Cryptosporidiosis in patients at a large teaching hospital.

In 1984, all stool samples submitted to a microbiology laboratory (n = 2,197) from a predominantly hospital-based population were examined for Cryptosporidium oocysts after Ziehl-Neelsen staining, but only 0.5% were positive. We suggest that in low-incidence areas, individual laboratories examine stool samples for this organism only in immunosuppressed patients and those in whom symptoms persist.

The isolation of Moellerella wisconsensis from stool samples in the U.K.

Three strains of Moellerella wisconsensis were isolated from a total of 400 stool specimens screened for this organism by means of a new selective medium developed in this laboratory. This is the first report of the isolation of this organism in the U.K. The exact role of M. wisconsensis in causing diarrhoea remains to be elucidated.

Clostridium difficile isolation in neonates in a special care unit. Lack of correlation with necrotizing enterocolitis.

The stools of 78% of 45 infants in a Special Care Baby Unit yielded Clostridium difficile on culture, and in 67% of these it was possible to detect C. difficile toxin by means of a tissue culture technique. The stools of six of the seven infants with necrotizing enterocolitis were positive for C. difficile, but neither of the two most severely affected contained C. difficile toxin. The incidence of C. difficile isolation was similar in infants treated by exchange transfusion, those treated with antibiotics, those of low birth weight, and those with respiratory distress. The serum of only 2 of 28 infants and 1 of 20 mothers contained a neutralizing factor to C. difficile toxin. The present study does not support a role for C. difficile in neonatal disorders and in particular necrotizing enterocolitis. The reason for the apparent tolerance of the neonatal bowel to C. difficile toxin remains to be explained.

Incidence and origin of Clostridium difficile in neonates.

The stools of 65 of 92 (71%) infants in a special care nursery yielded Clostridium difficile on culture. Ninety percent of stools collected after 6 to 35 days in the unit were positive, and 36% of these also contained toxin. When tested in vitro, 94% of the isolates produced toxin. Of 110 swabs collected from the environment of the unit, 9% were positive for C. difficile, but the stools of 12 nurses working on the unit were negative. Thirty-five vaginal swabs collected from mothers just before delivery were negative for C. difficile on culture, but 16 of their infants had C. difficile in their stools. It was concluded that there is a high carriage rate in the stools of neonates of C. difficile acquired progressively during the course of their stay in the special care unit. Infection is mainly from environmental sources rather than maternal transmission.

Antitoxin production in antibiotic-associated colitis?

The production of antitoxin after Clostridium difficile-induced diarrhoea has not been reported previously. The stool of a patient with prolonged antibiotic-associated diarrhoea contained C. difficile toxin, and the serum neutralised the cytopathic effect of C. difficile toxin in tissue culture.

Simple method for isolation and presumptive identification of Clostridium difficile.

Clostridium difficile can be isolated from stools and presumptively identified by inoculating the stools onto CCCBA and the culture looked at under ultra violet light where the golden-yellow fluorescent colonies show up against a dark background. The colonies are then identified by testing them using the API ZYM system and the results further confirmed by testing the culture for toxin production using the tissue culture technique employing Cl. sordellii antitoxin.

Spectrum of antibiotic-associated diarrhoea.

In an attempt to find the extent to which Clostridium difficile could be implicated as the cause of antibiotic-associated diarrhoea, the stools of 53 patients who had diarrhoea after a course of antibiotics were investigated for the presence of C. difficile toxin. Ten of the patients (19%) were found to be positive, but the stools of four out of 53 patients without diarrhoea after a course of antibiotics were also found to contain C. difficile toxin (7.5%). The titre of toxin in patients both with and without diarrhoea fell within the same range (up to 10(-5)). Neither the organism nor its toxin was found in the stool of 26 patients with ulcerative colitis, eight with Crohn's disease, 49 with non-specific diarrhoea, and 27 normal controls. We conclude that, while C. difficile is responsible for a proportion of cases of antibiotic-associated diarrhoea, the concentration of toxin is not the sole factor affecting the severity of this disorder.

Bacteriocine typing of Providencia isolates.

A method of typing isolates of Providencia is described based on the sensitivities of the organisms to bacteriocines. Twelve standard bacteriocine producing strains were selected from a large number of isolates tested and from these liquid bacteriocine preparations were obtained. The activities of these preparations on over 300 isolates were determined and from this information a bacteriocine typing system has been developed.

An investigation into factors affecting proteocine production by Proteus isolates.

Proteocine production was found to the influenced by the medium used, the temperature of incubation and the length of incubation. Different amounts of proteocine were produced in different media. Production in liquid media was maximum in proteose-peptone No. 3 (Difco). This medium gave a very high titre (1:10000) of proteocine when inoculated with the producer strain, and the aerated culture was induced with mitomycin C. Therefore a method of producing a high titre proteocine was developed using proteosepeptone No. 3 (Difco) and cultural conditions of 25 degrees C for 24 hours.

Physical properties and the fine structure of proteocines.

Proteocines derived from twelve previously described bacteriocinogenic strains of Proteus mirabilis and Proteus vulgaris were investigated. All proteocine preparations were particulate, unaffected by trypsin, and destroyed by freezing and thawing or by heating at 60 degrees C for 30 minutes. Proteocine activity was removed by adsorption with the appropriate sensitive organisms. The active principles of all preparations were partially purified by precipitation with 70% (w/v NH4(SO4)2 followed by ultra-centrifugation. Column chromatography showed that proteocine activity was associated with only one of the peaks of material which absorbed strongly at 257 mu. All twelve proteocine preparations were revealed by electron microscopy as a phage-tail-like structure and each particle had a sheath, a core, and a base-plate from which spine-like fibres extend. Adsorption of these particles to the cell wall of sensitive strains did not disrupt the bacterial cell wall, but the cytoplasmic membrane and the cell contents shrank, with consequent death of the "infected" cell.

Bacteriocine typing of Proteus.

A method of typing isolates of Proteus mirabilis and Proteus vulgaris is described based on the sensitivity of the organisms to bacteriocine. Twelve standard proteocine producing strains were selected from a large number of isolates tested, and from these liquid proteocine preparations were prepared. The sensitivities of 1805 isolates to these 12 preparations were then determined and none was found to be untypable. From the results a bacteriocine typing system has been developed.

An evaluation of two methods of bacteriocine typing of organisms of the genus Proteus.

An assessment of two simple methods of typing Proteus isolates using bacteriocines is described. The methods chosen were those of Cradock-Watson and Al-Jumaili. It has been shown that the use of liquid bacteriocine preparations is more satisfactory than methods involving proteocine production in solid media. For example, no non-typable isolates were encountered and a large number of strains were demonstrated. This method is therefore proposed for use in a routine diagnostic laboratory.