David R. Telford

Lethal challenge of gnotobiotic weanling rats with bacterial isolates from cases of sudden infant death syndrome (SIDS).

An attempt was made to produce an animal model of sudden infant death syndrome (SIDS). The experimental animals (germ free weanling rats) were exposed to nasopharyngeal isolates from cases of SIDS to test the hypothesis that common bacteria may have an aetiological role in the disease. Negative results were obtained when the strains were tested in isolation, but certain combinations of organisms (specifically some Staphylococcus aureus and Escherichia coli) killed the animals rapidly (less than 18 hours) without prolonged terminal illness. Post mortem histological findings were consistent with those of SIDS. The lethal toxigenic potential of nasopharyngeal bacteria, which are regarded as harmless in adults, should be reconsidered in respect of the aetiology of SIDS.

Lethal synergy between toxins of staphylococci and enterobacteria: implications for sudden infant death syndrome.

AIM--To test the hypothesis that lethal synergy occurs between toxin preparations of nasopharyngeal staphylococci and enterobacteria from sudden infant death syndrome (SIDS) victims and matched healthy infants. METHODS--SIDS and matched healthy babies were studied if both staphylococcal and enterobacterial strains were isolated from the nasopharynx. The lethality of toxin preparations from each bacterial isolate (separately and combined) was assessed over a range of dilutions using the chick embryo assay system. RESULTS--Staphylococci and enterobacteria were isolated together from the nasopharynx of seven SIDS babies but from only one normal healthy infant. Enterobacterial toxins were lethal at high dilutions. Staphylococcal toxins were less toxic. Simultaneous testing in the chick assay of staphylococcal and enterobacterial toxins, from each baby, at non-lethal concentrations enhanced lethality levels by 177 to 1011% compared with lethality expected by an additive effect alone. CONCLUSIONS--Synergy occurs between the toxins of nasopharyngeal staphylococci and enterobacteria. This combination of strains is more likely to occur in the nasopharynx of SIDS victims than that of healthy infants.

Lethal synergistic action of toxins of bacteria isolated from sudden infant death syndrome.

AIM: To test the hypothesis that lethal toxins of bacteria associated with sudden infant death syndrome (SIDS) can act synergistically. METHODS: Bacteria occurring together in the nasopharynx of cases of cot death were studied. The lethal toxicity of crude toxin preparations was determined over a range of dilutions by injections into the chorioallantoic vein of the chick embryo. Toxin preparations of low lethality for the chick embryo SIDS model were then tested in combination. RESULTS: Staphylococcus aureus toxin preparations showed low lethality when tested alone, even at low dilution. At 1 in 100 dilution S aureus toxin was lethal to one out of 15 chick embryos. Escherichia coli toxin preparations showed high lethality except on high dilution (1 in 80) when lethality fell to two out of 15 of chick embryos. When the same toxin preparations were tested simultaneously in combination, lethality rose to 14 out of 15. Similar findings were observed over a range of toxin dilutions. This finding was highly significant (p = 0.0012). CONCLUSIONS: That synergy between toxins can enhance the lethality of toxins elaborated by bacteria associated with SIDS.

Bacterial toxins: a possible cause of cot death.

AIM: To test the hypothesis that sudden infant death syndrome (SIDS) may be caused by toxins of commonly occurring bacteria in infants lacking developed immunity. METHODS: Nasopharyngeal microbial isolates from 22 pairs of SIDS cases and healthy infants matched for age (by month), sex, and sampling time (by month) were compared for lethal toxigenicity. Crude toxin preparations were made from isolates cultured on dialysis membrane overlaid on agar, and these preparations were then tested for lethality by intravenous injection into 11 day old chick embryos. RESULTS: Fifteen (68%) of the SIDS cases were each found to have at least one lethally toxigenic organism in their nasopharyngeal flora; only eight (36%) of the flora of normal infants included a lethally toxigenic species. CONCLUSION: Infants who have died of SIDS have a significantly higher (p less than 0.05) probability than matched healthy infants of having a lethally toxigenic bacterial species in their nasopharyngeal flora.

Significance of endotoxin in lethal synergy between bacteria associated with sudden infant death syndrome: follow up study.

AIM: To investigate the role of endotoxin in synergy between bacterial toxins associated with sudden infant death syndrome (SIDS). METHODS: Extracellular toxins of 13 isolates of Staphylococcus from SIDS victims and matched healthy infants were tested for lethal toxicity in chick embryos with and without standard endotoxin (used at 1.00 ng/embryo). Endotoxin and toxins from staphylococci were used at dilutions with negligible lethality. RESULTS: Simultaneous injection of non-lethal levels of endotoxin and toxins from 11 of the 13 staphylococcal isolates tested produced lethal toxicity that was 111 to 613% greater than expected by an additive effect alone. This was highly significant and occurred even in the absence of staphylococcal enterotoxins or toxic shock syndrome toxin-1. CONCLUSION: Endotoxin enhancement of staphylococcal toxin lethality could be partly responsible for the clinical outcome in SIDS.

