Antimicrobial surveillance in idiopathic parkinsonism: indication-specific improvement in hypokinesia following Helicobacter pylori eradication and non-specific effect of antimicrobials for other indications in worsening rigidity.

BACKGROUND: Following Helicobacter pylori eradication in a placebo-controlled trial, the hypokinesia of idiopathic parkinsonism improved but flexor rigidity worsened. METHODS: We surveyed the effect of all antimicrobial prescriptions in 66 patients with idiopathic parkinsonism over a median of 1.9 (interquartile range 0.4, 3.5) years. Initial Helicobacter screening was followed (where positive) by gastric biopsy. Serial lactulose hydrogen breath tests (364 tests) for small intestinal bacterial overgrowth monitored the need to encourage fluid intake and bulk/osmotic laxatives. We measured hypokinesia (401 assessments of mean stride length at free walking speed in 58 patients) and upper limb flexor rigidity (396 assessments in 49). RESULTS: Following successful H. pylori eradication (12 cases) but not failed (2), stride increased in entire group (including those receiving levodopa), core group (those receiving only longer-t½ antiparkinsonian medication or untreated) and untreated (p = .001 each case). The effect was greater with less antiparkinsonian medication (19 (95% CI, 14, 25) cm/year in untreated). Flexor rigidity was unchanged. Following antimicrobials for other indications (75 courses), hypokinesia was unchanged. However, flexor rigidity increased cumulatively. It increased in core group only after a first course (by (10 (0, 20)%/year, p = .05)), but then in entire, core and untreated after a second course (18 (6, 31), 33 (19, 48) and 29 (12, 48)%/year respectively; p = .002, .001 and .001) and further still after a third (17 (2, 34), 23 (8, 41) and 38 (15, 65)%/year; p = .02, .003 and .001). Initially, 40/66 were lactulose hydrogen breath test positive. Odds for positivity fell with time (by 59 (46, 75)%/year, p = .001) and tended to be lower with Helicobacter positivity (28 (8, 104)%, p = .06), but were unrelated to other antimicrobial interventions. CONCLUSIONS: Improved hypokinesia following antimicrobials appeared unique to Helicobacter eradication. Rigidity increased following successive antimicrobial exposures for other indications, despite diminishing lactulose hydrogen breath test positivity.

Characterization of the structurally diverse N-linked glycans of Campylobacter species.

The Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In this study, we have demonstrated that only Campylobacter species closely related to C. jejuni produce glycoproteins that interact with both a C. jejuni N-linked-glycan-specific antiserum and a lectin known to bind to the C. jejuni N-linked glycan. In order to further investigate the structure of Campylobacter N-linked glycans, we employed an in vitro peptide glycosylation assay combined with mass spectrometry to demonstrate that Campylobacter species produce a range of structurally distinct N-linked glycans with variations in the number of sugar residues (penta-, hexa-, and heptasaccharides), the presence of branching sugars, and monosaccharide content. These data considerably expand our knowledge of bacterial N-linked glycan structure and provide a framework for investigating the role of glycosyltransferases and sugar biosynthesis enzymes in glycoprotein biosynthesis with practical implications for synthetic biology and glycoengineering.

Imaging the Rex vein preoperatively using wedged hepatic venous portography.

BACKGROUND: Mesentericoportal bypass, the Rex shunt, restores the physiological hepatic portal flow and reduces the clinical sequelae of portal hypertension in children with extrahepatic portal vein obstruction (EHPVO). The preoperative evaluation includes an accurate assessment of the portal venous inflow and outflow. The former is readily assessed by ultrasound and MRI, while the outflow intrahepatic portal vein is harder to assess. PURPOSE: To assess the efficacy of wedged hepatic venous portography (WHVP) at detecting a patent Rex vein preoperatively in children with EHPVO who were considered for mesenterico-portal bypass. MATERIALS AND METHODS: A single-institution retrospective chart review was undertaken for children with EHPVO who had been considered for a mesoportal bypass between January 2001 and January 2010. RESULTS: Sixteen children were considered for mesoportal bypass, including four post reduced-size liver transplant patients. Ten children (63%) underwent WHVP. The Rex vein was clearly identified in 8/10 (80%). One Rex vein was seen at surgery but not demonstrated at WHVP. Six mesoportal bypasses were performed without WHVP, of which three (50%) were successful. CONCLUSION: WHVP had a sensitivity of more than 80% and specificity of 100% in the preoperative patency assessment of the Rex vein.

