- Volume 12, Issue 3, 1979

Cultures of Salmonella typhimurium(551 strains) of phage type 141 isolated in Scotland from 1965 to 1977 were examined for biotype and colicine type. Three main biotype clones were recognised: If(30 strains), 9f(507 strains) and 31bd(11 strains) with subtype variants 9bf(1 strain), 9cf(1 strain) and 31b(1 strain). The contribution made by each biotype clone to outbreaks in Scotland was analysed. The findings confirmed the distinctness of the three biotype clones within the single phage type and indicated possible origins of strains of biotypes of 9f and 31 bd.

The occurrence and nature of passive protective antibody in 100 samples of human serum was investigated in mice challenged with strains of Staphylococcus aureus capsular types A (Smith diffuse strain) and B (strain NS58D). Sixty of the sera passively protected mice against the capsular type-A strain, three against type B, and one against both types. Rabbit antisera against human IgG, IgA and IgM could remove the protective activity from a human serum of high potency, and the activity was also sensitive to 2-mercaptoethanol. Absorption with Smith surface antigen removed protective activity and reduced the concentration of IgG 7-fold, IgA 2.7 fold and of IgM 3-fold more than in a non-protective serum. Consequently, the protective activity of human serum is believed to be associated with antibodies to the S. aureus capsular antigen in the three immunoglobulin classes.

The protective effects of monospecific gonococcal antisera on 11-day chick embryos challenged with a known lethal dose of gonococci were assessed. The monospecific antisera were prepared by immunisation of rabbits with purified gonococcal antigens, and removal of trace amounts of unwanted antibodies was achieved by absorption with antigen covalently bound to cyanogen bromide-activated Sepharose beads. The antisera were standardised for IgG by solid-phase radioimmunoassay.

Antiserum raised against whole outer membrane was protective and antiserum raised against the lipopolysaccharide (LPS) was moderately protective. Outer-membrane antiserum from which the LPS component was removed by absorption was less protective than either of these sera. Investigation of the protective mechanism of anti-LPS antibodies indicated that in addition to any antitoxic effect, these anti bodies inhibited the mu1 tiplica tio n of gonococci. Antisera raised against individual outer-membrane proteins offered no protection in this test. Out of five antisera tested, antipilus serum gave the strongest protection when piliate gonococci were used as the challenge in this model; antipilus serum did not protect against challenge with non-piliate gonococci.

Infant rabbits were shown to respond to Escherichia coli heat-labile enterotoxin by a consistent increase in intestinal fluid content, which was maximal 5 h after oral dosing. Infant rabbits could be used in a simple quantitative assay for heat-labile E. coli enterotoxin based on the ratios of gut weight to remaining body weight 5 h after oral dosing. Infant rabbits remained responsive to heat-labile enterotoxin up to 14 days of age, after which their gastric pH became low enough to destroy the enterotoxin. Rabbits that had been deprived of food before being dosed had a reduced gastric pH and a reduced response to the enterotoxin.

Lincomycin and mitomycin C were found not to increase the yield of heat-labile enterotoxin from E. coli strain P307.

Of 422 clinical strains of Pseudomonas aeruginosa, 23 (5.5%) were resistant to gentamicin; 19 were also resistant to tobramycin and sisomycin, and one was resistant to to bramycin, sisomycin and amikacin. Of the gentamicin-resistant strains, 20 were also resistant to kanamycin. Sixteen strains with a high level of resistance to gentamicin (MIC > 160 µg/ml) transferred all their resistance determinants to recipient strains of P. aeruginosa and four transferred some resistance determinants to P. aeruginosa but none transferred resistance to a recipient strain of E. coli K12. These results show that gentamicin resistance in strains of P. aeruginosa isolated in Auckland is mediated by R plasmids.

A strain of Staphylococcus aureus (M7) contains a transmissible element determining production of penicillinase, and resistance to cadmium ions, neomycin, streptomycin and kanamycin (CPNS). This element was transferred either in toto or in fragments at low frequency from strain M7. The fragment (NS) possesses features typical of chromosomal genes and the fragment (CP), like (CPNS) itself, exhibits plasmid features. The element (CPNS) is transferred in mixed culture at high frequency, up to 10−3, between other strains of staphylococci. Lysogenisation of the recipient increases the frequency of transfer. The frequency of transduction of (CPNS), (CP) and (NS) from cell-free lysates corresponds well with the transfer frequency of each of these elements in mixed culture. A possible mechanism for the evolution of (CPNS) is discussed.

Strains of Neisseria gonorrhoeae from a variety of sources were examined for sensitivity to 11 partially purified R-type pyocines from Pseudomonas aeruginosa. Selective inhibition of gonococci by pyocines of Kageyama groups Rl and R5 was observed. “Matched isolates”, those from consorts or different body sites of individual patients, usually had very similar pyocine-sensitivity patterns and identical sensitivities to five antibiotics tested. This study included local isolates, strains from diverse geographic regions, and strains from disseminated gonococcal infections. It also proposed a relationship between pyocinereceptor sites in the lipopolysaccharide of Ps. aeruginosa and N. gonorrhoeae. Topics needing further evaluation are discussed.

