The Anti-Listeria Activity of Pseudomonas fluorescens Isolated from the Horticultural Environment in New Zealand.

Beneficial bacteria with antibacterial properties are attractive alternatives to chemical-based antibacterial or bactericidal agents. Our study sourced such bacteria from horticultural produce and environments to explore the mechanisms of their antimicrobial properties. Five strains of Pseudomonas fluorescens were studied that possessed antibacterial activity against the pathogen Listeria monocytogenes. The vegetative culture of these strains (Pseudomonas fluorescens-PFR46I06, Pseudomonas fluorescens-PFR46H06, Pseudomonas fluorescens-PFR46H07, Pseudomonas fluorescens-PFR46H08 and Pseudomonas fluorescens-PFR46H09) were tested against Listeria monocytogenes (n = 31), Listeria seeligeri (n = 1) and Listeria innocua (n = 1) isolated from seafood and horticultural sources and from clinical cases (n = 2) using solid media coculture and liquid media coculture. All Listeria strains were inhibited by all strains of P. fluorescens; however, P. fluorescens-PFR46H07, P. fluorescens-PFR46H08 and P. fluorescens-PFR46H09 on solid media showed good inhibition, with average zones of inhibition of 14.8 mm, 15.1 mm and 18.2 mm, respectively, and the other two strains and P. fluorescens-PFR46H09 had a significantly greater zone of inhibition than the others (p < 0.05). There was no inhibition observed in liquid media coculture or in P. fluorescens culture supernatants against Listeria spp. by any of the P. fluorescens strains. Therefore, we hypothesized that the structural apparatus that causes cell-to-cell contact may play a role in the ejection of ant-listeria molecules on solid media to inhibit Listeria isolates, and we investigated the structural protein differences using whole-cell lysate proteomics. We paid special attention to the type VI secretion system (TSS-T6SS) for the transfer of effector proteins or bacteriocins. We found significant differences in the peptide profiles and protein summaries between these isolates' lysates, and PFR46H06 and PFR46H07 possessed the fewest secretion system structural proteins (12 and 11, respectively), while PFR46H08 and PFR46H09 had 18 each. P. fluorescens-PFR46H09, which showed the highest antimicrobial effect, had nine tss-T6SS structural proteins compared to only four in the other three strains.

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. Whole-genome 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.

Campylobacter novaezeelandiae sp. nov., isolated from birds and water in New Zealand.

Six isolates of Campylobacter with similar non-standard colonial morphologies were identified during studies isolating Campylobacter from bird faeces and rivers in New Zealand. Genomic (16S rRNA gene sequencing and whole genome analysis) and phenotypic (MALDI-TOF analysis and conventional biochemical tests) showed that the isolates form a monophyletic clade with genetic relationships to Campylobacter coli/Campylobacter jejuni and Campylobacter peloridis/Campylobacter amoricus. They may be distinguished from other Campylobacter by their MALDI-TOF spectral pattern, their florid α-haemolysis, their ability to grow anaerobically at 37 °C, and on 2 % NaCl nutrient agar, and their lack of hippuricase. This study shows that these isolates represent a novel species within the genus Campylobacter for which the name Campylobacter novaezeelandiae sp. nov. is proposed. The presence of C. novaezeelandiae in water may be a confounder for freshwater microbial risk assessment as they may not be pathogenic for humans. The type strain is B423bT (=NZRM 4741T=ATCC TSD-167T).

Identifying Suitable Listeria innocua Strains as Surrogates for Listeria monocytogenes for Horticultural Products.

