Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system.

Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg(2+) induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A.

Dispositional optimism and stress-induced changes in immunity and negative mood.

Evidence suggests that optimism may be protective for health during times of heightened stress, yet the mechanisms involved remain unclear. In a double-blind placebo-controlled study, we recently showed that acute psychological stress and an immune stimulus (Typhim-Vi typhoid vaccine) synergistically increased serum levels of interleukin-6 (IL-6) and negative mood in 59 healthy men. Here we carried out further analysis of this sample to investigate the relationship between dispositional optimism and stress-induced changes in immunity and mood. Volunteers were randomly assigned to one of four experimental conditions in which they received either typhoid vaccine or saline placebo, and then rested or completed two mental tasks. In the stress condition, optimism was inversely related to IL-6 responses, independent of age, BMI, trait CES-D depression and baseline IL-6. This relationship was present across both stress groups (combining vaccine and placebo) and was not present in the vaccine/stress group alone, suggesting that optimism protects against the inflammatory effects of stress rather than vaccine per se. Typhoid vaccine induced a significant increase in participants' circulating anti-Vi antibody levels. Stress had no effect on antibody responses overall. However, in the vaccine/stress group, there was a strong positive association between optimism and antibody responses, indicating that stress accentuated the antibody response to vaccine in optimists. Across the complete sample, more optimistic individuals had smaller increases in negative mood and less reduction in mental vigour. Together these findings suggest that optimism may promote health, by counteracting stress-induced increases in inflammation and boosting the adjuvant effects of acute stress.

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli.

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli, had lipopolysaccharide profiles quite distinct from the respective strain of E. coli. It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.

Human infections with verocytotoxin-producing Escherichia coli O157--10 years of E. coli O157 serodiagnosis.

From 1997 to 2007, the Laboratory of Enteric Pathogens (LEP), Health Protection Agency, UK, received sera from 2148 patients for testing for antibodies to the LPS of verocytotoxin-producing Escherichia coli (VTEC) O157. A total of 676 (31.5 %) sera had antibodies binding the LPS of E. coli O157 and the majority of patients were below the age of 10 years, a trend observed for both males and females. Antibody-positive patients had haemolytic uraemic syndrome (HUS) in 79.3 % of cases and most of these presented with the atypical (D-) form of HUS. Nine patients were shown to have antibodies to the LPS of E. coli belonging to serogroups O26 (4), O103 (2), O111 (1) and O145 (2) and one patient had antibodies to the somatic antigens of both E. coli O26 and O103. The serodiagnosis of infections with E. coli O157 and other VTEC continues to be an important adjunct to bacteriology. Where clinicians suspect the involvement of a VTEC in disease, patients' sera should be submitted to the LEP for analysis without delay.

Serodiagnosis of Salmonella enterica serovar Typhi and S. enterica serovars Paratyphi A, B and C human infections.

The aim of this study was to evaluate an immunoassay for the detection of human serum antibodies to the LPS and flagellar antigens of Salmonella Typhi and Salmonella Paratyphi A, B and C, and to the Vi capsular polysaccharide of S. Typhi and S. Paratyphi C. A total of 330 sera were used; these originated from 15 patients who were culture-positive for S. Typhi and 15 healthy controls, together with 300 sera submitted to the Laboratory of Enteric Pathogens for Salmonella serodiagnosis. By SDS-PAGE/immunoblotting, all 15 sera from culture-positive patients had serum antibodies to the 9,12 LPS antigens and 10 had antibodies to the 'd' flagellar antigens. Of the 300 reference sera, 22 had antibodies to the 9,12 LPS antigens, one to the 1,4,5,12 LPS antigens and 12 to the 6,7 LPS antigens. Only two sera had antibodies to flagellar antigens, one of which bound to the 'b' and the other to the 'd' antigen. An ELISA was developed that successfully detected serum antibodies to the Vi capsular polysaccharides, but because of the kinetics of serum antibody production to the Vi, these antibodies may be of limited value in the serodiagnosis of acute infection with S. Typhi and S. Paratyphi C. The immunoassays described here provide a sensitive means of detecting serum antibodies to the LPS, flagellar and Vi antigens of S. Typhi and S. Paratyphi, and constitute a viable replacement for the Widal assay for the screening of sera. The Salmonella serodiagnosis protocols described here are the new standard operating procedures used by the Health Protection Agency's National Salmonella Reference Centre based in the Laboratory of Enteric Pathogens, Colindale, UK.