Characterization of N-linked protein glycosylation in Helicobacter pullorum.

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.

Genetic relationships among reptilian and mammalian Campylobacter fetus strains determined by multilocus sequence typing.

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared approximately 90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two groups. The reptile group represents a possible separate genomospecies capable of infecting humans.

Differential effect of Helicobacter pylori eradication on time-trends in brady/hypokinesia and rigidity in idiopathic parkinsonism.

BACKGROUND: We examine the effect of eradicating Helicobacter in idiopathic parkinsonism (IP). Marked deterioration, where eradication-therapy failed, prompted an interim report in the first 20 probands to reach de-blinding. The null-hypothesis, "eradication has no effect on principal outcome, mean stride length at free-walking speed," was rejected. We report on study completion in all 30 who had commenced post-treatment assessments. METHODS: This is a randomized, placebo-controlled, parallel-group efficacy study of eradicating biopsy-proven (culture and/or organism on histopathology) Helicobacter pylori infection on the time course of facets of IP, in probands taking no, or stable long-t(1/2), anti-parkinsonian medication. Persistent infection at de-blinding (scheduled 1-year post-treatment) led to open active eradication-treatment. RESULTS: Stride length improved (73 (95% CI 14-131) mm/year, p = .01) in favor of "successful" blinded active over placebo, irrespective of anti-parkinsonian medication, and despite worsening upper limb flexor rigidity (237 (57-416) Nm x 10(-3)/year, p = .01). This differential effect was echoed following open active, post-placebo. Gait did not deteriorate in year 2 and 3 post-eradication. Anti-nuclear antibody was present in all four proven (two by molecular microbiology only) eradication failures. In the remainder, it marked poorer response during the year after eradication therapy, possibly indicating residual "low-density" infection. We illustrate the importance of eradicating low-density infection, detected only by molecular microbiology, in a proband not receiving anti-parkinsonian medication. Stride length improved (424 (379-468) mm for 15 months post-eradication, p = .001), correction of deficit continuing to 3.4 years. Flexor rigidity increased before hydrogen-breath-test positivity for small intestinal bacterial overgrowth (208 (28-388) Nm x 10(-3), p = .02), increased further during (171 (67-274), p = .001) (15-31 months), and decreased (136 (6-267), p = .04) after restoration of negativity (32-41 months). CONCLUSION: Helicobacter is an arbiter of progression, independent of infection-load.

Creation of a large deletion mutant of Campylobacter jejuni reveals that the lipooligosaccharide gene cluster is not required for viability.

Deletion of the lipooligosaccharide biosynthesis region (Cj1132c to Cj1152c) from the genome of Campylobacter jejuni NCTC11168 shows that the core is not required for viability. The mutant was attenuated for growth and has increased sensitivity to antibiotics and detergents. Natural transformation and invasion of cultured host cells was abolished.

Amoxicillin therapy of poultry flocks: effect upon the selection of amoxicillin-resistant commensal Campylobacter spp.

BACKGROUND: The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance. METHODS: Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined. RESULTS: Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates. CONCLUSIONS: Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Investigation of the biological relevance of Helicobacter pylori cagE locus diversity, presence of CagA tyrosine phosphorylation motifs and vacuolating cytotoxin genotype on IL-8 induction in gastric epithelial cells.

Isolates of Helicobacter pylori from dyspeptic patients in England and South Africa were tested for ability to induce interleukin-8 (IL-8) in gastric cells. All isolates were cagA-positive, which was used as a marker for the presence of the cag pathogenicity island. The aims were to determine if activities were related to diversity within cagE (HP0544), a locus encoding a key component in the Type IV secretion system, and if disease severity might be linked to a combination of strain features. We found that isolates were heterogeneous in ability to induce IL-8 activity with the 23 positive isolates (59%) showing activities ranging from 260 to 3200 pg ml(-1). The cagE locus was detected in most isolates and RFLP analysis of a 1.52-kb internal fragment showed interstrain diversity with 12 combined (MboI/NlaIII) types. Most cagE genotypes were not associated with IL-8 induction, however two genotypes were found only in IL-8-inducing strains and one genotype was associated with lack of IL-8 induction. IL-8 activity was not associated with either the number or composition of cagA tyrosine phosphorylation motifs and vacA m-type. Although we found a weak association between cagE type and the ability to induce IL-8, our results imply that gastric cell factors or bacterial factors other than vacA, cagA and cagE are involved in the induction of IL-8 and the development of severe gastric disease.

