- Volume 47, Issue 5, 1998

Sorbarod biofilms were investigated for their suitability in establishing continuous culture biofilms for the study of bacterial vaginosis. Two important organisms in the condition, Gardnerella vaginalis and Lactobacillus acidophilus, were studied. In contrast to growth in broth culture, both organisms were maintained for at least 96 h in a steady state on the biofilms. With G. vaginalis, the haemolytic activity was consistently maintained in the biofilms in contrast to short-term activity in broth culture which matched the bacterial titre. The simple Sorbarod system appears to be suitable for studying the growth conditions of bacteria in continuous culture and has potential for investigating interactions between micro-organisms.

The pathogenesis of neonatal necrotising enterocolitis (NEC) remains unclear. Gnotobiotic quails fed a lactose diet have been used to investigate the role of clostridial strains originating from faecal specimens of neonates through the intestinal lesions, the changes in microflora balance and the production of bacterial metabolites, i.e., short-chain fatty acids and hydrogen. Bifidobacteria are thought to exert various beneficial effects on host health, including interaction with the colonic microflora. Therefore, it was hypothesised that a protective role could be exercised through bifidobacterial colonisation. A Clostridium butyricum strain (CB 155–3) and a whole faecal flora including three clostridial species (C. butyricum, C. perfringens, C. difficile), each from premature infants suffering from NEC, caused caecal lesions in quails similar to those observed in man, i.e., thickening of the caecal wall with gas cysts, haemorrhagic ulceration and necrotic areas. Conversely, a whole faecal flora including bifidobacteria (identified as Bifidobacterium pseudo-catenulatum) and no clostridia, isolated from a healthy premature infant, was unable to produce NEC-like lesions. When the two clostridial groups were associated with a Bifidobacterium strain (B. infantis-longum, CUETM 89–215, isolated from a healthy infant), bifidobacterial colonisation suppressed all pathological lesions. This study is the first demonstration of a protective role for bifidobacteria against NEC via the inhibition of growth of C. butyricum or the disappearance of C. perfringens. C. difficile was not found to be responsible for the aetiology of the caecal lesions in quails. The main effect of bifidobacteria on lactose fermentation was either a dramatic decrease or a disappearance of butyric acid. The protective role was not associated with changes in H2 production. Therefore, a new step between colonic colonisation and its relevance to NEC is thought to involve the fermentation of unabsorbed lactose into butyric acid at the onset of the disease.

In 1995, Haemophilus influenzae became the first free-living organism to have its entire genome sequence published. Since then, many similar projects have been started and, by the millennium, the genomes of a significant number of important human pathogens will have been sequenced. During this period of increasing access to microbial sequence data, parallel advances have occurred in techniques that allow the large-scale study of the entire genetic complement of micro-organisms. In the near future, these approaches will enable researchers to unravel further the complexity of microbial pathogenesis and identify new virulence determinants. Many of these will be suitable targets for development as diagnostic reagents, antimicrobial agents and vaccine candidates. Although it is difficult to predict the full impact that this almost overwhelming volume of information will have on the practice of microbiology, it is clear that it will result ultimately in new ways of diagnosing and combating infectious diseases.

Several hydrolytic and reductive bacterial enzymes (β-glucuronidase, GN; β-glucosidase, GS; arylsulphatase, AS; azoreductase, AR; nitroreductase, NR) involved in production of mutagenic or genotoxic metabolites were measured in human colonic contents. Cell-associated AS and extracellular GS were approximately twice as high in the distal colon compared with the proximal bowel, while AR changed little throughout the gut. Measurements of these enzymes in faeces from seven healthy donors confirmed that the majority were cell-associated, and demonstrated high levels of inter-individual variability. NR decreased four-fold between the proximal and distal colon while extracellular GN was reduced by 50%. Most probable number (MPN) analysis on faeces obtained from six healthy donors showed that counts of intestinal bacteria producing GS and AR were c. 1010 and 1011/g, respectively, in all samples tested. Numbers of GN- and AS-forming organisms were between two and three orders of magnitude lower. Inter-individual carriage rates of bacterial populations synthesising NR were highly variable. Screening of 20 pure cultures of intestinal bacteria, belonging to six different genera, showed that Bacteroides ovatus, in particular, synthesised large amounts of GS, whereas B. fragilis, B. vulgatus and Bifidobacterium pseudolongum formed the highest cell-associated levels of GN. In general, bifidobacteria and Lactobacillus acidophilus did not produce significant amounts of AR. All five clostridia studied (Clostridium bifermentans, C. septicum, C. perfringens, C. sporogenes and C. butyricum) produced NR and AR, as did the bacteroides (B. fragilis, B. ovatus and B. vulgatus). Escherichia coli and C. perfringens formed large amounts of NR. Levels of AS production were invariably low and few of the organisms screened synthesised this enzyme. In-vitro studies investigating the effect of intestinal transit time on enzyme production, in a three-stage (V1–V3) continuous culture model of the colon operated at system retention times (R) of either 31.1 or 68.4 h, showed that specific activities of GS were up to four-fold higher (V3) at R = 31.1 h. Bacteriological analysis demonstrated that representative populations of colonic micro-organisms were maintained in the fermentation system, and indicated that changes in GS activity were not related to numbers of the predominant anaerobic or facultative anaerobic species within the model, but were explainable on the basis of substrate-induced modulation of bacterial metabolism.

