Effect of Endogenous Clostridioides difficile Toxin Antibodies on Recurrence of C. difficile Infection.

BACKGROUND: Endogenous antibodies (eAbs) against Clostridioides (Clostridium) difficile toxins may protect against recurrence of C. difficile infection (rCDI). This hypothesis was tested using placebo group data from MODIFY (Monoclonal Antibodies for C. difficile Therapy) I and II (NCT01241552 and NCT01513239, respectively), global, randomized phase 3 trials that assessed the efficacy and safety of the antitoxin monoclonal antibodies bezlotoxumab and actoxumab in participants receiving antibiotic therapy for CDI. METHODS: A placebo infusion (normal saline) was administered on study day 1. Serum samples were collected on day 1, week 4, and week 12, and eAb-A and eAb-B titers were measured by 2 validated electrochemiluminescence immunoassays. Rates of initial clinical cure and rCDI were summarized by eAb titer category (low, medium, high) at each time point. RESULTS: Serum eAb titers were available from a total of 773 participants. The proportion of participants with high eAb-A and eAb-B titers increased over time. Rates of initial clinical cure were similar across eAb titer categories. There was no correlation between eAb-A titers and rCDI rate at any time point. However, there was a negative correlation between rCDI and eAb-B titer on day 1 and week 4. rCDI occurred in 22% of participants with high eAb-B titers at baseline compared with 35% with low or medium titers (P = .015). CONCLUSIONS: Higher eAb titers against toxin B, but not toxin A, were associated with protection against rCDI. These data are consistent with the observed efficacy of bezlotoxumab, and lack of efficacy of actoxumab, in the MODIFY trials. CLINICAL TRIALS REGISTRATION: NCT01241552 and NCT01513239.

Bezlotoxumab for Prevention of Recurrent Clostridium difficile Infection.

BACKGROUND: Clostridium difficile is the most common cause of infectious diarrhea in hospitalized patients. Recurrences are common after antibiotic therapy. Actoxumab and bezlotoxumab are human monoclonal antibodies against C. difficile toxins A and B, respectively. METHODS: We conducted two double-blind, randomized, placebo-controlled, phase 3 trials, MODIFY I and MODIFY II, involving 2655 adults receiving oral standard-of-care antibiotics for primary or recurrent C. difficile infection. Participants received an infusion of bezlotoxumab (10 mg per kilogram of body weight), actoxumab plus bezlotoxumab (10 mg per kilogram each), or placebo; actoxumab alone (10 mg per kilogram) was given in MODIFY I but discontinued after a planned interim analysis. The primary end point was recurrent infection (new episode after initial clinical cure) within 12 weeks after infusion in the modified intention-to-treat population. RESULTS: In both trials, the rate of recurrent C. difficile infection was significantly lower with bezlotoxumab alone than with placebo (MODIFY I: 17% [67 of 386] vs. 28% [109 of 395]; adjusted difference, -10.1 percentage points; 95% confidence interval [CI], -15.9 to -4.3; P<0.001; MODIFY II: 16% [62 of 395] vs. 26% [97 of 378]; adjusted difference, -9.9 percentage points; 95% CI, -15.5 to -4.3; P<0.001) and was significantly lower with actoxumab plus bezlotoxumab than with placebo (MODIFY I: 16% [61 of 383] vs. 28% [109 of 395]; adjusted difference, -11.6 percentage points; 95% CI, -17.4 to -5.9; P<0.001; MODIFY II: 15% [58 of 390] vs. 26% [97 of 378]; adjusted difference, -10.7 percentage points; 95% CI, -16.4 to -5.1; P<0.001). In prespecified subgroup analyses (combined data set), rates of recurrent infection were lower in both groups that received bezlotoxumab than in the placebo group in subpopulations at high risk for recurrent infection or for an adverse outcome. The rates of initial clinical cure were 80% with bezlotoxumab alone, 73% with actoxumab plus bezlotoxumab, and 80% with placebo; the rates of sustained cure (initial clinical cure without recurrent infection in 12 weeks) were 64%, 58%, and 54%, respectively. The rates of adverse events were similar among these groups; the most common events were diarrhea and nausea. CONCLUSIONS: Among participants receiving antibiotic treatment for primary or recurrent C. difficile infection, bezlotoxumab was associated with a substantially lower rate of recurrent infection than placebo and had a safety profile similar to that of placebo. The addition of actoxumab did not improve efficacy. (Funded by Merck; MODIFY I and MODIFY II ClinicalTrials.gov numbers, NCT01241552 and NCT01513239 .).