Detection of antibodies to Pseudomonas aeruginosa in serum and oral fluid from patients with cystic fibrosis.

Cystic fibrosis (CF) patients who are chronically infected with Pseudomonas aeruginosa make serum antibodies to bacterial surface LPS as well as other pseudomonas antigens. This study investigated the feasibility of using oral fluid samples for the detection of pseudomonas antibodies in CF patients and compared these results with corresponding serum antibodies. Most strains of P. aeruginosa produce two forms of LPS molecule, termed A-band (described as a common antigen) and B-band (O-serotype-specific antigen), apparently bound to a common core oligosaccharide moiety. A-band LPS was demonstrated in 45 out of 49 clinical isolates of P. aeruginosa by SDS-PAGE and immunoblotting with a specific antibody. Oral fluids were collected from 17 adult CF patients, all of whom were sputum culture positive for P. aeruginosa (13 also provided serum samples), 11 primary ciliary dyskinesia (PCD) patients and 37 healthy volunteers. Antibodies to A-band LPS were detected by immunoblotting in all of the CF patients' oral fluids but 10 of the volunteer samples gave weak reactions with immunoblotting. Six of the PCD patients gave a weak reaction with A-band antibodies and only one demonstrated antibodies to core LPS. In a quantitative ELISA, 15 of the 17 CF patients' oral fluids were shown to contain antibodies to A-band LPS, whilst none of the volunteer samples contained antibodies to A-band LPS. All serum samples from the CF patients were positive by both methods. Thus this is a sensitive procedure for the detection of antibodies to A-band LPS of P. aeruginosa in oral fluid and serum from patients with CF.

Detection of enteroaggregative Escherichia coli in faecal samples from patients in the community with diarrhoea.

The aim of this study was to assess the usefulness of a multiplex PCR assay targeting the aat, aaiA and astA genes for the detection of typical and atypical enteroaggregative Escherichia coli (EAEC) in bacterial cultures from faecal samples from patients with community-acquired diarrhoea. The isolates harbouring these genes were also tested using the HEp-2 cell-adhesion assay to clarify their EAEC status. aat, aai or astA was found in E. coli faecal isolates from 39 (7.8 %) of 500 patients, and 20 of these strains adhered to HEp-2 cells in a pattern characteristic of EAEC. Eight isolates carrying the aai or astA gene but not the aat gene were shown to be HEp-2 cell test positive, although 12 strains with this genotype were HEp-2 cell test negative. Using the HEp-2 adhesion assay as the gold standard, the addition of primers detecting aaiA and astA to the aat PCR increased the number of EAEC isolates detected, but identified strains of E. coli that were not EAEC. The variety of genotypes exhibiting aggregative adherence highlights the problems associated with developing a molecular diagnostic test for EAEC. This PCR assay detects a variety of strains exhibiting characteristics of the EAEC group, making it a useful tool for identifying both typical and atypical EAEC.

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis.

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.

Human serum amyloid P component protects against Escherichia coli O157:H7 Shiga toxin 2 in vivo: therapeutic implications for hemolytic-uremic syndrome.

Shiga toxin (Stx) 2 causes hemolytic-uremic syndrome (HUS), an intractable and often fatal complication of enterohemorrhagic Escherichia coli O157:H7 infection. Here, we show that serum amyloid P component (SAP), a normal human plasma protein, specifically protects mice against the lethal toxicity of Stx2, both when injected into wild-type mice and when expressed transgenically; in the presence of human SAP, there was greatly reduced in vivo localization of Stx2 to the kidneys, suggesting a possible mechanism of protection. In humans, circulating SAP concentrations did not differ between patients with suspected enterohemorrhagic E. coli infection with antibodies to E. coli O157:H7 lipopolysaccharide and those without antibodies or between patients with HUS and those without it. However, the potent protection conferred by human SAP in the mouse model suggests that infusion of supplemental SAP may be a useful novel therapeutic approach to the treatment of this devastating condition.

Genotyping of enteroaggregative Escherichia coli and identification of target genes for the detection of both typical and atypical strains.