This study investigated whether gender or smoking has an impact on immune responses to Chlamydia pneumoniae in generally healthy adults. A total of 129 twins (46 twin pairs and 37 single twins) from the Finnish Twin Cohort who had previously reported the highest discordance for smoking with their co-twins participated. C. pneumoniae-specific serum IgA and IgG antibody levels were measured by the micro-immunofluorescence test (micro-IF) at admission and 3 months later if the IgA level in the first sample was elevated. Cell-mediated immune (CMI) responses to C. pneumoniae and control antigens from heparinised blood samples were assessed by the lymphoproliferation (LP) assay. When all the subjects were pooled and analysed by gender and smoking status, marked differences in the humoral immune response between the genders were observed, irrespective of smoking status. When twin pairs solely were analysed, significantly elevated IgA antibody levels suggestive of persistent infection were found among the currently or formerly smoking men compared to their non-smoking co-twins. The CMI response showed a reciprocal trend with respect to humoral immunity. In conclusion, specific antibody levels were found to be higher in men than in women irrespective of smoking status, although smoking may further enhance the humoral response and depress the CMI response in men.

The gastrointestinal pathogen Aeromonas hydrophila strain A186 produces a collagen-binding protein (CNBP) which is found extracellularly and loosely associated with the cell surface. The cell-associated CNBP was purified by sequential ammonium sulphate precipitation, size-exclusion chromatography and ion-exchange chromatography, or by sequential ammonium sulphate precipitation and affinity chromatography with collagen-Sepharose. The purified CNBP was homogeneous in SDS-PAGE, and had a mol. wt of c. 98 kDa. Cyanogen bromide cleavage of the CNBP destroyed collagen-binding activity; however, enzymic digestion with Staphylococcus aureus V8 protease generated > 10 polypeptide fragments, from which a 30-kDa polypeptide contained the strongest collagen-binding activity. Binding of collagen by the CNBP was restricted to the α1 (I) chain of the collagen molecule and binding seemed to involve both the carbohydrate moieties and certain peptide sequences on the collagen. Collagen-saccharides generated by alkaline hydrolysis inhibited collagen binding by A. hydrophila. Also, glycosidase digestion and chemical alteration of the carbohydrate residues of collagen reduced its ability to be bound by the CNBP. Collagen-homologous synthetic peptides inhibited binding of 125I-collagen by the bacteria.

The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or ‘PS ribotyping’, was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and SphI, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PT11 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that ‘PS ribotyping’ was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.

Although a number of studies have shown that various free fatty acids (FFAs) and monoacylglycerides (MGs) have bactericidal properties in vitro, the role of these compounds in vivo has not been determined. This study evaluated the antibacterial properties of medium-chain MGs and FFAs for different bacterial enteropathogens with an in-vitro bacterial killing assay and an in-vivo model of intestinal colonisation. Incubation of test bacteria with medium-chain MGs for 4 h led to 100–10 000-fold reductions in numbers of viable cells of Vibrio cholerae, Salmonella typhi, Shigella sonnei and enterotoxigenic Escherichia coli (ETEC). Lauric acid was the only medium-chain FFA to show comparable in-vitro bactericidal activity. The ability of dietary MGs to reduce or eliminate bacterial colonisation of the intestinal tract was evaluated in mice that were predisposed to bacterial colonisation by treatment with streptomycin (STR+). Mice were treated with streptomycin, challenged intragastrically with V. cholerae or ETEC, and given monocaprin (C10:0 MG) either concurrently or as part of the daily diet. Control mice given STR+ without MGs and challenged with V. cholerae or ETEC showed high numbers of challenged bacteria in gastrointestinal contents by 1 h after administration. Concurrent administration of V. cholerae and C10:0 MG (2.5 mg/ml) caused > 1000-fold reduction in numbers of V. cholerae recovered from the gastrointestinal tracts of STR+ mice. Concurrent administration of C10:0 MG with ETEC did not cause a reduction in the number of viable ETEC present in the intestinal tract of STR+ mice. Administration of C10:0 MG in the diet had no effect on the number of viable V. cholerae or ETEC associated with caecal or ileal tissue of STR+ mice when C10:0 MG in the diet was started 1 day before, the same day, or 2 days after bacterial challenge. Collectively, these results suggested that dietary MGs may prevent intestinal colonisation by bacterial enteropathogens if administered at the time of exposure, but have little effect on established intestinal infections.

