- Volume 2, Issue 1, 1969

By testing some of the many methods that have been described for the identification of Ps. aeruginosa with a series of typical strains an identification scheme was evolved and subsequently applied in an epidemiological investigation of Ps. aeruginosa respiratory tract infections. Its use led to the rapid identification of typical strains, but even atypical strains were usually identified within 48 hr of their isolation. The scheme has been evolved with the needs of the routine medical diagnostic laboratory in mind.

Further experience of the typing of strains of Pseudomonas pyocyanea (Ps. aeruginosa) by their production of pyocines has confirmed the value of pyocine typing for epidemiological studies.

The introduction of five new indicator strains has made it possible to distinguish eight subtypes of the commonly occurring pyocine type 1. The subtyping of the type-1 strains shows the same dependence on use of the correct temperature and duration of incubation of the producer strain as does the main typing procedure.

In studies of the reliability of the typing and subtyping methods for epidemiological purposes it was noted that not all strains of Ps. pyocyanea isolated from the same site in the same patient were of the same pyocine type or subtype. When several colonies of Ps. pyocyanea from individual diagnostic plates were typed it was found that more than one pyocine type or subtype was more frequently present in the same lesion in patients in hospital than in patients being treated at home. This finding suggests that the occurrence of more than one pyocine type or subtype of Ps. pyocyanea in a patient is generally due to his having been separately infected with strains of more than one type, rather than to instability of pyocine production in a single infecting strain.

In preliminary studies in a hospital in Manchester in which tissue culture was used for cultivation, Hartmannella castellanii was isolated from the air on two occasions and from surface samples on one. Water agar plates with a lawn of Klebsiella aerogenes were exposed in a slit sampler, or as settle plates, in and outside a laboratory in London and the following amoebae were isolated: 12 strains of H. castellanii, 7 of H. leptocnemusjagricola, 5 of H. glebae, 1 of H. agricola, 3 of Naegleria sp. and 1 of Schizopyrenus sp. In outdoor air an average of one amoebic cyst of any species was found in 322 cu. ft (9-1 m3) and one cyst of H. castellanii in 644 cu. ft (18·2 m3). All the isolates of H. castellanii damaged HeLa cells. Four that grew poorly or not at all at 37°C showed a marked cytopathic effect at 33°C. An identification key is given.

The toxins and other active products of Clostridium sordellii hitherto described include a lethal, oedema-producing toxin, an oxygen-labile haemolysin, a fibrinolysin, a collagenase and a non-toxic phospholipase C.

Using cultures of a strain of Cl. sordellii that did not produce phospholipase C grown in a peptone-glycerophosphate medium, we have re-examined the pathogenesis of Cl. sordellii gas gangrene in relation to the toxins produced by this organism. Toxic extracts were prepared from washed cells by sonic disintegration or by suspension in saline.

We have obtained evidence that oedema and internal haemorrhages produced by this organism are due to two independent toxins, both of which are, however, dermonecrotising and haemorrhagic in the skin of rats and guinea-pigs. The two skin reactions are distinguishable by their appearances. The toxin responsible for the internal haemorrhages is extractable from sporulating cells and the evidence suggests that it is protein in nature. It is neutralised by Cl. sordellii antitoxin, but not by normal horse serum. This haemorrhagic toxin produces characteristic localised brownish haemorrhages in the skin, which can be used to assay the toxin. Whilst petechial haemorrhages are produced locally after intramuscular injection, intravenous or intraperitoneal injection produces haemorrhages in the omentum and mesentery and in contiguous viscera. The adipose tissue in these sites often shows fat necrosis.

The lesions in rat or guinea-pig skin produced by the venom of the snake Agkistrodon piscivorus and by lysolecithin are very similar to the lesions produced by the haemorrhagic toxin. Though it is possible that this toxin produces its effects indirectly through the mediation of toxic secondary products such as lysophosphatides, attempts to detect such products in toxin-tissue mixtures have failed.

The requirements for X and V factors and for CO2, and the biochemical reactions of the Khairat and Hunka strains of Haemophilus aphrophilus were re-examined. The four Khairat strains were found to be dependent on X factor and CO2 although they produced a relatively high proportion of X-independent variants. Gas was not produced during fermentation of carbohydrates by the Khairat strains in tests made by two methods. The presence or absence of 0°6 per cent. NaCl in Yeastrel agar made no difference to the growth requirements of the four strains in cultures grown in air or in cultures grown in air with added CO2, although better growth was obtained on NaCl-containing media.

Toshach’s Hunka strain showed no dependence upon either X factor or V factor and in our view does not belong to the genus Haemophilus.

Our findings suggest that retention of the taxon Haemophilus aphrophilus is justified, and we designate the Khairat strain NCTC no. 5906 as the holotype.

The cytopathic effects of standard inocula (30,000 blastospores per ml) of Candida albicans, C. tropicalis, C. stellatoidea, C. krusei, C. pseudotropicalis, C. parapsilosis and C. guilliermondii (isolated from man) on murine renal epithelial cells in culture were compared with those of C. cacaoi, C. diddensii, C. kefyri, C. blankii, C. ingens and C. shehatae (isolated from non-human sources). The nature of the changes in the mammalian cells, and the extent of their destruction, were shown to be correlated with the growth rate and with the proportion of mycelium in the growing fungi. The presence of mammalian cells induced filamentous change in some species.

These experiments demonstrate the greater importance of the M phase in the progression of Candida lesions, because of the more widespread involvement of cells by organisms with rapid growth rates, but they do not demonstrate a qualitative difference in the cytopathic effects of the M and Y phases. They suggest that in vivo the Y phase may initiate infection, as it does experimentally, and that the M phase is associated with the more rapidly growing species and with extension of the lesion. The filamentous phase may be induced by exposure of the fungi to cellular exudates or transudates.

There is good evidence for the existence of two groups of bacteria that differ from Haemophilus suis and H. gallinarum only in not requiring the supply of X factor for growth. It is proposed that these groups should be established as new species named, respectively, Haemophilus parasuis and H. paragallinarum.