Variation in the OC locus of Acinetobacter baumannii genomes predicts extensive structural diversity in the Lipooligosaccharide

Lipooligosaccharide (LOS) is a complex surface structure that is linked to many pathogenic properties of Acinetobacter baumannii. In A. baumannii, the genes responsible for the synthesis of the outer core (OC) component of the LOS are located between ilvE and aspS. The content of the OC locus is usually variable within a species, and examination of 6 complete and 227 draft A. baumannii genome sequences available in GenBank non-redundant and Whole Genome Shotgun databases revealed nine distinct new types, OCL4-OCL12, in addition to the three known ones. The twelve gene clusters fell into two distinct groups, designated Group A and Group B, based on similarities in the genes present. OCL6 (Group B) was unique in that it included genes for the synthesis of L-Rhamnosep. Genetic exchange of the different configurations between strains has occurred as some OC forms were found in several different sequence types (STs). OCL1 (Group A) was the most widely distributed being present in 18 STs, and OCL6 was found in 16 STs. Variation within clones was also observed, with more than one OC locus type found in the two globally disseminated clones, GC1 and GC2, that include the majority of multiply antibiotic resistant isolates. OCL1 was the most abundant gene cluster in both GC1 and GC2 genomes but GC1 isolates also carried OCL2, OCL3 or OCL5, and OCL3 was also present in GC2. As replacement of the OC locus in the major global clones indicates the presence of sub-lineages, a PCR typing scheme was developed to rapidly distinguish Group A and Group B types, and to distinguish the specific forms found in GC1 and GC2 isolates.

5,7-di-N-acetyl-acinetaminic acid: A novel non-2-ulosonic acid found in the capsule of an Acinetobacter baumannii isolate

An Acinetobacter baumannii global clone 1 (GC1) isolate was found to carry a novel capsule biosynthesis gene cluster, designated KL12. KL12 contains genes predicted to be involved in the synthesis of simple sugars, as well as ones for N-acetyl-l-fucosamine (l-FucpNAc) and N-acetyl-d-fucosamine (d-FucpNAc). It also contains a module of 10 genes, 6 of which are required for 5,7-di-N-acetyl-legionaminic acid synthesis. Analysis of the composition of the capsule revealed the presence of N-acetyl-d-galactosamine, l-FucpNAc and d-FucpNAc, confirming the role of fnlABC and fnr/gdr genes in the synthesis of l-FucpNAc and d-FucpNAc, respectively. A non-2-ulosonic acid, shown to be 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-altro-non-2-ulosonic acid, was also detected. This sugar has not previously been recovered from biological source, and was designated 5,7-di-N-acetyl-acinetaminic acid (Aci5Ac7Ac). Proteins encoded by novel genes, named aciABCD, were predicted to be involved in the conversion of 5,7-di-N-acetyl-legionaminic acid to Aci5Ac7Ac. A pathway for 5,7-di-N-acetyl-8-epilegionaminic acid biosynthesis was also proposed. In available A. baumannii genomes, genes for the synthesis of 5,7-di-N-acetyl-acinetaminic acid were only detected in two closely related capsule gene clusters, KL12 and KL13, which differ only in the wzy gene. KL12 and KL13 are carried by isolates belonging to clinically important clonal groups, GC1, GC2 and ST25. Genes for the synthesis of N-acyl derivatives of legionaminic acid were also found in 10 further A. baumannii capsule gene clusters, and three carried additional genes for production of 5,7-di-N-acetyl-8-epilegionaminic acid.

Developing an epidemic forecasting model for Influenza A in Brisbane, Australia, based on climate and Hong Kong Influenza A surveillance data

Influenza is associated with substantial disease burden [ 1]. Development of a climate-based early warning system for in fluenza epidemics has been recommended given the signi fi - cant association between climate variability and influenza activity [2]. Brisbane is a subtropical city in Australia and offers free in fluenza vaccines to residents aged ≥65 years considering their high risks in developing life-threatening complications, especially for in fluenza A predominant seasons. Hong Kong is an international subtropical city in Eastern Asia and plays a crucial role in global infectious diseases transmission dynamics via the international air transportation network [3, 4]. We hypothesized that Hong Kong in fluenza surveillance data could provide a signal for in fluenza epidemics in Brisbane [ 4]. This study aims to develop an epidemic forecasting model for influenza A in Brisbane elders, by combining climate variability and Hong Kong in fluenza A surveillance data. Weekly numbers of laboratoryconfirmed influenza A positive isolates for people aged ≥65 years from 2004 to 2009 were obtained for Brisbane from Queensland Health, Australia, and for Hong Kong from Queen Mary Hospital (QMH). QMH is the largest public hospital located in Hong Kong Island, and in fluenza surveillance data from this hospital have been demonstrated to be representative for influenza circulation in the entirety of Hong Kong [ 5]. The Brisbane in fluenza A epidemics occurred during July –September, whereas the Hong Kong in fluenza A epidemics occurred during February –March and May –August.