Bezlotoxumab for prevention of Clostridium difficile infection recurrence: Distinguishing relapse from reinfection with whole genome sequencing.

BACKGROUND: Bezlotoxumab has been shown to prevent Clostridium difficile infection recurrence (rCDI) in high-risk patients. METHODS: We used whole genome sequencing to estimate the impact of bezlotoxumab on same-strain relapse or new-strain reinfection in MODIFY I/II trials. Reinfection with a new strain and relapse with the same strain were differentiated by the comparison of ribotype (RT) and pair-wise single-nucleotide whole genome sequencing (WGS) variations (PWSNV). Relapse was assigned if the baseline RT and the RT isolated during rCDI were the same, and if PWSNVs were ≤ 2. Reinfection was assigned if the baseline RT and the RT isolated during rCDI were different, or if the RT was the same but PWSNVs were > 10. Unknown status was assigned if the RT was the same but PWSNVs were 3-10. RESULTS: 259 rCDI events were evaluable (50 [19.3%] reinfection; 198 [76.4%] relapse). The proportion of relapses was higher for ribotype 027 (84.5%) compared with other ribotypes (74.1%). Cumulative incidence of relapse was significantly lower for bezlotoxumab versus no bezlotoxumab (p < 0.0001), with a non-significant trend towards reduction for reinfection (p = 0.14). CONCLUSION: Bezlotoxumab treatment significantly reduced the rate of CDI relapse versus a regimen without bezlotoxumab. (NCT01241552/NCT01513239).

Generation of Markerless Deletions in the Nosocomial Pathogen Clostridium difficile by Induction of DNA Double-Strand Breaks.

Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddAB mutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase.IMPORTANCE Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.

International Clostridium difficile animal strain collection and large diversity of animal associated strains.

BACKGROUND: Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria of one strain (PCR ribotype) per animal species per laboratory. RESULTS: Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates as well as in terms of number of different host species: 078 (14.3% of isolates; 4 hosts), 014/020 (11.6%; 8 hosts); 002 (5.4%; 4 hosts) and 012 (5.4%; 5 hosts). Two animal hosts were best represented; cattle with 31 isolates (20 PCR ribotypes; 7 countries) and pigs with 31 isolates (16 PCR ribotypes; 10 countries). CONCLUSIONS: This results show that although PCR ribotype 078 is often reported as the major animal C. difficile type, especially in pigs, the variability of strains in pigs and other animal hosts is substantial. Most common human PCR ribotypes (014/020 and 002) are also among most prevalent animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen.

The changing faces of Clostridium difficile: a personal reflection of the past 34 years.

Late in 1978 my boss gave me a folder with "Clostridium difficile (diffikilé)" written on it. Inside were a few recent and now classic papers by Bartlett, Larson and co. It was suggested that this might be an interesting research topic. So began a continuing adventure which has resulted in at least 50 publications from my group. Over the years we have made several important contributions to the field. Beginning in 1982 we showed that C. difficile was a common cause of community-acquired infection! During the next few years we did extensive structural studies on the bacterium. This culminated in 1984 with a fingerprinting study (by immunoblotting surface antigens), on Swedish strains supplied by Carl-Erik Nord, which was probably the first study to demonstrate that C. difficile was really an infectious agent. This was later reinforced with strains sent from Amsterdam by Ed Kuijper. Later in the 1980s, in a study of recurrent disease, we showed that ca. 50% of recurrences were due to infection with a different strain. During my term as chair of the European Study Group for C. difficile, we began to define the status of C. difficile infection (CDI) in Europe and develop guidance for diagnosis and treatment. Recently we utilised our extensive culture collection, with isolates from the 1970s to the present, to observe how epidemiology has been driven largely by antibiotic usage. We have now come full circle: in the early years C. difficile infection was caused by many different strains. Then in the period beginning in the 1990s, characterised by often-large outbreaks and poor infection control, disease was caused by a few endemic strains highlighted by the 027/NAP1/BI pandemic. Now in a much-improved local situation, we are seeing again that the majority of cases (largely sporadic) is caused by multiple types. Current studies range from molecular studies on toxin and spore production, immune responses, novel observations on CDI in children, to what is the best way of decontaminating the anaerobe laboratory.