A laboratory-based study testing 9 Listeria innocua strains independently and a cocktail of 11 Listeria monocytogenes strains was carried out. The aim was to identify suitable L. innocua strain(s) to model L. monocytogenes in inactivation experiments. Three separate inactivation procedures and a hurdle combination of the three were employed: thermal inactivation (55°C), UV-C irradiation (245 nm), and chemical sanitizer (TsunamiTM 100, a mixture of acetic acid, peroxyacetic acid, and hydrogen peroxide). The responses were strain dependent in the case of L. innocua with different strains responding differently to different regimes and L. innocua isolates generally responded differently to the L. monocytogenes cocktail. In the thermal inactivation treatment, inactivation of all strains including the L. monocytogenes cocktail plateaued after 120 min. In the case of chemical sanitizer, inactivation could be achieved at concentrations of 10 and 20 ppm with inactivation increasing with contact time up to 8 min, beyond which there was no significant benefit. All L. innocua strains except PFR16D08 were more sensitive than the L. monocytogenes cocktail to the hurdle treatment. PFR16D08 almost matched the resistance of the L. monocytogenes cocktail but was much more resistant to the individual treatments. A cocktail of two L. innocua strains (PFR 05A07 and PFR 05A10) had the closest responses to the hurdle treatment to those of the L. monocytogenes cocktail and is therefore recommended for hurdle experiments.

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

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.

Faeco-prevalence of Campylobacter jejuni in urban wild birds and pets in New Zealand.

BACKGROUND: Greater attention has been given to Campylobacter jejuni (C. jejuni) prevalence in poultry and ruminants as they are regarded as the major contributing reservoirs of human campylobacteriosis. However, relatively little work has been done to assess the prevalence in urban wild birds and pets in New Zealand, a country with the highest campylobacteriosis notification rates. Therefore, the aim of the study was to assess the faeco-prevalence of C. jejuni in urban wild birds and pets and its temporal trend in the Manawatu region of New Zealand. FINDINGS: A repeated cross-sectional study was conducted from April 2008 to July 2009, where faecal samples were collected from 906 ducks, 835 starlings, 23 Canadian goose, 2 swans, 2 pied stilts, 498 dogs and 82 cats. The faeco-prevalence of C. jejuni was 20% in ducks, 18% in starlings, 9% in Canadian goose, 5% in dogs and 7% in cats. The faeco-prevalence of C. jejuni was relatively higher during warmer months of the year in ducks, starlings and dogs while starlings showed increased winter prevalence. No such trend could be assessed in Canadian goose, swans, pied stilts and cats as samples could not be collected for the entire study period from these species. CONCLUSIONS: This study estimated the faeco-prevalence of C. jejuni in different animal species where the prevalence was relatively high during warmer months in general. However, there was relative increase in winter prevalence in starlings. The urban wild bird species and pets may be considered potential risk factors for human campylobacteriosis in New Zealand, particularly in small children.

Campylobacter jejuni colonization and population structure in urban populations of ducks and starlings in New Zealand.

A repeated cross-sectional study was conducted to determine the prevalence of Campylobacter spp. and the population structure of C. jejuni in European starlings and ducks cohabiting multiple public access sites in an urban area of New Zealand. The country's geographical isolation and relatively recent history of introduction of wild bird species, including the European starling and mallard duck, create an ideal setting to explore the impact of geographical separation on the population biology of C. jejuni, as well as potential public health implications. A total of 716 starling and 720 duck fecal samples were collected and screened for C. jejuni over a 12 month period. This study combined molecular genotyping, population genetics and epidemiological modeling and revealed: (i) higher Campylobacter spp. isolation in starlings (46%) compared with ducks (30%), but similar isolation of C. jejuni in ducks (23%) and starlings (21%), (ii) significant associations between the isolation of Campylobacter spp. and host species, sampling location and time of year using logistic regression, (iii) evidence of population differentiation, as indicated by FST , and host-genotype association with clonal complexes CC ST-177 and CC ST-682 associated with starlings, and clonal complexes CC ST-1034, CC ST-692, and CC ST-1332 associated with ducks, and (iv) greater genetic diversity and genotype richness in ducks compared with starlings. These findings provide evidence that host-associated genotypes, such as the starling-associated ST-177 and ST-682, represent lineages that were introduced with the host species in the 19th century. The isolation of sequence types associated with human disease in New Zealand indicate that wild ducks and starlings need to be considered as a potential public health risk, particularly in urban areas.

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.