Application of 3'-mismatched reverse primer PCR compared with real-time PCR and PCR-RFLP for the rapid detection of 23S rDNA mutations associated with clarithromycin resistance in Helicobacter pylori.

Helicobacter pylori clarithromycin (Cla) resistance dramatically reduces efficacy of eradication therapy. In this study, 3'-mismatched reverse primer PCR (3M-PCR), real-time PCR (LightCycler), and PCR-RFLP assays were investigated to determine their sensitivity for detecting clarithromycin resistance associated with 23S rDNA mutations (A2142G, A2142C, and A2143G). For 84.8% (123/145) of isolates, the same allelic type was detected by each method although methods differed in efficiency of detecting mutations in cultures either containing mixtures of two alleles (24 isolates), or that were dual allelic variants (two isolates). The novel 3M-PCR assay format was the most sensitive, detecting all alleles at > or =0.02 ng/microl in DNA mixtures, and thus provides more precise information to guide clinical management of patients at risk of treatment failure.

Discovering new pathogens: culture-resistant bacteria.

Recent advances in gene-amplification technology and molecular phylogenetics have provided the means of detecting and classifying bacteria directly from their natural habitats without the need for culture. These techniques have revolutionized environmental microbiology, and it is now apparent that the global diversity of microorganisms is much greater than previously thought. In the context of clinical microbiology, this molecular-based approach has facilitated the characterization of culture-resistant bacteria associated with human disease. Examples include Helicobacter heilmannii, a cause of gastritis, Tropheryma whippeli (the agent of Whipple's disease), and the agents of human ehrlichiosis and bacillary angiomatosis. Molecular-based techniques also provide a means of investigating complex bacterial flora within the human ecosystem, such as feces and dental plaque, without the bias of culture-based isolation. This has given a new perspective to the study of polymicrobial infections such as gingivitis, and offers the potential for the detection and identification of novel bacterial pathogens from among complex and numerous endogenous microbial flora.

Leukocyte-subset counts in idiopathic parkinsonism provide clues to a pathogenic pathway involving small intestinal bacterial overgrowth. A surveillance study.

BACKGROUND: Following Helicobacter pylori eradication in idiopathic parkinsonism (IP), hypokinesia improved but flexor-rigidity increased. Small intestinal bacterial-overgrowth (SIBO) is a candidate driver of the rigidity: hydrogen-breath-test-positivity is common in IP and case histories suggest that Helicobacter keeps SIBO at bay. METHODS: In a surveillance study, we explore relationships of IP-facets to peripheral immune/inflammatory-activation, in light of presence/absence of Helicobacter infection (urea-breath- and/or stool-antigen-test: positivity confirmed by gastric-biopsy) and hydrogen-breath-test status for SIBO (positivity: >20 ppm increment, 2 consecutive 15-min readings, within 2h of 25G lactulose). We question whether any relationships found between facets and blood leukocyte subset counts stand in patients free from anti-parkinsonian drugs, and are robust enough to defy fluctuations in performance consequent on short t½ therapy. RESULTS: Of 51 IP-probands, 36 had current or past Helicobacter infection on entry, 25 having undergone successful eradication (median 3.4 years before). Thirty-four were hydrogen-breath-test-positive initially, 42 at sometime (343 tests) during surveillance (2.8 years). Hydrogen-breath-test-positivity was associated inversely with Helicobacter-positivity (OR 0.20 (95% CI 0.04, 0.99), p<0.05).In 38 patients (untreated (17) or on stable long-t½ IP-medication), the higher the natural-killer count, the shorter stride, slower gait and greater flexor-rigidity (by mean 49 (14, 85) mm, 54 (3, 104) mm.s-1, 89 (2, 177) Nm.10-3, per 100 cells.µl-1 increment, p=0.007, 0.04 & 0.04 respectively, adjusted for patient characteristics). T-helper count was inversely associated with flexor-rigidity before (p=0.01) and after adjustment for natural-killer count (-36(-63, -10) Nm.10-3 per 100 cells.µl-1, p=0.007). Neutrophil count was inversely associated with tremor (visual analogue scale, p=0.01). Effect-sizes were independent of IP-medication, and not masked by including 13 patients receiving levodopa (except natural-killer count on flexor-rigidity). Cellular associations held after allowing for potentially confounding effect of hydrogen-breath-test or Helicobacter status. Moreover, additional reduction in stride and speed (68 (24, 112) mm & 103 (38, 168) mm.s-1, each p=0.002) was seen with Helicobacter-positivity. Hydrogen-breath-test-positivity, itself, was associated with higher natural-killer and T-helper counts, lower neutrophils (p=0.005, 0.02 & 0.008). CONCLUSION: We propose a rigidity-associated subordinate pathway, flagged by a higher natural-killer count, tempered by a higher T-helper, against which Helicobacter protects by keeping SIBO at bay.