Comparison of toxin and spore production in clinically relevant strains of Clostridium difficile.

Clostridium difficile is a major cause of nosocomial diarrhoea. The toxins that it produces (TcdA and TcdB) are responsible for the characteristic pathology of C. difficile infection (CDI), while its spores persist in the environment, causing its widespread transmission. Many different strains of C. difficile exist worldwide and the epidemiology of the strains is ever-changing: in Scotland, PCR ribotype 012 was once prevalent, but currently ribotypes 106, 001 and 027 are endemic. This study aimed to identify the differences among these ribotypes with respect to their growth, and toxin and spore production in vitro. It was observed that the hypervirulent ribotype 027 produces significantly more toxin than the other ribotypes in the exponential and stationary phases of growth. Further, the endemic strains produce significantly more toxins and spores than ribotype 012. Of note was the observation that tcdC expression did not decrease into the stationary phase of growth, implying that it may have a modulatory rather than repressive effect on toxin production. Further, the increased expression of tcdE in ribotype 027 suggests its importance in the release of the toxins. It can thus be concluded that several genotypic and phenotypic traits might synergistically contribute to the hypervirulence of ribotype 027. These observations might suggest a changing trend towards increased virulence in the strains currently responsible for CDI.

Monocytes heterozygous for the Asp299Gly and Thr399Ile mutations in the Toll-like receptor 4 gene show no deficit in lipopolysaccharide signalling.

Toll-like receptor 4 (TLR4)-mediated recognition of lipopolysaccharide (LPS) is required for efficient recognition of Gram-negative bacterial infections. Two commonly occurring mutations in the human TLR4 gene (Asp299Gly and Thr399Ile) have recently been shown to be associated with blunted physiological responses to inhaled LPS, and with increased risk of Gram-negative bacteraemia in sepsis patients and reduced risk of atherosclerosis in an Italian population. Here we show that monocytes from individuals heterozygous for both mutations in the TLR4 gene exhibit no deficit in recognition of LPS of Escherichia coli, Neisseria meningitidis, Bacteroides fragilis, Yersinia pestis, Chlamydia trachomatis, Porphyromonas gingivalis, or Pseudomonas aeruginosa. We propose that the relatively high frequency of these mutations in the Caucasian population may reflect modified responses of carriers to alternative TLR4 agonists.

Bacteroides fragilis signals through Toll-like receptor (TLR) 2 and not through TLR4.

Although it is desirable to identify the interactions between endotoxin/LPS and the innate immune mechanism, it is often not possible to isolate these interactions from other cell wall-related structures of protein or polysaccharide origin. There is no universally accepted method to extract different LPSs from different bacteria, and their natural state will be influenced by their interactions with the associated molecules in the bacterial outer membrane. It is now believed that Toll-like receptor (TLR) 4 is the main signal transducer of classical LPS (i.e. Escherichia coli LPS), while TLR2 is used by certain non-classical LPSs. There are contradictory reports as to whether Bacteroides fragilis LPS, a non-classical LPS, signals primarily through TLR2 or TLR4. This study was designed to address this problem. Different non-purified and purified B. fragilis LPSs extracted by different methods together with different heat-killed, whole-cell populations of B. fragilis were used to elucidate the TLR specificity. All of these B. fragilis preparations showed a significant signalling specificity for TLR2 but not for TLR4. This indicates that changing the extraction methods, with or without applying a repurification procedure, and varying the cell populations do not alter the TLR specificity of B. fragilis LPS.

