Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

A Multilocus Sequence Typing Scheme for Rapid Identification of Xanthomonas citri Based on Whole-Genome Sequencing Data.

Xanthomonas citri is a plant-pathogenic bacterium associated with a diverse range of host plant species. It has undergone substantial reclassification and currently consists of 14 different subspecies or pathovars that are responsible for a wide range of plant diseases. Whole-genome sequencing (WGS) provides a cutting-edge advantage over other diagnostic techniques in epidemiological and evolutionary studies of X. citri because it has a higher discriminatory power and is replicable across laboratories. WGS also allows for the improvement of multilocus sequence typing (MLST) schemes. In this study, we used genome sequences of Xanthomonas isolates from the NCBI RefSeq database to develop a seven-gene MLST scheme that yielded 19 sequence types (STs) that correlated with phylogenetic clades of X. citri subspecies or pathovars. Using this MLST scheme, we examined 2,911 Xanthomonas species assemblies from NCBI GenBank and identified 15 novel STs from 37 isolates that were misclassified in NCBI. In total, we identified 545 X. citri assemblies from GenBank with 95% average nucleotide identity to the X. citri type strain, and all were classified as one of the 34 STs. All MLST classifications correlated with a phylogenetic position inferred from alignments using 92 conserved genes. We observed several instances where strains from different pathovars formed closely related monophyletic clades and shared the same ST, indicating that further investigation of the validity of these pathovars is required. Our MLST scheme described here is a robust tool for rapid classification of X. citri pathovars using WGS and a powerful method for further comprehensive taxonomic revision of X. citri pathovars.

Genomic Acquisitions in Emerging Populations of Xanthomonas vasicola pv. vasculorum Infecting Corn in the United States and Argentina.

Xanthomonas vasicola pv. vasculorum is an emerging bacterial plant pathogen that causes bacterial leaf streak on corn. First described in South Africa in 1949, reports of this pathogen have greatly increased in the past years in South America and in the United States. The rapid spread of this disease in North and South America may be due to more favorable environmental conditions, susceptible hosts and/or genomic changes that favored the spread. To understand whether genetic mechanisms exist behind the recent spread of X. vasicola pv. vasculorum, we used comparative genomics to identify gene acquisitions in X. vasicola pv. vasculorum genomes from the United States and Argentina. We sequenced 41 genomes of X. vasicola pv. vasculorum and the related sorghum-infecting X. vasicola pv. holcicola and performed comparative analyses against all available X. vasicola genomes. Time-measured phylogenetic analyses showed that X. vasicola pv. vasculorum strains from the United States and Argentina are closely related and arose from two introductions to North and South America. Gene content comparisons identified clusters of genes enriched in corn X. vasicola pv. vasculorum that showed evidence of horizontal transfer including one cluster corresponding to a prophage found in all X. vasicola pv. vasculorum strains from the United States and Argentina as well as in X. vasicola pv. holcicola strains. In this work, we explore the genomes of an emerging phytopathogen population as a first step toward identifying genetic changes associated with the emergence. The acquisitions identified may contain virulence determinants or other factors associated with the spread of X. vasicola pv. vasculorum in North and South America and will be the subject of future work.

Tephritid-microbial interactions to enhance fruit fly performance in sterile insect technique programs.

