Andrew J. Lawson

Host Association of Campylobacter Genotypes Transcends Geographic Variation

ABSTRACT Genetic attribution of bacterial genotypes has become a major tool in the investigation of the epidemiology of campylobacteriosis and has implicated retail chicken meat as the major source of human infection in several countries. To investigate the robustness of this approach to the provenance of the reference data sets used, a collection of 742 Campylobacter jejuni and 261 Campylobacter coli isolates obtained from United Kingdom-sourced chicken meat was established and typed by multilocus sequence typing. Comparative analyses of the data with those from other isolates sourced from a variety of host animals and countries were undertaken by genetic attribution, genealogical, and population genetic approaches. The genotypes from the United Kingdom data set were highly diverse, yet structured into sequence types, clonal complexes, and genealogical groups very similar to those seen in chicken isolates from the Netherlands, the United States, and Senegal, but more distinct from isolates obtained from ruminant, swine, and wild bird sources. Assignment analyses consistently grouped isolates from different host animal sources regardless of geographical source; these associations were more robust than geographic associations across isolates from three continents. We conclude that, notwithstanding the high diversity of these pathogens, there is a strong signal of association of multilocus genotypes with particular hosts, which is greater than the geographic signal. These findings are consistent with local and international transmission of host-associated lineages among food animal species and provide a foundation for further improvements in genetic attribution.

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.

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.

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.

Molecular epidemiology of human Campylobacter jejuni shows association between seasonal and international patterns of disease

SUMMARY We sought to explain seasonality and other aspects of Campylobacter jejuni epidemiology by integrating population genetic and epidemiological analysis in a large 3-year longitudinal, two-centre, population-based study. Epidemiological information was collected for 1505 isolates, which were multilocus sequence-typed. Analyses compared pathogen population structure between areas, over time, and between clinical presentations. Pooled analysis was performed with published international datasets. Subtype association with virulence was not observed. UK sites had nearly identical C. jejuni populations. A clade formed by ST45 and ST283 clonal complexes showed a summer peak. This clade was common in a Finnish dataset but not in New Zealand and Australian collections, countries with less marked seasonality. The UK, New Zealand and Australian collections were otherwise similar. These findings map to known in-vitro differences of this clade. This identifies a target for studies to elucidate the drivers of the summer peak in human C. jejuni infection.

Persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline

OBJECTIVES The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline. METHODS Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined. RESULTS For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to >128 mg/L) contained tet(O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAbetaN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance. CONCLUSIONS Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population.

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

BackgroundFollowing 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.MethodsIn 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.ResultsOf 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).ConclusionWe 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.

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

Following Helicobacter pylori eradication in a placebo‐controlled trial, the hypokinesia of idiopathic parkinsonism improved but flexor rigidity worsened.

Genome Sequence of the Emerging Pathogen Helicobacter canadensis

We determined the genome sequence of the type strain of Helicobacter canadensis, an emerging human pathogen with diverse animal reservoirs. Potential virulence determinants carried by the genome include systems for N-linked glycosylation and capsular export. A protein-based phylogenetic analysis places H. canadensis close to Wolinella succinogenes.

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.