Enhancement of the cytotoxic activity of Clostridium difficile toxin A by surface-associated antigens.

Cell-surface antigens of Clostridium difficile and LPS from Escherichia coli were investigated for modulating effects on the activity of C. difficile toxin A on Vero and Caco2 cells. The antigens of C. difficile tested comprised: (i) an EDTA extract, which contained several major and minor cell-surface proteins and the membrane-associated lipocarbohydrate (LC); (ii) a guanidine hydrochloride extract, which mainly contained the surface-layer proteins; (iii) an aqueous phenol-extracted, protein-free LC. On their own, none of the antigens had a detrimental effect on the cells, with the EDTA extract and LC having a marginally protective effect. When these antigens were added to suboptimal levels of toxin A, there was significant enhancement of its cytotoxicity by the EDTA and LC preparations on both cell types. LPS showed some enhancement of the effect of toxin on Vero cells at the lowest levels of toxin investigated. It was concluded that this effect seen in vitro may have a role to play in the colon during infection with C. difficile.

Systemic antibody response to Clostridium difficile in colonized patients with and without symptoms and matched controls.

It has been proposed that patients who develop Clostridium difficile-associated disease (CDAD) do so because they are unable to mount an adequate immune response. Serum was collected from three groups of elderly in-patients: (i) cases (n=21) of CDAD, being toxin A/B-positive; (ii) carriers (n=21) asymptomatic for CDAD (no diarrhoea) but at least toxin or culture positive; and (iii) controls (n=26) asymptomatic for CDAD and negative for both C. difficile toxin and culture. The age and gender of each group were compared, and the colonizing strains were ribotyped and toxinotyped. Serum antibodies (IgG and IgM) were measured by ELISA using different antigen preparations: EDTA extract (containing cell-surface proteins and carbohydrates), guanidine hydrochloride extract (surface-layer proteins), aqueous phenol-extracted lipocarbohydrate (LC); crude toxin (dialysis culture supernatant) and purified toxin A. LPS from Escherichia coli was used as a control antigen. Antibodies were also tested for toxin neutralization on tissue monolayers and for binding to EDTA-extracted antigens by Western blotting. IgG antibody measurements to cytomegalovirus (CMV) were included as an indicator of potential immunosenescence. Results showed that the patient groups were well matched by age and gender, and the colonizing strains were similar in cases and carriers, being predominantly ribotype 001 and toxinotype 0. By ELISA, IgG levels to most of the antigens were highest in the cases and lowest in the controls, with the exception of antibodies to the LC, which were higher in the controls than the cases. Levels in the carriers tended to be of intermediate level or similar to the controls. For all antigens, the levels of IgM were not significantly different among cases, carriers and controls. Serum from all groups was able to neutralize the cytotoxic action of toxin on both Vero and Caco2 cells, and all to a similar extent. Western blots showed an overall higher level of IgG antibodies to the EDTA-extracted antigens in the cases. The results of the CMV ELISA showed that specific IgG was detected in more cases (78%) than carriers and controls (both 65%), but this difference in seropositivity was not significant. The conclusion is that, during symptomatic infection, patients respond to protein antigens of C. difficile in a manner typical of a secondary antibody response, with no evidence that an inability to respond predisposes to the appearance of symptoms.

A PCR approach to determine the distribution of toxin genes in closely related Clostridium species: Clostridium botulinum type C and D neurotoxins and C2 toxin, and Clostridium novyi alpha toxin.