The effect of Staphylococcus aureus carriage in late pregnancy on antibody levels to staphylococcal toxins in cord blood and breast milk

We investigated the effect of carriage of Staphylococcus aureus in the later stages of pregnancy on levels of antibody specific to the S. aureus toxins, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin-1 (TSST-1), in cord blood and breast milk and also explored the relationship between levels of antibody in antenatal serum and cord blood. Nasopharyngeal swabs and stool samples were collected on two occasions, from 96 women, during the last 6 weeks of pregnancy. Samples were cultured and S. aureus isolates were identified. Antenatal and cord blood samples from the same women and their infants were analysed for IgG antibody to SEB, SEC and TSST-1 by enzyme-linked immunosorbent assay. Breast milk samples were analysed for IgA antibody to the same toxins. We found that S. aureus carriage in pregnancy is common and exposure to a toxin-producing isolate boosts immunity. Over 89% of women and infants have some protective antibody to the toxins, and antitoxin IgG levels are higher in cord blood samples compared with antenatal samples. Levels of cord blood IgG and breast milk IgA specific for the staphylococcal toxins vary. Some infants lack protection and could be at risk of toxin-induced disease.

Staphylococcal pyrogenic toxins in infant urine samples; a possible marker of transient bacteraemia.

Aim: To screen infant urine for staphylococcal pyrogenic toxins as a possible marker for a toxigenic, transient bacteraemia. Methods: Nasopharyngeal swabs, skin swabs, stool and urine samples were collected from 30 infants at 2 weeks, 10 weeks and 7 months of age when the infants were healthy, and from infants of 7 months of age when they had a cold. Samples were cultured and Staphylococcus aureus isolates identified. Isolates were tested for the production of staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC) and toxic shock syndrome toxin (TSST-1). Urine samples were analysed for the presence of these toxins by ELISA. Results: Nasopharyngeal carriage of S aureus decreased with age from 50% at 2 weeks of age to 13% in healthy infants at 7 months of age. Carriage was increased in infants over 7 months of age with a cold (36%). Stool carriage remained constant (37–40%) in healthy infants but increased significantly in infants over 7 months of age with a cold (82%). 13.9% of the isolates produced SEB, 16.7% produced SEC and 18% produced TSST-1. Some isolates produced more than one toxin. 43% of infants were colonised at some time with a toxigenic S aureus strain. S aureus toxins were detected in 9/101 urine samples. The proportion of positive samples was increased with infection and at 10 weeks of age. Conclusions: Infants are exposed early in life to S aureus pyrogenic toxins, which can be detected in infant urine samples. Age and infection affect the proportion of positive samples. The pattern of results can be explained by episodes of transient bacteraemia.

Postmortem Cerebrospinal Fluid Pleocytosis: A Marker of Inflammation or Postmortem Artifact?

The aim of this paper is to reassess the significance of postmortem cerebrospinal fluid pleocytosis. Published articles of CSF changes after death were reviewed, and reanalysis, in the light of modern views on the significance of bacterial postmortem isolates, was undertaken. There is theoretical and experimental evidence that the blood brain barrier to the movement of protein and cells is preserved in the first few hours after death. The number of mononuclear cells in the cerebrospinal fluid does rise in the first 24 hours after death, and this is most probably due to detachment of leptomeningeal lining cells. But the marked increase in lymphocyte counts seen in some cases of sudden infant death syndrome (SIDS) and in other deaths in the paediatric age range could well be a marker of inflammation.

Low dose, early mucosal exposure will minimize the risk of microbial disease

In the 21st century we will rediscover the germ theory of disease: germs not only cause infection as described in standard textbooks but also have a pathogenic role in autoimmunity, atherosclerosis, cancer and even acute psychiatric conditions. In order to reduce morbidity and mortality caused by common organisms we should ensure that exposure is early, often, by the mucosal route and in low dose. Micro-organisms should be delivered daily throughout life by respiratory mucosal spray or enteric coated pill, in precise dose and in a predetermined schedule.

Possibility of separating toxins from bacteria associated with sudden infant death syndrome using anion exchange chromatography.

AIMS: To develop techniques for the characterisation of toxins elaborated by a strain of Escherichia coli associated with sudden infant death syndrome (SIDS). METHODS: E coli SIDS 04, isolated from the nasopharynx of a case of SIDS, was studied. Cell-free toxin preparations were standardised, their protein measured, and analytical separation of proteins achieved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Acetone precipitation of proteins was required prior to Coomassie blue staining of bands. Preparative separation was achieved on an anion exchange column using a programmed concentration gradient of NaCl in TRIS buffer. Fractions were tested individually or pooled for presence of lethal toxin including endotoxin. Lethal toxin was detected with the chick embryo test system. Endotoxin was measured using a chromogenic modification of the Limulus amoebocyte assay. RESULTS: Twenty one peaks were detected by chromatography. Ten individual, or pooled, fractions were assayed for endotoxin which ranged from 27-33 pg/ml. Much greater variation was found when the same fractions were assayed in chick embryos. E coli fractions varied considerably in lethal toxicity, from 0/10 to 10/10 chick embryos killed/tested. Certain E coli fractions tested individually (lethality four out of 10 to eight out of 10) proved more lethal (10 out of 10) if pooled prior to testing. CONCLUSIONS: In E coli infection associated with SIDS relatively low concentrations of extracellular protein are lethally toxigenic for the chick embryo model of SIDS. These proteins can be separated analytically by SDS-PAGE and preparatively by anion exchange chromatography. Toxicity of individual fractions is not correlated with endotoxin concentrations in samples tested.