Blood profile holds clues to role of infection in a premonitory state for idiopathic parkinsonism and of gastrointestinal infection in established disease.

The two-stage neuroinflammatory process, containment and progression, proposed to underlie neurodegeneration may predicate on systemic inflammation arising from the gastrointestinal tract. Helicobacter infection has been described as one switch in the pathogenic-circuitry of idiopathic parkinsonism (IP): eradication modifies disease progression and marked deterioration accompanies eradication-failure. Moreover, serum Helicobacter-antibody-profile predicts presence, severity and progression of IP. Slow gastrointestinal-transit precedes IP-diagnosis and becomes increasingly-apparent after, predisposing to small-intestinal bacterial-overgrowth (SIBO). Although IP is well-described as a systemic illness with a long prodrome, there has been no comprehensive overview of the blood profile. Here, it is examined in relation to Helicobacter status and lactulose-hydrogen-breath-testing for SIBO. A robust finding of reduced lymphocyte count in 126 IP-probands and 79 spouses (without clinically-definite IP), compared with that in 381 controls (p < 0.001 in each case), was not explained by Helicobacter-status or breath-hydrogen. This complements a previous report that spouses were 'down-the-pathway' to 'clinically-definite' disease. In 205 other controls without clinically-definite IP, there were strong associations between sporadic cardinal features and immunoglobulin class concentration, not explained by Helicobacter-status. Premonitory states for idiopathic parkinsonism associated with relative lymphopenia, higher serum immunoglobulin concentrations and evidence of enteric-nervous-system damage may prove viral in origin.Although only 8% of the above 79 spouses were urea-breath-test-positive for Helicobacter, all 8 spouses with clinically-definite IP were (p < 0.0001). Transmission of a 'primer' to a Helicobacter-colonised recipient might result in progression to the diagnostic threshold. Twenty-five percent of the 126 probands were seropositive for anti-nuclear autoantibody. In 20 probands, monitored before and serially after anti-Helicobacter therapy, seropositivity marked a severe hypokinetic response (p = 0.03). It may alert to continuing infection, even at low-density. Hyperhomocysteinemia is a risk factor for dementia and depression. Serum homocysteine exceeded the target in 43% of the 126 IP-probands. It was partially explained by serum B12 (12% variance, p < 0.001), but not by Helicobacter-status (gastric-atrophy uncommon in IP) or levodopa treatment. Immune-inflammatory activation increases homocysteine production. Since an estimated 60% of probands are hydrogen-breath-test positive, SIBO, with its increased bacterial utilisation of B12, is a likely cause. Thus, two prognostic indicators in established IP fit with involvement of Helicobacter and SIBO.

Real-time PCR detection and frequency of 16S rDNA mutations associated with resistance and reduced susceptibility to tetracycline in Helicobacter pylori from England and Wales.