The closely related Clostridium novyi and Clostridium botulinum types C and D are of current interest because of their association with serious infections in injecting drug users (C. novyi type A) and equine and feline dysautonomias (C. botulinum types C/D). The species are defined by the major toxins they produce: the alpha toxin of C. novyi, and the type C and D neurotoxins of C. botulinum (BoNT/C and BoNT/D). The other major toxin produced by this group, and previously thought to be restricted to the botulinum types, is the chromosomally encoded C2--a binary toxin consisting of two components, I and II. In the current study 44 of these clostridia from the authors' culture collection were investigated--most of which had been identified previously by conventional biochemical tests as 'C. novyi type A'. The aim was to check the distribution of toxin genes by PCR to see if the identities were consistent with the genes carried, and to ascertain if the C2 gene was only found in authentic C. botulinum strains. Several combinations of the species-defining genes and the two components of the C2 genes were detected. Only the authentic BoNT/C- and BoNT/D-positive C. botulinum strains and one of two non-neurotoxic variants of type C carried genes for both components of the C2 toxin. Of the remaining 40 C. novyi type A-like strains, the gene for the alpha toxin was found in 22, with 19 of these also possessing the gene for component I (16) or component II (3) but not both. In the alpha toxin-negative strains (22), both of the C2 genes were detected in 5 strains (3 C. botulinum), with component I in 11 strains and neither gene in 6 strains.

Acinetobacter baumannii lipopolysaccharides are potent stimulators of human monocyte activation via Toll-like receptor 4 signalling.

Acinetobacter baumannii is a major nosocomial pathogen and frequent cause of hospital-acquired pneumonia, surgical wound infections and sepsis. As very little is known of the endotoxic potential of A. baumannii lipopolysaccharide (LPS) with respect to human cells or of its ability to stimulate inflammatory signalling via human Toll-like receptors (TLRs), the biological activity of these endotoxins was investigated in human monocytic THP-1 cells and in TLR-deficient HEK-293 cells transfected with human TLR2 and TLR4 constructs. Endotoxins derived from five clinical isolates of A. baumannii and one of Acinetobacter 'genomospecies 9' showed high potency, which was comparable to that of Escherichia coli strain R1 NCTC 13114 LPS, in the induction of the Limulus amoebocyte reaction and interleukin 8 and tumour necrosis factor alpha release from THP-1 cells. Whole UV-killed cells of A. baumannii and Acinetobacter 'genomospecies 9' stimulated both TLR2- and TLR4-dependent signalling, whereas pure endotoxins of all investigated strains induced signalling via TLR4, but not TLR2.

Molecular characterization and antimicrobial susceptibility patterns of Clostridium difficile strains isolated from hospitals in south-east Scotland.

Clostridium difficile isolates (n=149) collected in south-east Scotland between August and October 2005 were typed by four different methods and their susceptibility to seven different antibiotics was determined. The aims were to define the types of strain occurring in this region and to determine whether there were any clonal relationships among them with respect to genotype and antibiotic resistance pattern. Ribotyping revealed that 001 was the most common type (n=113, 75.8 %), followed by ribotype 106 (12 isolates, 8.1 %). The majority of the isolates (96.6 %, n=144) were of toxinotype 0, with two toxinotype V isolates and single isolates of toxinotypes I, IV and XIII. PCR and restriction analysis of the fliC gene from 147 isolates gave two restriction patterns: 145 of pattern VII and two of pattern I. Binary toxin genes were detected in only three isolates: two isolates of ribotype 126, toxinotype V, and one isolate of ribotype 023, toxinotype IV. S-types showed more variation, with 64.5 % (n=40) of the common S-type (4,939) and 21 % (n=13) of S-type 4,741, with six other S-types (one to three isolates each). All ribotype 001 isolates were of the same S-type (4,939), with three isolates of other ribotypes being this S-type. No resistance was found to metronidazole or vancomycin, with resistance to tetracycline only found in 4.3 % of the isolates. A high proportion of isolates were resistant to clindamycin (62.9 %), moxifloxacin, ceftriaxone (both 87.1 %) and erythromycin (94.8 %). Resistance to three antibiotics (erythromycin, clindamycin and ceftriaxone) was seen in 66 isolates, with erythromycin, ceftriaxone and moxifloxacin resistance seen in 96 isolates. Resistance to all four of these antibiotics was found in 62 isolates and resistance to five (the above plus tetracycline) in one isolate: a ribotype 001, toxinotype 0 strain. Whilst ribotype 001 was the most commonly encountered type, there was no evidence of clonal relationships when all other typing and antibiotic resistance patterns were taken into account.