Enteroaggregative Escherichia coli (EAggEC) is an important cause of diarrhea worldwide, and there is a need for better detection methods in diagnostic laboratories. The aims of this study were i) to characterize strains of EAggEC by assigning each isolate a genotypic profile and (ii) to determine target genes for the detection of both typical and atypical EAggEC. The heterogeneity of the EAggEC group makes selection of a single target gene difficult. The plasmid-encoded genes, aat, aggR, and aap, are all appropriate targets for the detection of typical EAggEC. Of the chromosomally encoded genes, aaiA would be the most suitable target gene to identify typical and atypical EAggEC. The astA gene, encoding the enteroaggregative heat stable toxin, although not specific for EAggEC, may be used effectively in combination with other specific EAggEC genes. A polymerase chain reaction test based on the detection of characteristic EAggEC virulence genes, such as aat, astA, and aaiA, would improve EAggEC diagnosis.

Use of a microarray to assess the distribution of plasmid and chromosomal virulence genes in strains of enteroaggregative Escherichia coli.

A DNA microarray was used to analyze the distribution of plasmid and chromosomal genes among strains of enteroaggregative Escherichia coli (EAEC) isolated from a prospective diarrhoea surveillance study in the United Kingdom. Target genes were extracted from existing databases and from the genome sequence of prototype EAEC strain 042. We found that strains exhibiting the aggregative adherence (AA) phenotype could be broadly divided into two groups depending upon whether they harboured genes from the EAEC virulence plasmid (pAA) and a set of chromosomal genes found in EAEC strain 042. Several chromosomal loci were inherited en bloc, and were more common in strains which we designated Group 1; genes at the pheU locus were particularly conserved. Genes encoded on the pAA plasmid and those under control of the master regulator AggR were also concentrated in the Group 1 EAEC. A gene encoding a type 1 pilin allele was detected more frequently in Group 2 EAEC. Our data suggest that strains previously designated as typical EAEC harbour a large number of conserved plasmid and chromosomal loci, further illuminating a package of virulence genes common to the most important EAEC.

Childhood hemolytic uremic syndrome, United Kingdom and Ireland.

We conducted prospective surveillance of childhood hemolytic uremic syndrome (HUS) from 1997 to 2001 to describe disease incidence and clinical, epidemiologic and microbiologic characteristics. We compared our findings, where possible, with those of a previous study conducted from 1985 to 1988. The average annual incidence of HUS for the United Kingdom and Ireland (0.71/100,000) was unchanged from 1985 to 1988. The overall early mortality had halved, but the reduction in mortality was almost entirely accounted for by improved outcome in patients with diarrhea-associated HUS. The principal infective cause of diarrhea-associated HUS was Shiga toxin-producing Escherichia coli O157 (STEC O157), although in the 1997-2001 survey STEC O157 phage type (PT) 21/28 had replaced STEC O157 PT2 as the predominant PT. The risk of developing diarrhea-associated HUS was significantly higher in children infected with STEC O157 PT 2 and PT 21/28 compared with other PTs. Hypertension as a complication of HUS was greatly reduced in patients with diarrhea-associated HUS.

Analysis of saliva for antibodies to the LPS of Escherichia coli O157 in patients with serum antibodies to E. coli O157 LPS.

The salivary antibody response to the Escherichia coli O157 LPS antigen was assessed in 44 patients with serum antibodies binding to the LPS of E. coli O157. Saliva from 477 controls was also examined to assess the specificity of the immunoassay used. Twenty of the 44 patients had salivary antibodies to E. coli O157 LPS, giving the salivary antibody test a sensitivity of 0.45 and a predictive positive value for seropositivity of 1.00. The presence of these antibodies appeared not to relate to the time interval between serum sampling and saliva sampling. None of the 477 volunteers had salivary antibodies binding to the LPS of E. coli O157 alone; however, 15 had antibodies which bound non-specifically to both O157 LPS and BSA.

Interactions of the gastrotropic bacterium Helicobacter pylori with the leukocyte-endothelium adhesion molecules, the selectins--a preliminary report.

The deleterious effects of Helicobacter pylori infection of the stomach are largely the result of a vigorous chronic inflammatory response, and include chronic gastritis, peptic ulceration and gastric cancer. We are exploring the possibility that carbohydrate components on H. pylori contribute to the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. Lipopolysaccharides of most H. pylori strains contain sequences related to the Lewis (Le(x) or Le(a)) antigens. Carbohydrate sequences of this family encompass ligands for the leukocyte-endothelium adhesion molecules of the host, namely, the E- and P-selectins, which are expressed on inflamed endothelia, and L-selectin, which is constitutively expressed on leukocytes. Here we investigate H. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer for their interactions with the selectins. Our results provide unequivocal evidence of interactions of isolates from each of the diagnostic groups with E- and L-selectins.