Phase-contrast and electron microscopic, serological and biochemical evidence is advanced to show that a strain of Yersinia enterocolitica can pass through a developmental cycle interpretable as an L-cycle.

Cell-free culture filtrates and crude enterotoxin preparations from six strains of Aeromonas hydrophiza caused the accumulation of fluid in rabbit ileal loops. This activity was due to a non-dialysable, heat and acid-labile antigenic protein and was lost when culture filtrates and crude enterotoxin preparations were heated at 60°C for 20 min. or 56°C for 30 min. respectively. Maximum activity was observed at pH 8.0–10.0; there was a gradual loss at lower pH and activity was abolished in culture filtrates held at pH 3.0 and crude enterotoxin preparations held at pH 4.0. Titration of the crude enterotoxin preparations in rabbit ileal loops showed that the ED50 (equivalent to 1 unit of toxin) was contained in 25 μg of protein; a logarithmic plot of the neutralisation coefficients against antiserum concentrations showed that one unit of antitoxin was contained in 42 × 10−4 ml of the antiserum.

Inhibitors of trophozoite motility and phagocytosis were used to investigate the mechanism of Naegleria fowleri cytopathogenicity in mouse-embryo (ME)-cell cultures. Amoebae that were immobilised and agglutinated by specific antiserum exhibited no cytopathic activity, although they remained alive and were in constant contact with the ME cells. Mammalian-cell damage occurred only when the organisms recovered pseudopodium function and began to migrate over the monolayers as they overcame the inhibitory effects of the antiserum. Cytochalasin B at a concentration of 10 μg/ml, shown to prevent the engulfment of chick erythrocytes by amoebae, also inhibited the cytopathogenicity of Naegleria when incorporated in ME-cell culture medium. Despite repeated contact with active trophozoites, the ME cells showed only those morphological changes characteristically induced by cytochalasin B itself. The amoebae in turn showed signs of starvation after 3 or 4 days’ incubation, suggesting that the feeding activity of trophozoites was suppressed. Colchicine, on the other hand, inhibited neither the ingestion of erythrocytes nor the destruction of ME cells by amoebae. It was concluded that the cytopathogenicity of N. fowleri in ME-cell cultures was due to physical rather than biochemical or cytotoxic mechanisms and was associated with the phagocytic activity of trophozoites.

The destruction of secondary mouse-embryo (ME) cells by Naegleria fowleri was studied by indirect immunofluorescence with ME-cell antiserum as a specific label to trace the fate of mammalian-cell cytoplasm. The appearance of naegleria-induced cytopathic effect in the cultures coincided with the accumulation of discrete particles containing granules of ME-cell antigen within the cytoplasm of amoebae, suggesting that the organisms ingested host-cell material. In cultures containing cytochalasin B, a non-lethal inhibitor of phagocytosis by N. fowleri trophozoites failed to acquire any granular fluorescence and were not cytopathogenic. The engulfment of mammalian-cell cytoplasm by the organisms was confirmed when thin sections of naegleria-infected ME-cell cultures were examined by electron microscopy. Amoebae were seen in the process of detaching portions of cytoplasm from whole ME cells by means of distinctive ingesting pseudopodia, and fragments of mammalian-cell cytoplasm were identified within the food vacuoles of trophozoites. There was no evidence for cytotoxic disruption of ME cells before or during engulfment of these fragments. It is concluded that N. fowleri trophozoites attack and destroy cultured ME cells by a phagocytosis-like mechanism alone, without the aid of any amoeba-associated cytotoxic or cytolytic agents. The possible significance of these findings with respect to the in-vivo pathocity of N. fowleri is discussed.

A preliminary examination has been made of the adherence to human buccal cells of 3H-thymidine labelled strains of Enterobacteriaceae isolated from sputa. The radioadherence method was found to correlate well with the conventional light-microscope adherence technique. Saturation of buccal-cell binding sites with bacteria occurred when less than 10% of the buccal-cell surface was occupied. The adherence of Enterobacter aerogenes to buccal cells was impaired by the prior adherence of bacilli of either the same strain, or of a strain of Klebsiella pneumoniae.

Eighty-six clinical isolates of fluorescent pseudomonads that did not produce pyocyanin on Diagnostic Sensitivity Test Agar or Cetrimide Agar were identified on the basis of their antibiotic sensitivity, production of pigment on King’s “A” medium, growth at 42°C, production of lecithinase and hydrolysis of gelatin. The identity of the strains was confirmed in tests with the ammonium salt sugars ethanol, glucose and mannitol. These tests were adequate for distinguishing between the three important fluorescent pseudomonads.

The detection of casein hydrolysis on milk agar was assessed as a rapid method of distinguishing P. aeruginosa from the other species of fluorescent pseudomonads but proved unhelpful when compared with, or included in, a small set of tests. Most strains of P. aeruginosa and P. fluorescens hydrolysed casein.

Stools from 56 patients with gastroenteritis were cultured for Campylobacter jejuni. The five strains isolated were examined by electron microscopy. The campylobacter cells were pleomorphic and most displayed appearances similar to those of V. fetus. Morphological changes were observed in cultures subjected to prQlonged incubation.