Various serotypes reported in three serotyping systems for the ‘Streptococcus milleri’ group, i.e., Ottens I–IV, Osano I–IV and serotypes a–k, were compared with each other in a capillary precipitation test and by the double immunodiffusion test. Only two of the 19 serotypes, f and Ottens-III, were identical, and the other 17 serotypes were independent. Thus, 18 serotypes and another three candidates (a-, b- and g-cross-reactive) were found in the ‘S. milleri’ group. Of the 248 strains tested, 197 were serotypable, and 23 strains carried two type antigens. Generally, the Ottens I, II, III (serotype f), IV and Osano IV antigens, often together with the Lancefield group F antigen, were found in S. anginosus and S. constellatus. In addition, the serotype a, c, d, e and k antigens with the group A, C or G antigen were distributed in S. anginosus, the serotype b antigen in S. constellatus and the g, h, i, j, Osano I, II and III antigens in S. intermedius. Twenty-five of the untypable strains were Lancefield groupable (mostly F).

Most aminoglycoside resistance in Acinetobacter spp. involves production of aminoglycoside-modifying enzymes. Previous studies have shown that the genes encoding these enzymes can be present on plasmids, transposons or within integron-type structures. To determine whether particular mechanisms of aminoglycoside resistance have developed in strains from specific geographical locations (with subsequent clonal spread), or whether common mechanisms have been acquired by genotypically distinct clinical isolates of Acinetobacter spp. throughout the world, a genotypically heterogeneous collection of 24 multiresistant clinical isolates of Acinetobacter spp. from 15 hospitals in 11 countries worldwide was studied. All were resistant to two or more aminoglycoside antibiotics. The full aminoglycoside resistance profile was determined for each isolate, allowing a putative enzyme content to be inferred, with subsequent confirmation of enzyme content and genetic location by polymerase chain reaction (PCR) and hybridisation techniques. All produced at least one aminoglycoside-modifying enzyme, most commonly AAC(3)-I and ANT(3′)-I in various combinations. Other enzymes found were AAC(3)-II, AAC(6′)-I, ANT(2′), APH(3′)-I and APH(3′)-VI. None was confined to strains from a particular geographical area. Nine isolates transferred resistance mediated by AAC(3)-I, ANT(2′)-I, APH(3′)-I or APH(3)′-VI by conjugation to a sensitive strain of A. baumannii, but most resistance was non-transferable. PCR mapping revealed an integron location in six isolates for the aac(3)-Ia gene and in three isolates for the ant(3′)-Ia gene. Overall, the study demonstrated that similar aminoglycoside-modifying enzymes are found in unrelated isolates of Acinetobacter spp., and that particular genes are not restricted to specific areas of the world. The demonstration of certain genes on plasmids and integrons emphasises the probable importance of these structures in the dissemination of certain types of aminoglycoside resistance in Acinetobacter spp.

In-vivo administration of the culture supernates from Yersinia enterocolitica resulted in thymus atrophy in C3H/HeJ mice, known to be lipopolysaccharide (LPS)-non-responders. The thymocytes underwent apoptosis as characterised by fragmented DNA ladders on agarose gel electrophoresis, a cell death detection ELISA and a morphological study by the TUNEL reaction. As a control, LPS treatment did not induce thymocyte apoptosis in C3H/HeJ mice. Flow cytometric analysis indicated that thymus atrophy was due predominantly to the deletion of CD4+CD8+T cells. When cells were undergoing apoptosis, an elevation in the percentage of T-cell receptor (TCR)-αβhigh cells was observed at 24 h, which was correlated with the increase in the percentages of cells expressing high levels of the Vβ6 and Vβ8 TCR. Gel electrophoretic analysis demonstrated the presence of protein bands with mol. wts ranging from 17 to 65 kDa in Y. enterocolitica culture supernates.