Production of serum antibodies that recognise epitopes located on the R3 region of Escherichia coli core lipopolysaccharides by patients infected with strains of enterohaemorrhagic E. coli.

Antibody-antigen cross-reactions were examined with sera from patients with Escherichia coli O157 infection and lipopolysaccharide (LPS) purified from a range of enterohaemorrhagic E. coli (EHEC) including those belonging to serogroups O26, O103, O111, O145 and O157. Six of 10 patients infected with an O157 EHEC produced serum antibodies that cross-reacted with common LPS-core epitopes, which were expressed by 23 of 33 strains of EHEC examined. These common LPS-core epitopes were also present on strains of E. coli O26 which did not produce verocytotoxin. These cross-reacting antibodies did not influence the basic immunoblotting procedures used for the routine serodiagnosis of infections with E. coli O157.

The kinetics of antibody production to antigens of Escherichia coli O157 in a pregnant woman with haemolytic uraemic syndrome.

Sequential blood samples taken from a pregnant woman with haemolytic uraemic syndrome caused by verocytotoxin (VT)-producing Escherichia coli O157 were used to examine the kinetics of serum antibody production to E. coli O157 lipopolysaccharide (LPS), intimin and the conserved region of the translocated intimin receptor (Tir-M). Umbilical cord blood and two samples of blood from the newborn baby were also examined for antibodies to these antigens. In the mother, antibodies of the IgM class, specific for E. coli O157 LPS, were produced in the initial stages of the infection, reaching a peak at 9 days after onset of diarrhoea and subsiding 3 days later. High levels of IgG class antibodies, specific for E. coli O157 LPS, were detected 8 days after the onset of diarrhoea and were present at high titres on day 18. Serum antibodies of the IgA class to E. coli O157 LPS were not detected. Antibodies binding to Tir-M were detected 8 days after the onset of diarrhoea and high titres of these antibodies were still present on day 18. Serum antibodies to intimin were not detected in the mother and no antibodies to any of the antigens tested were detected in either the baby's blood or cord blood. This study describes for the first time the kinetics of serum antibody production during pregnancy, to selected antigens expressed by E. coli O157.

Invasiveness of Salmonella serotypes Typhimurium and Enteritidis of human gastro-enteritic origin for rabbit ileum: role of LPS, plasmids and host factors.

An organ culture system involving explants of distal rabbit ileum was used to study the roles of lipopolysaccharide (LPS) and plasmids in primary invasiveness for enterocytes in situ of strains of Salmonella serotypes Typhimurium and Enteritidis. Long-chain LPS per se does not confer invasiveness on Typhimurium, as known avirulent, hypo-invasive strains express smooth LPS. However, the invasiveness of a naturally occurring rough isogenic derivative of Salmonella serotype Enteritidis PT 4 was about half that of its wild-type parent. Therefore, smooth LPS appears to play a secondary role in maximising invasiveness. No evidence was found to correlate primary invasiveness for gut of 18 strains of Typhimurium with plasmid profiles in general or with the 60-MDa serovar-specific virulence plasmid in particular. Evidence is presented that strongly suggests a seasonal variability in susceptibility of rabbit gut to invasion by Typhimurium. Although no explanation is given for this summer insusceptibility, the data indicate the importance of the physiological status of the host in relation to susceptibility to invasion by Salmonella.

Haemolysin production by strains of Verocytotoxin-producing Escherichia coli.

Twenty-one strains of Verocytotoxin-producing Escherichia coli (VTEC) that hybridized with DNA probe CVD419 were examined for the ability to produce haemolysin. With solid media, all strains produced most haemolysin when grown in blood agar tubes and least when grown on blood agar plates incubated in air. Haemolysin production was increased considerably by incubating blood agar plates in an atmosphere comprising 8% carbon dioxide, 40% hydrogen and 52% nitrogen at 37 degrees C for 16 h, followed by 6 h at 21 degrees C in air. Haemolysin production was also increased when strains were grown on L-agar containing the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) prior to subculture on blood agar. Intracellular haemolysin was detected in five out of the 21 strains of E. coli grown on L-agar in the atmosphere described above, but haemolysin was not detected in L-broth culture supernatants. The haemolysins lysed guinea pig, mouse and ferret erythrocytes, but not human, rabbit, rat, turkey or chicken erythrocytes. Also, the addition of calcium ions to culture media was not required for haemolytic activity. It was concluded that haemolysins produced by VTEC appear to be quite distinct from E. coli alpha-haemolysin and resemble a form of beta-haemolysin.