BACKGROUND: The Sterile Insect Technique (SIT) is being applied for the management of economically important pest fruit flies (Diptera: Tephritidae) in a number of countries worldwide. The success and cost effectiveness of SIT depends upon the ability of mass-reared sterilized male insects to successfully copulate with conspecific wild fertile females when released in the field. METHODS: We conducted a critical analysis of the literature about the tephritid gut microbiome including the advancement of methods for the identification and characterization of microbiota, particularly next generation sequencing, the impacts of irradiation (to induce sterility of flies) and fruit fly rearing, and the use of probiotics to manipulate the fruit fly gut microbiota. RESULTS: Domestication, mass-rearing, irradiation and handling, as required in SIT, may change the structure of the fruit flies' gut microbial community compared to that of wild flies under field conditions. Gut microbiota of tephritids are important in their hosts' development, performance and physiology. Knowledge of how mass-rearing and associated changes of the microbial community impact the functional role of the bacteria and host biology is limited. Probiotics offer potential to encourage a gut microbial community that limits pathogens, and improves the quality of fruit flies. CONCLUSIONS: Advances in technologies used to identify and characterize the gut microbiota will continue to expand our understanding of tephritid gut microbial diversity and community composition. Knowledge about the functions of gut microbes will increase through the use of gnotobiotic models, genome sequencing, metagenomics, metatranscriptomics, metabolomics and metaproteomics. The use of probiotics, or manipulation of the gut microbiota, offers significant opportunities to enhance the production of high quality, performing fruit flies in operational SIT programs.

Commensal microbiota modulates larval foraging behaviour, development rate and pupal production in Bactrocera tryoni.

BACKROUND: Commensal microbes can promote survival and growth of developing insects, and have important fitness implications in adulthood. Insect larvae can acquire commensal microbes through two main routes: by vertical acquisition from maternal deposition of microbes on the eggshells and by horizontal acquisition from the environment where the larvae develop. To date, however, little is known about how microbes acquired through these different routes interact to shape insect development. In the present study, we investigated how vertically and horizontally acquired microbiota influence larval foraging behaviour, development time to pupation and pupal production in the Queensland fruit fly ('Qfly'), Bactrocera tryoni. RESULTS: Both vertically and horizontally acquired microbiota were required to maximise pupal production in Qfly. Moreover, larvae exposed to both vertically and horizontally acquired microbiota pupated sooner than those exposed to no microbiota, or only to horizontally acquired microbiota. Larval foraging behaviour was also influenced by both vertically and horizontally acquired microbiota. Larvae from treatments exposed to neither vertically nor horizontally acquired microbiota spent more time overall on foraging patches than did larvae of other treatments, and most notably had greater preference for diets with extreme protein or sugar compositions. CONCLUSION: The integrity of the microbiota early in life is important for larval foraging behaviour, development time to pupation, and pupal production in Qflies. These findings highlight the complexity of microbial relations in this species, and provide insights to the importance of exposure to microbial communities during laboratory- or mass-rearing of tephritid fruit flies.

Diet and irradiation effects on the bacterial community composition and structure in the gut of domesticated teneral and mature Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

BACKGROUND: Mass-rearing, domestication and gamma irradiation of tephritid fruit flies used in sterile insect technique (SIT) programmes can negatively impact fly quality and performance. Symbiotic bacteria supplied as probiotics to mass-reared fruit flies may help to overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known. RESULTS: We have used next-generation sequencing to characterise the bacterial community composition and structure within Queensland fruit fly, Bactrocera tryoni (Froggatt), by generating 16S rRNA gene amplicon libraries derived from the guts of 58 individual teneral and mature, female and male, sterile and fertile adult flies reared on artificial larval diets in a laboratory or mass-rearing environment, and fed either a full adult diet (i.e. sugar and yeast hydrolysate) or a sugar only adult diet. Overall, the amplicon sequence read volume in tenerals was low and smaller than in mature adult flies. Operational taxonomic units (OTUs), belonging to the families Enterobacteriaceae (8 OTUs) and Acetobacteraceae (1 OTU) were most prevalent. Enterobacteriaceae dominated laboratory-reared tenerals from a colony fed a carrot-based larval diet, while Acetobacteraceae dominated mass-reared tenerals from a production facility colony fed a lucerne chaff based larval diet. As adult flies matured, Enterobacteriaceae became dominant irrespective of larval origin. The inclusion of yeast in the adult diet strengthened this shift away from Acetobacteraceae towards Enterobacteriaceae. Interestingly, irradiation increased 16S rRNA gene sequence read volume. CONCLUSIONS: Our findings suggest that bacterial populations in fruit flies experience significant bottlenecks during metamorphosis. Gut bacteria in teneral flies were less abundant and less diverse, and impacted by colony origin. In contrast, mature adult flies had selectively increased abundances for some gut bacteria, or acquired these bacteria from the adult diet and environment. Furthermore, irradiation augmented bacterial abundance in mature flies. This implies that either some gut bacteria were compensating for damage caused by irradiation or irradiated flies had lost their ability to regulate bacterial load. Our findings suggest that the adult stage prior to sexual maturity may be ideal to target for probiotic manipulation of fly microbiota to increase fly performance in SIT programmes.

Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease.

BACKGROUND: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. RESULTS: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. CONCLUSIONS: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc.

Comparative genomic analysis of a multiple antimicrobial resistant enterotoxigenic E. coli O157 lineage from Australian pigs.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) are a major economic threat to pig production globally, with serogroups O8, O9, O45, O101, O138, O139, O141, O149 and O157 implicated as the leading diarrhoeal pathogens affecting pigs below four weeks of age. A multiple antimicrobial resistant ETEC O157 (O157 SvETEC) representative of O157 isolates from a pig farm in New South Wales, Australia that experienced repeated bouts of pre- and post-weaning diarrhoea resulting in multiple fatalities was characterized here. Enterohaemorrhagic E. coli (EHEC) O157:H7 cause both sporadic and widespread outbreaks of foodborne disease, predominantly have a ruminant origin and belong to the ST11 clonal complex. Here, for the first time, we conducted comparative genomic analyses of two epidemiologically-unrelated porcine, disease-causing ETEC O157; E. coli O157 SvETEC and E. coli O157:K88 734/3, and examined their phylogenetic relationship with EHEC O157:H7. RESULTS: O157 SvETEC and O157:K88 734/3 belong to a novel sequence type (ST4245) that comprises part of the ST23 complex and are genetically distinct from EHEC O157. Comparative phylogenetic analysis using PhyloSift shows that E. coli O157 SvETEC and E. coli O157:K88 734/3 group into a single clade and are most similar to the extraintestinal avian pathogenic Escherichia coli (APEC) isolate O78 that clusters within the ST23 complex. Genome content was highly similar between E. coli O157 SvETEC, O157:K88 734/3 and APEC O78, with variability predominantly limited to laterally acquired elements, including prophages, plasmids and antimicrobial resistance gene loci. Putative ETEC virulence factors, including the toxins STb and LT and the K88 (F4) adhesin, were conserved between O157 SvETEC and O157:K88 734/3. The O157 SvETEC isolate also encoded the heat stable enterotoxin STa and a second allele of STb, whilst a prophage within O157:K88 734/3 encoded the serum survival gene bor. Both isolates harbor a large repertoire of antibiotic resistance genes but their association with mobile elements remains undetermined. CONCLUSIONS: We present an analysis of the first draft genome sequences of two epidemiologically-unrelated, pathogenic ETEC O157. E. coli O157 SvETEC and E. coli O157:K88 734/3 belong to the ST23 complex and are phylogenetically distinct to EHEC O157 lineages that reside within the ST11 complex.

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Fluoroquinolone-resistant extraintestinal pathogenic Escherichia coli, including O25b-ST131, isolated from faeces of hospitalized dogs in an Australian veterinary referral centre.

OBJECTIVES: To determine rates of carriage of fluoroquinolone-resistant Escherichia coli and extraintestinal pathogenic E. coli (ExPEC) among dogs in a specialist referral hospital and to examine the population structure of the isolates. METHODS: Fluoroquinolone-resistant faecal E. coli isolates (n = 232, from 23 of 123 dogs) recovered from hospitalized dogs in a veterinary referral centre in Sydney, Australia, over 140 days in 2009 were characterized by phylogenetic grouping, virulence genotyping and random amplified polymorphic DNA (RAPD) analysis. RESULTS: The RAPD dendrogram for representative isolates showed one group B2-associated cluster and three group D-associated clusters; each contained isolates with closely related ExPEC-associated virulence profiles. All group B2 faecal isolates represented the O25b-ST131 clonal group and were closely related to recent canine extraintestinal ST131 clinical isolates from the east coast of Australia by RAPD analysis. CONCLUSIONS: Hospitalized dogs may carry fluoroquinolone-resistant ExPEC in their faeces, including those representing O25b-ST131.

