid die-off cannot be accounted for by the pH of the yoghurt we compared the survival rates of C. jejuni in milk, whose pH had been adjusted by lactic, propionic and hydrochloric acid respectively, with that of yoghurt. Even for an inoculum of 10(5)-10(8) cfu/ml propionic acid was bactericidal in minutes. Lactic acid reduced the bacterial populations by 3-5 logs in 30 min. Strong inorganic acid HCl, by contrast, had little or no effect on the populations. Although lactic acid is quite bactericidal for C. jejuni, it is apparently not the only factor to which the prompt elimination of this pathogen from yoghurt could be attributed.

Some biological characteristics of Escherichia coli strains isolated from acute diarrhoeal diseases of infants.

The aim of this study was to evaluate the methods of biological characterization of Escherichia coli strains, in order to use them as screening tests in clinical microbiological laboratories. In two thirds of the 45 E. coli, isolated from acute intestinal infection of infants, different factors of virulence were found. The strains showed prevalently an enterotoxigenic character (66,6%), which was in correlation with the increased permeability (62,2%). Adhesive properties were present in 20% of E. coli independent of their enterotoxigenicity; only 11,1% of the strains had penetrating abilities. The rabbit skin model seems to be accurate and quick for ETEC screening purposes, as is the guinea-pig eye for the EIEC. The mouse intestinal loop has been found sensitive but time-consuming. The infant mouse model is simple and quick, but in this study it has not given optimal results. The Vero cell culture system is simple and easy to perform, but at least for some strains the preparations should be concentrated. The investigation of adhesiveness by haemagglutination is possible to conduct in every small clinical laboratory. Our opinion is, however, that after the isolation of E. coli from different pathological materials, the determination of the antigenic structure of the strains is necessary and later on the additional biological characterization should be performed.