Whole-Genome Comparison of Two Campylobacter jejuni Isolates of the Same Sequence Type Reveals Multiple Loci of Different Ancestral Lineage

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in 1.659 Mb (H22082) and 1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of 2 kb. This includes a 12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates

Molecular epidemiology of campylobacteriosis and evolution of Campylobacter jejuni ST-474 in New Zealand : a thesis presented in partial fullfilment [sic] of the requirements for the degree of Doctor of Philosophy at Massey University, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand

Population genetics and phylogenetics have the potential to provide enormous insights into the epidemiology and ecology of disease causing pathogens. Molecular datasets are the basis to infer population structure, gene flow (between host populations and between different geographical locations) and to predict the evolutionary dynamics of pathogens. Campylobacter colonisation in food producing animals has been extensively studied and the population structure and host association of C. jejuni, the most commonly reported gastro-enteric pathogen, has also been well defined. In contrast, host-pathogen relationships and the population structure of C. jejuni in urban wild birds and pets have not been well defined on a wide range of spatial and/or temporal scales. A greater understanding of these details should allow disease control authorities to track the transmission of pathogens from one host species to another, identify the origin of pathogens and to better understand environmental factors influencing underlying molecular mechanisms. In the first study in this thesis the presence of C. jejuni in mallard ducks and starlings within five playgrounds in Palmerston North, New Zealand was studied. The prevalence of Campylobacter and C. jejuni in both species showed a bimodal seasonal pattern. The population structure and population differentiation of C. jejuni in these species were examined using multilocus sequence typing (MLST). Rarefaction analyses showed that the C. jejuni populations within mallard ducks were more diverse than starlings, particularly during the winter. Pairwise fixation indices showed that the population of C. jejuni in ducks was significantly different from that of starlings and that it differed over time. Conspicuous host association was evident with clonal complexes of C. jejuni such as ST-1034, ST-692 and ST-1332 specific to ducks and ST-177 and ST-682 specific to starlings. In addition, a larger proportion of C. jejuni genotypes that could not be assigned a clonal complex were found in both ducks and starlings, particularly during the winter. In the second study, C. jejuni from domestic pets (dogs and cats) were characterised using MLST and by typing the cell surface antigens, porA and flaA. The ST-45 complex, a clonal complex predominantly reported in human campylobacteriosis cases, was found to be the predominant clone present in both species. These findings shed some light on the contribution of pets as a putative source of human campylobacteriosis cases in New Zealand. In the third study, the ST-474 C. jejuni genotype, considered to be the endemic strain in New Zealand, was isolated from human cases and poultry carcasses from the Manawatu region from 2005 to 2009. Seven samples of ST-474 were sequenced and a subset of 50 full length genes were studied. These analyses demonstrated molecular differences between full length genes that were identical in the region used for MLST. Further, alleles characteristic of the ST-474 genome within the investigated metabolic housekeeping genes (n = 25) were identified. Our findings were that ST-474 genome is genetically distinct from other C. jejuni reference genomes with respect to certain alleles. In addition, MLST alleles were found to be robust predictors of the most recent common ancestors of a genome. The fourth study investigated the genetic stability and vulnerability of the informational genes to various evolutionary forces within the seven ST- 474 genomes. Twenty five genes comprised of nucleotide metabolism, repair and ribosomal functions were investigated showing a high level of genetic diversity in the DNA repair as well as nucleotide metabolic genes such as gidA, ogt, recJ, ssb, uvrA, uvrB and xseA. In contrast, the ribosomal genes were stable and identical across the seven genomes. The insertion of selenocysteine in three of the 25 genes indicates the presence of horizontal gene transfer within the ST-474 genomes. It is hypothesised that the genetic uniqueness of ST-474 may have arisen due to the geographic isolation of New Zealand, its poultry industry and an absence of exchange of sequence types which might typically occur through international trade of fresh poultry meat. Collectively, the studies presented in this thesis provide a better understanding of the dynamism of C. jejuni as a species and ST-474’s adaptational capacity and evolutionary potential (within the investigated set of genes) in response to changing intracellular and extracellular environments. This thesis has introduced the idea of using individual full length gene analysis, demonstrating the molecular differences between genes that contained identical alleles at the MLST loci. The research approaches implemented in this thesis can be readily applied to any pathogenic bacteria, particularly foodborne and emerging pathogens such as E. coli and Salmonella. This, in turn should provide new opportunities for bacterial drug targets and vaccine candidates

Multilocus sequence typing (MLST), porA and flaA typing of Campylobacter jejuni isolated from cats attending a veterinary clinic

Abstract Objective Campylobacter is a major cause of gastroenteritis in humans and pet ownership is a risk factor for infection. To study the occurrence, species distribution and sequence-based types of Campylobacter spp. in pet cats, 82 faecal samples were collected from cats in New Zealand. The PCR positive samples of Campylobacter jejuni were characterized by multilocus sequence typing (MLST), major outer membrane protein gene (porA) and flagellin A gene (flaA) sequence typing. Results Seven faecal samples were tested positive for Campylobacter spp. (9%, or 4–17% at 95% confidence interval), of which six were identified as C. jejuni, and one was C. upsaliensis. The six C. jejuni isolates were characterised by MLST; four belonged to ST-45 clonal complex and two of the isolates could not be typed. Two flaA-SVR types were identified: three samples were flaA-SVR type 8 and one belonged to 239. By combining all data, three isolates were indistinguishable with allelic combinations of ST-45, flaA-SVR 8, porA 44, although no epidemiological connection between these isolates could be established. To conclude, healthy cats can carry C. jejuni, whose detected genetic diversity is limited. The isolated sequence type ST-45 is frequently reported in human illnesses

Guanine-cytosine (GC) and GC3 contents of the individual metabolic housekeeping and informational genes.