High-quality full genome assembly of historic Xylella fastidiosa strains from ICMP collection using a hybrid sequencing approach.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

Population structure of Bactrocera dorsalis s.s., B. papayae and B. philippinensis (Diptera: Tephritidae) in southeast Asia: evidence for a single species hypothesis using mitochondrial DNA and wing-shape data.

BACKGROUND: Bactrocera dorsalis s.s. is a pestiferous tephritid fruit fly distributed from Pakistan to the Pacific, with the Thai/Malay peninsula its southern limit. Sister pest taxa, B. papayae and B. philippinensis, occur in the southeast Asian archipelago and the Philippines, respectively. The relationship among these species is unclear due to their high molecular and morphological similarity. This study analysed population structure of these three species within a southeast Asian biogeographical context to assess potential dispersal patterns and the validity of their current taxonomic status. RESULTS: Geometric morphometric results generated from 15 landmarks for wings of 169 flies revealed significant differences in wing shape between almost all sites following canonical variate analysis. For the combined data set there was a greater isolation-by-distance (IBD) effect under a 'non-Euclidean' scenario which used geographical distances within a biogeographical 'Sundaland context' (r(2) = 0.772, P < 0.0001) as compared to a 'Euclidean' scenario for which direct geographic distances between sample sites was used (r(2) = 0.217, P < 0.01). COI sequence data were obtained for 156 individuals and yielded 83 unique haplotypes with no correlation to current taxonomic designations via a minimum spanning network. beast analysis provided a root age and location of 540kya in northern Thailand, with migration of B. dorsalis s.l. into Malaysia 470kya and Sumatra 270kya. Two migration events into the Philippines are inferred. Sequence data revealed a weak but significant IBD effect under the 'non-Euclidean' scenario (r(2) = 0.110, P < 0.05), with no historical migration evident between Taiwan and the Philippines. Results are consistent with those expected at the intra-specific level. CONCLUSIONS: Bactrocera dorsalis s.s., B. papayae and B. philippinensis likely represent one species structured around the South China Sea, having migrated from northern Thailand into the southeast Asian archipelago and across into the Philippines. No migration is apparent between the Philippines and Taiwan. This information has implications for quarantine, trade and pest management.

Molecular characterization of Escherichia coli strains that cause symptomatic and asymptomatic urinary tract infections.

The differences between Escherichia coli strains associated with symptomatic and asymptomatic urinary tract infections (UTIs) remain to be properly determined. Here we examined the prevalence of plasmid types and bacteriocins, as well as genetic relatedness, in a defined collection of E. coli strains that cause UTIs. Comparative analysis identified a subgroup of strains with a high number of virulence genes (VGs) and microcins M/H47. We also identified associations between microcin genes, VGs, and specific plasmid types.

Molecular characterization of commensal Escherichia coli adapted to different compartments of the porcine gastrointestinal tract.

The role of Escherichia coli as a pathogen has been the focus of considerable study, while much less is known about it as a commensal and how it adapts to and colonizes different environmental niches within the mammalian gut. In this study, we characterize Escherichia coli organisms (n = 146) isolated from different regions of the intestinal tracts of eight pigs (dueodenum, ileum, colon, and feces). The isolates were typed using the method of random amplified polymorphic DNA (RAPD) and screened for the presence of bacteriocin genes and plasmid replicon types. Molecular analysis of variance using the RAPD data showed that E. coli isolates are nonrandomly distributed among different gut regions, and that gut region accounted for 25% (P < 0.001) of the observed variation among strains. Bacteriocin screening revealed that a bacteriocin gene was detected in 45% of the isolates, with 43% carrying colicin genes and 3% carrying microcin genes. Of the bacteriocins observed (H47, E3, E1, E2, E7, Ia/Ib, and B/M), the frequency with which they were detected varied with respect to gut region for the colicins E2, E7, Ia/Ib, and B/M. The plasmid replicon typing gave rise to 25 profiles from the 13 Inc types detected. Inc F types were detected most frequently, followed by Inc HI1 and N types. Of the Inc types detected, 7 were nonrandomly distributed among isolates from the different regions of the gut. The results of this study indicate that not only may the different regions of the gastrointestinal tract harbor different strains of E. coli but also that strains from different regions have different characteristics.

