- Volume 46, Issue 7, 1997

One hundred isolates of Salmonella serotype Eimsbuettel from various human, animal and environmental sources in six countries were typed and shown to belong to five ribotypes, five biotypes and eight different ribotype/biotype groups, one of which, ribotype 3/biotype 5, was represented among isolates from all six countries. Most of the Eimsbuettel isolates from Scotland belonged to ribotype 1/biotype 3, which was the epidemic strain involved in a large outbreak centred in a Glasgow maternity hospital in 1986. That strain was also responsible for almost all the human infections that occurred in the west of Scotland in the years of this study. However, isolates from human cases in the east of Scotland belonged to either ribotype 2/biotype 1 or ribotype 3/biotype 5, groups not found in the west of Scotland. Representatives of all three ribotype/biotype groups causing human infection in Scotland were also found among isolates from poultry or poultry-associated materials. Plasmids were carried by only 14% of isolates and so provided little additional strain discrimination. However, plasmid analysis suggested that Salmonella Eimsbuettel of ribotype 2/biotype 1 had the potential to enter the human food chain in the UK via meat or bone meal, animal feed and poultry.

Bacteroides splanchnicus in common with several members of the B. fragilis group constitutively produced a number of protein and peptide hydrolysing enzymes. Amongst the most active was an arylamidase, which specifically hydrolysed the dipeptidyl chromogenic substrates glycylprolyl p-nitroanilide (GPRPNA), glycylprolyl β-naphthylamide (GPβNA) and valylalanine p-nitroanilide (VAPNA), and had some proteolytic activity towards azocasein. No activity was detected against proline β-naphthylamide, glycine, valanine or alanine p-nitroanilides. Physiological studies showed that the enzyme was largely cell-associated during exponential growth in batch culture, but was progressively released by the bacteria before the cells entered stationary phase. Glycylprolyl arylamidase (GPA) was completely cell-bound during growth in continuous culture, where synthesis increased concomitantly with dilution rate (specific growth rate) in both carbon- and nitrogen-limited chemostats. Gel-filtration chromatography of B. splanchnicus cell extracts yielded a single peak of GPA activity, with an apparent molecular mass of c. 160 kDa, while one peak of enzyme activity was eluted by 0.3 M NaCl during cation-exchange chromatography. Activity staining of SDS polyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded during preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possible existence of GPA isoenzymes. GPRPNA, VAPNA and azocasein were hydrolysed by the major fraction (fraction 3), while only the p-nitroanilide substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relatively broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated that while aspartic (pepstatin A), thiol (iodoacetate) and metalloprotease (EDTA, cysteine) inhibitors had little effect on hydrolysis of glycylproline p-nitroanilide, GPA was strongly inhibited (c. 80%) by 5 mm phenyl-methylsulphonyl fluoride (PMSF), indicating it to be a serine enzyme.

An exotoxin lethal to cells in culture (cytolethal toxin, CLT) was identified in culture filtrates of Burkholderia pseudomallei, the causative organism of melioidosis. CLT could pass through a 10-kDa cut-off ultrafilter and its properties suggest that it is a peptide. Isolates from soil, animals and man showed differential cytolethality in vitro. The isolates were divided into low, medium and high CLT producers with soil isolates being low producers and isolates from patients with melioidosis encephalitis being high producers. CLT levels are subject to regulation, as a strain isolated from an infected goat was one of the highest producers whereas the same strain isolated from soil was a low producer. In addition to CLT, all isolates produced a protein with cell-elongating activity which was also present in culture filtrates.

Burkholderia pseudomallei is isolated frequently from the soil in regions where the disease melioidosis occurs. However, recent surveys in Thailand have shown that the frequency of isolation of the organism from soil samples is not directly related to the incidence of melioidosis in an area. To determine whether strain populations of B. pseudomallei prevalent in soil are gentypically related to strains causing clinical disease, rRNA BamHI restriction fragment length polymorphisms (RFLP) of 139 soil environmental isolates and 228 human isolates were compared. Two groups of ribotype patterns were found. Group I comprised 37 different ribotype patterns which were characterised by five to eight hybridisation bands of 2.8-> 23 kb. All of these ribotypes were identified among the clinical isolates, and 18 of them were also found in 59 environmental isolates. Group II was represented by 12 ribotypes found only in environmental strains. These ribotype patterns comprised one to five bands in the size range 9-> 23 kb. All but one of the 73 isolates in this group grew on a minimal medium supplemented with L-arabinose. In contrast, only 3% of the 66 isolates from the environment with group I ribotype patterns could utilise this sugar as their sole energy source. These findings suggest that B. pseudomallei strains that utilise arabinose constitute a population that is genetically distinct from other environmental and clinical strains.