<p>The data provides the guanine-cytosine (GC) contents for individual metabolic housekeeping genes and the individual genes investigated in the study from 19 C. jejuni genomes. It also provides the guanine-cytosine contents at the third codon position (GC3) for individual metabolic housekeeping genes and individual informational genes investigated in the study from 19 C. jejuni genomes.</p

Nucleotide sequences of metabolic housekeeping genes from 19 C. jejuni genomes

<p>Metabolic housekeeping genes from 19 <i>C. jejuni</i> genomes that include seven <i>C. jejuni</i> ST-474 genomes sequenced at Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. These are nucleotide sequences presented in FASTA format. CJ11168: <i>C. jejuni</i> subsp. <i>jejuni</i> 11168, CJ260.94: <i>C. jejuni</i> subsp. <i>jejuni</i> 260.90, CJ81116: <i>C. jejuni</i> subsp. <i>jejuni</i> 81116, CJ81176: <i>C. jejuni</i> subsp. <i>jejuni</i> 81-176, CJ8421: <i>C. jejuni</i> subsp. <i>jejuni</i> 84-21, CJ8425: <i>C. jejuni</i> subsp. <i>jejuni</i> 84-25, CJ8486: <i>C. jejuni</i> subsp. <i>jejuni</i> 84-26, CJ9313: <i>C. jejuni</i> subsp. <i>jejuni</i> 93-13, CJ936: <i>C. jejuni</i> subsp. <i>jejuni</i> 93-6, CJD: <i>C. jejuni</i> subsp. doylei, CJIA3902: <i>C. jejuni</i> subsp. <i>jejuni</i> IA3902, CJRM1221: <i>C. jejuni</i> subsp. <i>jejuni</i> 1221, H22082: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 AEIO00000000.1 (H: from human clinical case), H704: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (H: from human clinical case), P110b: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 000242395.2 (P: from poultry), P179a : <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (P: from poultry), P569a: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (P: from poultry), P694a: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (P: from poultry), H73020: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (H: from human clinical case).</p

Nucleotide sequences of informational genes from 19 C. jejuni genomes.

<p>Informational genes from 19 <i>C. jejuni</i> genomes that include seven <i>C. jejuni</i> ST-474 genomes sequenced at Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand. These are nucleotide sequences presented in FASTA format. CJ11168: <i>C. jejuni</i> subsp. <i>jejuni</i> 11168, CJ260.94: <i>C. jejuni</i> subsp. <i>jejuni</i> 260.90, CJ81116: <i>C. jejuni</i> subsp. <i>jejuni</i> 81116, CJ81176: <i>C. jejuni</i> subsp. <i>jejuni</i> 81-176, CJ8421: <i>C. jejuni</i> subsp. <i>jejuni</i> 84-21, CJ8425: <i>C. jejuni</i> subsp. <i>jejuni</i> 84-25, CJ8486: <i>C. jejuni</i> subsp. <i>jejuni</i> 84-26, CJ9313: <i>C. jejuni</i> subsp. <i>jejuni</i> 93-13, CJ936: <i>C. jejuni</i> subsp. <i>jejuni</i> 93-6, CJD: <i>C. jejuni</i> subsp. doylei, CJIA3902: <i>C. jejuni</i> subsp. <i>jejuni</i> IA3902, CJRM1221: <i>C. jejuni</i> subsp. <i>jejuni</i> 1221, H22082: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 AEIO00000000.1 (H: from human clinical case), H704: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (H: from human clinical case), P110b: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 000242395.2 (P: from poultry), P179a : <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (P: from poultry), P569a: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (P: from poultry), P694a: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (P: from poultry), H73020: <i>C. jejuni</i> subsp. <i>jejuni</i> ST 474 (H: from human clinical case).</p

Genomic diversity of Listeria monocytogenes isolates from seafood, horticulture and factory environments in New Zealand

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Wholegenome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.Peer reviewe

Efficacy of Preemptive Dexamethasone versus Methylprednisolone in the Management of Postoperative Discomfort and Pain after Mandibular Third Molar Surgery: A Systematic Review and Meta-Analysis

The corticosteroids have been used for preemptive management of surgical sequelae after mandibular third molar extraction. The aim of this article was to review the efficacy of methylprednisolone versus dexamethasone in the management of postsurgical pain, swelling, and trismus after mandibular third molar surgery. Randomized, double-blinded studies from PubMed, CINAHL, Scopus, DOSS, Cochrane central, and Web of Science were identified by using a search strategy. Randomized controlled trials evaluating the efficacy of use of dexamethasone versus methylprednisolone for mandibular third molar extraction were only considered. The studies involving the use of any other corticosteroid agent were excluded. Outcomes assessed were postoperative pain, the number of rescue analgesics required, swelling, trismus, and adverse events. The search strategy yielded 1046 articles for title and abstract screening, out of which only seven studies were included in the systematic review after full text screening. There was considerable heterogeneity between the studies with regards to the method as well as the parameters assessed. Risk of bias was low in three studies and unclear in other four studies. On pooled analyses, there was no significant difference with respect to pain, rescue analgesics, and swelling in the test and the control group. Forest plot analysis showed that dexamethasone had lesser trismus in early postoperative period (postoperative day 2) as compared to methylprednisolone. None of the included studies reported any adverse effects. Both the corticosteroids have similar efficacy in reducing the postoperative pain and swelling; however, dexamethasone showed statistically significant difference from methylprednisolone in reducing trismus (estimated standardized mean difference of −0.69 mm; 95% CI: −1.01 to −0.38; p<0.0001) in the early postoperative period. However, due to statistical heterogeneity, quality of the evidence for the review was low to moderate. Hence, more studies with larger study sample and low risk of bias are needed to confirm these results