Dynamics of the Queensland Fruit Fly Microbiome through the Transition from Nature to an Established Laboratory Colony.

The transition from nature to laboratory or mass rearing can impose significant physiological and evolutionary impact on insects. The Queensland fruit fly (also known as 'Qfly'), Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), is a serious economic pest that presents major challenges for horticulture industries in Australia. The sterile insect technique (SIT) is being developed to manage outbreaks in regions that remain free of Qfly and to suppress populations in regions where this species is endemic. The biology of Qfly is intimately connected to its microbiome. Therefore, changes in the microbiome that occur through domestication have implications for SIT. There are numerous studies of the microbiome in Qfly larvae and adults, but there is little information on how the microbiome changes as Qfly laboratory colonies are established. In this study, high-throughput Illumina sequencing was used to assess the Qfly microbiome in colonies reared from wild larvae, collected from fruit, for five generations, on a gel-based larval diet. Beta diversity analysis showed that the bacterial communities from Generation 5 (G5) clustered separately from earlier generations. At the genus level, bacterial communities were significantly different between the generations and mostly altered at G5. However, communities were found similar at phyla to family taxonomic levels. We observed high abundance of Morganella and Burkholderia at the genus level in the larval and pupal stages respectively at G5, but these were not detected in earlier generations. Overall, our findings demonstrate that the domestication process strongly affects the Qfly microbiome and prompts questions about the functional relationship between the Qfly and its microbiome, as well as implications for the performance of insects that have been domesticated and mass-reared for SIT programs.

Building More Resilient Culture Collections: A Call for Increased Deposits of Plant-Associated Bacteria.

Biological collections preserve our past, while helping protect our future and increase future knowledge. Plant bacterial culture collections are our security for domestic and global biosecurity. This feature article will provide an introduction to the global position of plant bacterial collections. The role of collections in monitoring plant pathogenic bacteria will be explored through the presentation of five cases studies. These case studies demonstrate why culture collections were imperative for the outcome in each situation. We discuss what we believe should be the best practices to improve microbial preservation and accessioning rates, and why plant bacterial culture collections must increase deposits to be prepared for future emerging pathogens. This is not only the case for global culture collections, but on a much bigger scale, our future scientific successes, our biosecurity decisions and responses, and our knowledge are contingent upon preserving our valuable bacterial strains. It is hoped that once you read this article, you will see the need to deposit your strains in registered public collections and make a concerted effort to build better bacterial culture collections with us.

Using Genomics to Design a Pathovar-Specific Loop-Mediated Isothermal Amplification (LAMP) Assay, for the Improved Detection of Xanthomonas citri pv. citri.

The ability to swiftly respond to pathogen incursions relies heavily on fast and accurate diagnostics. Current published assays for citrus bacterial canker do not target Xanthomonas citri pv. citri, the causative agent, with high specificity when testing Australian samples. While the current diagnostics are useful in countries where canker is endemic, the detection of canker in Australia requires an emergency response. Close relatives to X. citri pv. citri found in Australia may generate false positives with the current recommended diagnostic assays. Therefore, we developed a more specific detection tool for citrus bacterial canker to provide greater diagnostic confidence for surveillance and eradication efforts. We used genomic comparisons of 161 Xanthomonad genomes and identified and confirmed genomic regions specific for X. citri pv. citri by performing local alignments of unique regions to reference genomes. We then developed loop-mediated isothermal amplification primers and validated them against a panel of 190 isolates to confirm specificity. Our diagnostic assay showed 100% corroboration with the concurrently developed multiplex primers and represents an improved diagnostic method capable of effective citrus bacterial canker identification.