"Dirty little secrets"--endotoxin contamination of recombinant proteins.

The identification of Toll-like receptors has revolutionised our understanding of innate immunity. TLR4 transduces the LPS signal and that of a number of structurally and functionally unrelated agonists. However, recent evidence adds to longstanding concerns that endotoxin contamination of bacterially derived recombinant TLR4 agonists is responsible for effects attributed to these molecules. We highlight key factors in differentiating specific agonist effects from those of endotoxin and emphasize why conventional methods of detecting and eliminating LPS may lead to erroneous results. We propose that considerable caution is needed in the investigation of TLR4 agonists, particularly when using proteins produced in a bacterium that also houses the most ideal TLR4 agonist, LPS.

The biological activity of a liposomal complete core lipopolysaccharide vaccine.

A vaccine that induces humoral immunity to lipopolysaccharide (LPS), while remaining non-pyrogenic should be beneficial, as high levels of antibodies against LPS are associated with a reduced risk of adverse outcome. However, pure LPS or bacteria expressing LPS are generally considered too toxic to be used as vaccines. Recently, a novel, immunogenic complete core lipopolysaccharide vaccine has been described, which has been designed to prevent endotoxin-related inflammatory reactions in surgical and high-risk hospitalized patients. In vivo studies have shown that while administration of the vaccine to rabbits results in no toxicity over 7 days, it does induce significantly enhanced antibody responses towards a broad range of clinically relevant Gram-negative LPSs. Here we show that encapsulation of the four complete core LPS types Escherichia coli K12, Escherichia coli R1, Bacteroides fragilis and Pseudomonas aeruginosa into liposomes greatly reduces the ability of a given amount of LPS to induce TNF-alpha production in vitro from human monocytes. In contrast to previous studies of liposomal LPS, we demonstrate a reduction in activity of approximately 100,000-fold; a reduction approximately 100-1,000-fold more than that previously described. The signalling by the liposomal LPS appears to be entirely dependent on serum factors, though this can be partially restored by soluble CD14 or, to a lesser extent, by lipopolysaccharide binding protein. Time-course experiments reveal that liposomal LPS signalling shows similar kinetics to pure LPS signalling. Therefore, as well as inducing specific antibody responses, liposomal LPS demonstrates characteristics suitable for use as a vaccine to be used in human beings.

Structure and function of lipopolysaccharides.

The lipopolysaccharides of Gram-negative bacteria have a profound effect on the mammalian immune system and are of great significance in the pathophysiology of many disease processes. Consideration is given in this review to the relationship between structure and function of these lipopolysaccharides.

The distribution of, and antibody response to, the core lipopolysaccharide region of Escherichia coli isolated from the faeces of healthy humans and cattle.

There are five different core types of Escherichia coli lipopolysaccharide (LPS), and enterohaemorrhagic E. coli tend to have the R3 core type. It has been hypothesized that increased carriage of bacteria with a specific core type will induce higher levels of antibodies and protect against disease caused by bacteria carrying that specific LPS core. Approximately 320 isolates of E. coli, half from healthy human faeces and half from healthy bovine faeces have been core typed both by core-specific monoclonal antibodies, and by PCR for genes encoding the enzymes responsible for the biosynthesis of the specific core structures. Results showed that E. coli possessing R1 core LPS were most frequently detected in both human and cattle populations (63 and 49%, respectively). Compared to the human isolates a significantly higher level of bacteria with R3 core LPS was detected among the bovine commensal E. coli (11% compared to 4%; P < 0.05). Antibody levels to each of the specific core types were measured in serum samples from healthy humans (n = 91) and healthy cattle (n = 39). In each population the highest level of antibody detected was reactive to the R4 core. In cattle the level of anti-R3 core antibody was significantly higher than the level of anti-R1, -R2 and -K12 antibodies (P < 0.01). In summary there was a greater proportion of E. coli with R3 core type in cattle, together with a corresponding higher anti-R3 antibody level. This suggests that cattle may have greater immunity to E. coli strains with an LPS of R3 core type.