Adult conventional rats were starved for 48 h with or without haemorrhage at 24 h, and translocation of caecal coliforms to mesenteric lymph nodes (MLNs) was measured. Translocation was detected in three of 11 rats without haemorrhage, in 6 of 11 starved and sham-operated rats and in 12 of 22 rats after haemorrhage. In contrast, only one of 13 non-instrumented and fed control rats showed translocation. Translocation was associated with more coliforms adhering to caecal epithelium in rats. Coliform isolates from caecum, caecal epithelium and MLNs were characterised and grouped into different biochemical phenotypes (BPTs) by a biochemical fingerprinting method. Of 291 BPTs detected in the caecum of all rats, 108 were also found on caecal epithelium; 36 BPTs were detected in MLNs, of which 17 were not detected either in the caecum or on the caecal epithelium of the corresponding rats. One isolate from each of these 36 BPTs was selected and compared to the others. Four common (C) BPTs (i.e., C1–C4) were identified among them. Strains of C1 formed the majority of isolates from the caecum (79%), caecal epithelium (71%) and MLNs (91%). In contrast, C2–C4 had a significantly lower incidence both in the caecum and on the caecal epithelium, but not in the MLNs. These findings indicate that not all caecal coliforms adhere to the epithelium during catabolic stress and that for translocation to occur, other bacterial properties besides adhesion are needed. It is also concluded that coliforms with a low incidence in the caecum can translocate with the same efficiency as those with a high incidence.

This study evaluated the detection of antibodies, circulating antigens and parasite DNA by polymerase chain reaction (PCR) in the diagnosis of toxoplasma encephalitis. The detection of antibody classes and IgG avidity were not useful diagnostically. The detection of circulating antigens by the ELISA system described was not sufficiently sensitive. The detection of DNA by PCR was the most useful test especially in untreated patients, with a sensitivity of 62% overall, 81% in untreated patients and only 20% in treated patients. The use of non-isotopic probes makes the use of this technique feasible in routine diagnostic parasitology laboratories.

Two outer-membrane proteins (OMPs) of Pasteurella multocida serotype D, designated H and W, possess potentially important serotype D-specific antigens. Antigenicity as well as toxigenicity of 55 strains of P. multocida representing various serotypes, geographic origins and host species were studied by SDS-PAGE, enzyme-linked immunosorbent assay (ELISA), immunoblot and polymerase chain reaction (PCR) assays. Based on the electrophoretic mobility of protein H, different OMP patterns were observed within different capsular serotypes. Three monoclonal antibodies (MAbs) designated MT1, MT2 and MT3 were produced against H and W proteins of P. multocida in BALB/c mice. MAbs MT2 and MT3 reacted with two distinct epitopes on W protein of serotype D in competitive ELISA. MAb MT1 reacted with all serotype D-I strains but not with D-II strains, whereas MAb MT2 reacted with both serotype D-I and D-II strains in dot-ELISA and immunoblot assay. MAb MT3 reacted with all P. multocida strains belonging to different capsular serotypes in dot-ELISA. None of the MAbs reacted with other gram-negative bacteria tested, indicating that protein H has a serotype D-I specific epitope and protein W has both serotype and species-specific epitopes. PCR assay was used to identify toxigenic strains of P. multocida; 92% of P. multocida strains possess both toxA gene and MAb MT2 reacting epitope, suggesting a strong association between MAb MT2 reacting epitopes and toxA gene. Rapid dot-ELISA with MAb was found to be specific, sensitive and easy to perform and thus suitable for routine serotyping of P. multocida serotype D strains which might be potentially pathogenic.