Phylogenetic diversity analysis of shotgun metagenomic reads describes gut microbiome development and treatment effects in the post-weaned pig.

Intensive farming practices can increase exposure of animals to infectious agents against which antibiotics are used. Orally administered antibiotics are well known to cause dysbiosis. To counteract dysbiotic effects, numerous studies in the past two decades sought to understand whether probiotics are a valid tool to help re-establish a healthy gut microbial community after antibiotic treatment. Although dysbiotic effects of antibiotics are well investigated, little is known about the effects of intramuscular antibiotic treatment on the gut microbiome and a few studies attempted to study treatment effects using phylogenetic diversity analysis techniques. In this study we sought to determine the effects of two probiotic- and one intramuscularly administered antibiotic treatment on the developing gut microbiome of post-weaning piglets between their 3rd and 9th week of life. Shotgun metagenomic sequences from over 800 faecal time-series samples derived from 126 post-weaning piglets and 42 sows were analysed in a phylogenetic framework. Differences between individual hosts such as breed, litter, and age, were found to be important contributors to variation in the community composition. Host age was the dominant factor in shaping the gut microbiota of piglets after weaning. The post-weaning pig gut microbiome appeared to follow a highly structured developmental program with characteristic post-weaning changes that can distinguish hosts that were born as little as two days apart in the second month of life. Treatment effects of the antibiotic and probiotic treatments were found but were subtle and included a higher representation of Mollicutes associated with intramuscular antibiotic treatment, and an increase of Lactobacillus associated with probiotic treatment. The discovery of correlations between experimental factors and microbial community composition is more commonly addressed with OTU-based methods and rarely analysed via phylogenetic diversity measures. The latter method, although less intuitive than the former, suffers less from library size normalization biases, and it proved to be instrumental in this study for the discovery of correlations between microbiome composition and host-, and treatment factors.

Green fluorescent protein-based biosensor to detect and quantify stress responses induced by DNA-degrading colicins.

Here we report the development of a whole-cell biosensor to detect and quantify the induction of the SOS response activated by DNA-degrading colicins. This biosensor utilizes the SOS-responsive cda promoter to regulate the expression of green fluorescent protein. The biosensor assay revealed induction of stress for all DNA-degrading reference colicins (E2, E7, and E8).

Virulence characteristics of translocating Escherichia coli and the interleukin-8 response to infection.

Four efficiently translocating Escherichia coli (TEC) strains isolated from the blood of humans (HMLN-1), pigs (PC-1) and rats (KIC-1 and KIC-2) were tested for their ability to adhere and translocate across human gut epithelial Caco-2 and HT-29 cells, to elicit a proinflammatory response and for the presence of 47 pathogenic E. coli virulence genes. HMLN-1 and PC-1 were more efficient in adhesion and translocation than rat strains, had identical biochemical phenotype (BPT) and serotype (O77:H18) and phylogenetic group (D). KIC-2 adhered more than KIC-1, belonged to different BPT and serotype but the same phylogenetic group as KIC-1. TEC strains elicited significantly higher IL-8 response in both cell lines (P < 0.05) and monocytic THP-1 (P < 0.0001) cells than non-TEC strains. KIC-2 induced the highest IL-8 response which may be associated with its immunostimulatory flagellin. Apart from adhesin genes fimH and bmaE that were carried by all strains, HMLN-1 and PC-1 carried capsule synthesis gene kpsMT III and KIC-2 carried the EAST1 toxin gene. The lack of known virulence genes and the ability of TEC to efficiently adhere and translocate whilst causing proinflammatory response suggests that these strains may carry as yet unidentified genes that enable their translocating ability.