Mucosal and systemic antibody responses to the lipopolysaccharide of Escherichia coli O157 in health and disease.

Mucosal immunity in the gastrointestinal (GI) tract is a primary defence against GI pathogens. We hypothesise that a mucosal response to lipopolysaccharide (LPS), especially to the common (core) determinants of GI pathogenic Escherichia coli strains, is protective. The aims of this study were to investigate the specificities, levels and development of humoral responses in health and GI disease to the R3 LPS core and O-polysaccharide of E. coli O157. The purpose was to try to predict whether vaccination or passive immunisation might induce protection. Wherever possible, paired whole gut lavage fluid (WGLF) and serum samples were collected for comparison of the mucosal and systemic responses. Matched saliva samples were also collected from some study groups. The patient groups included those with acute E. coli O157 disease (serum only), patients convalescing after E. coli O157 infections, and patients undergoing routine investigation for GI conditions but subsequently shown to be immunologically normal. Some samples of WGLF from patients with Crohn's disease (CRO) and ulcerative colitis (UC) were included to allow comparisons with patients with inflammatory conditions known to alter antibody secretion in the GI tract. The healthy groups from whom serum and saliva only were taken included blood donors, healthy volunteers and a group of slaughterhouse workers. This latter group was likely to have been exposed regularly to faecal bacteria from animals and antibody specificities might have been expected to be different from other healthy individuals. Levels and classes of antibodies were determined by ELISA with microtitration plates coated with polymyxin complexes of whole LPS extracted from E. coli O157 and LPS from the E. coli R3 rough mutant. Antibodies of IgG and IgM classes were measured in serum and IgA was measured in WGLF and saliva. IgG antibodies to the O157 LPS and the R3 core oligosaccharide were detected in the serum of healthy blood donors. Patients with acute E. coli O157 disease showed elevated levels of serum IgM to O157 LPS and R3, with IgG levels raised only to R3. In serum from convalescent patients, IgG to O157 LPS was significantly above the control groups only in the period 6-16 weeks after infection. Total IgA levels were similar in WGLF specimens from all groups, except the patients with UC, whose levels were much higher. Specific IgA levels were higher in the E. coli O157 convalescent group, but there were no significant correlations overall. UC patients had significantly lower levels of IgA to O157 and CRO patients had higher O157 IgA levels than UC patients and healthy volunteers. In serum, inhibition of ELISA showed that the response to the O157 LPS was due in part to a response to the R3 oligosaccharide component. This response was much more pronounced in the healthy and non-O157 groups than in convalescent patients. There was no correlation between specific IgA antibody levels in saliva and matched specimens of WGLF, and levels in sequential saliva specimens fluctuated widely. The significant IgG and IgA responses to the R3 core suggest that there is immunological memory to this oligosaccharide LPS component which may have a role in protection against E. coli LPS both systemically and locally in the GI tract. Boosting of this mucosal response to the LPS core, either naturally through exposure or by active or passive immunisation, may confer protection. Finally, antibody responses to E. coli O157 must be interpreted with caution, as the response detected is a sum of responses to the O-specific polysaccharide and the R3 core.

Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via toll-like receptor 2.

Recognition of bacterial lipopolysaccharide (LPS) is critical in the host defence against Gram-negative infection. While enterobacterial LPS signals via Toll-like receptor 4 (TLR4), it has recently been reported that the LPS of Leptospira interrogans, Legionella pneumophila, Rhizobium species Sin-1 and at least one strain of Porphyromonas gingivalis are capable of signalling via TLR2. Using a TLR transfection assay and measurement of an NF-kappaB-sensitive promoter region, the results show that the LPS of Bacteroides fragilis NCTC-9343, Chlamydia trachomatis LGV-1 and Pseudomonas aeruginosa PAC-611 also signal via TLR2 and it is pointed out that all TLR2-signalling LPS discovered to date demonstrate relatively weak endotoxicity in some models and structural features distinct from those LPS shown to signal via TLR4.