OBJECTIVES: To investigate the occurrence of 16S rDNA mutations associated with resistance or reduced susceptibility to tetracycline in Helicobacter pylori isolated in England and Wales, and to develop a real-time PCR assay to detect these DNA polymorphisms from culture and gastric biopsies. METHODS: Tetracycline susceptibility was determined by disc diffusion. The MIC of isolates with reduced susceptibility was determined by Etest and agar dilution methods. The 16S rDNA of these isolates was sequenced and resistance-associated mutations identified. A LightCycler assay developed to detect these mutations was applied to DNA extracted from culture and gastric biopsies. RESULTS: From 1006 isolates of H. pylori examined, 18 showed reduced susceptibility to tetracycline. Of these, three were resistant (>or=4 mg/L). Mutations in 16S rDNA were detected in 10 of the reduced susceptibility isolates: one double base mutation of A926T/A928C, one A926C, one A928C; and seven A926G. The first two polymorphisms were novel and had not been reported from clinical isolates previously. The LightCycler assay identified each of the 10 isolates with 16S rDNA mutations, but did not detect polymorphisms in 100 tetracycline-susceptible H. pylori isolates. The assay correctly determined the tetracycline susceptibility of H. pylori in 20 gastric biopsy samples. CONCLUSIONS: Mutations in 16S rDNA were detected in H. pylori isolated in England and Wales with reduced susceptibility to tetracycline, but resistance to this antibiotic was uncommon. We show molecular-based susceptibility testing for tetracycline is possible direct from biopsy material.

16S rRNA gene sequences of 'Candidatus Campylobacter hominis', a novel uncultivated species, are found in the gastrointestinal tract of healthy humans.

Although some Campylobacter species are agents of gastroenteritis and periodontal disease in humans, little is known of the variety of campylobacters in the gastrointestinal tract of healthy individuals. This paper provides evidence for the existence of a previously undescribed, uncultivated Campylobacter species that may be a commensal in the healthy human gut. Saliva and faeces from 20 healthy individuals were examined by PCR assays specific for nine species of campylobacter (C. sputorum, C. concisus, C. upsaliensis, C. helveticus, C. lari, C. fetus, C. hyointestinalis, C. jejuni and C. coli) and for the genus as a whole. Genus-specific amplicons were produced from 19 of 20 saliva samples and from 18 of 20 faecal samples. C. concisus species-specific amplicons were produced from 19 of 20 saliva samples and 3 of 20 faecal samples. The faecal samples were all PCR-negative for other Campylobacter species. Three unidentified 16S rRNA Campylobacter genus-specific amplicons of faecal origin were sequenced. Phylogenetic analysis showed that these sequences were 99% similar, and clustered within the genus as a novel group which was termed HS (HS = healthy subject). A PCR primer pair specific for the HS group was designed from the sequence data and used to reexamine the original samples. Although it was not possible to culture the organism from faeces, specific PCR assay detected it in 10 of the 20 faecal samples, but not in any corresponding saliva samples. The authors propose that the source of the amplicons is a previously undescribed and so far uncultivated species, which they term 'Candidatus Campylobacter hominis'.

Fluorescent amplified fragment length polymorphism subtyping of multiresistant Salmonella enterica serovar Typhimurium DT104.

Fluorescent amplified fragment length polymorphism (FAFLP) subtyping analysis was used to genotype multiresistant Salmonella enterica serovar Typhimurium definitive phage type 104. Thirteen distinct FAFLP profiles were found among 85 isolates exhibiting identical pulsed-field gel electrophoresis (PFGE) profiles. A single FAFLP profile was shared by 93% of outbreak-associated isolates and 82% of sporadic isolates. This study demonstrates the value of FAFLP as a high-resolution tool for epidemiological investigation of Salmonella.

Multiply resistant (MR) Salmonella enterica serotype Typhimurium DT 12 and DT 120: a case of MR DT 104 in disguise?

Multiresistant Salmonella enterica serotype Typhimurium definitive phage type (DT) 12 and DT 120 are more closely related to DT 104 than to non-multiresistant strains of their respective phage types. Multiresistant DT 12 and DT 120 appear to have arisen due to changes in phage susceptibility of DT 104 rather than horizontal transfer of resistance genes.

Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis.

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.

Role of electronic data exchange in an international outbreak caused by Salmonella enterica serotype Typhimurium DT204b.

From July through September 2000, patients in five European countries were infected with a multidrug-resistant strain of Salmonella Typhimurium DT204b. Epidemiologic investigations were facilitated by the transmission of electronic images (Tagged Image Files) of pulsed-field gel electrophoresis profiles. This investigation highlights the importance of standardized protocols for molecular typing in international outbreaks of foodborne disease.