Modified MLVA for genotyping Queensland invasive Streptococcus pneumoniae

Background Globally, over 800 000 children under five die each year from infectious diseases caused by Streptococcus pneumoniae. To understand genetic relatedness between isolates, study transmission routes, assess the impact of human interventions e.g. vaccines, and determine infection sources, genotyping methods are required. The ‘gold standard’ genotyping method, Multi-Locus Sequence Typing (MLST), is useful for long-term and global studies. Another genotyping method, Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA), has emerged as a more discriminatory, inexpensive and faster technique; however there is no universally accepted method and it is currently suitable for short-term and localised epidemiology studies. Currently Australia has no national MLST database, nor has it adopted any MLVA method for short-term or localised studies. This study aims to improve S. pneumoniae genotyping methods by modifying the existing MLVA techniques to be more discriminatory, faster, cheaper and technically less demanding than previously published MLVA methods and MLST. Methods Four different MLVA protocols, including a modified method, were applied to 317 isolates of serotyped invasive S. pneumoniae isolated from sterile body sites of Queensland children under 15 years from 2007–2012. MLST was applied to 202 isolates for comparison. Results The modified MLVA4 is significantly more discriminatory than the ‘gold standard’ MLST method. MLVA4 has similar discrimination compared to other MLVA techniques in this study). The failure to amplify particular loci in previous MLVA methods were minimised in MLVA4. Failure to amplify BOX-13 and Spneu19 were found to be serotype specific. Conclusion We have modified a highly discriminatory MLVA technique for genotyping Queensland invasive S. pneumoniae. MLVA4 has the ability to enhance our understanding of the pneumococcal epidemiology and the changing genetics of the pneumococcus in localised and short-term studies.

Comparison of human and animal Chlamydia pneumoniae responses to interferon gamma and penicillin treatment

This thesis has made a significant contribution to future chlamydial research by uncovering the chlamydial pathogenic mechanisms which will potentially help in the development of targeted vaccine against the pathogen. This thesis has made important new contributions to our understanding of Chlamydia pneumoniae specific adaptations to stress responses and has provided new perspectives on the survival of this successful pathogen. This thesis has used two well established microbial stressors and has identified major differences in stress responses between human and animal Chlamydia pneumoniae isolates.

Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

Molecular and pathological insights into Chlamydia pecorum-associated sporadic bovine encephalomyelitis (SBE) in Western Australia

Background Despite its global recognition as a ruminant pathogen, cases of Chlamydia pecorum infection in Australian livestock are poorly documented. In this report, a C. pecorum specific Multi Locus Sequence Analysis scheme was used to characterise the C. pecorum strains implicated in two cases of sporadic bovine encephalomyelitis confirmed by necropsy, histopathology and immunohistochemistry. This report provides the first molecular evidence for the presence of mixed infections of C. pecorum strains in Australian cattle. Case presentation Affected animals were two markedly depressed, dehydrated and blind calves, 12 and 16 weeks old. The calves were euthanized and necropsied. In one calf, a severe fibrinous polyserositis was noted with excess joint fluid in all joints whereas in the other, no significant lesions were seen. No gross abnormalities were noted in the brain of either calf. Histopathological lesions seen in both calves included: multifocal, severe, subacute meningoencephalitis with vasculitis, fibrinocellular thrombosis and malacia; diffuse, mild, acute interstitial pneumonia; and diffuse, subacute epicarditis, severe in the calf with gross serositis. Immunohistochemical labelling of chlamydial antigen in brain, spleen and lung from the two affected calves and brain from two archived cases, localised the antigen to the cytoplasm of endothelium, mesothelium and macrophages. C. pecorum specific qPCR, showed dissemination of the pathogen to multiple organs. Phylogenetic comparisons with other C. pecorum bovine strains from Australia, Europe and the USA revealed the presence of two genetically distinct sequence types (ST). The predominant ST detected in the brain, heart, lung and liver of both calves was identical to the C. pecorum ST previously described in cases of SBE. A second ST detected in an ileal tissue sample from one of the calves, clustered with previously typed faecal bovine isolates. Conclusion This report provides the first data to suggest that identical C. pecorum STs may be associated with SBE in geographically separated countries and that these may be distinct from those found in the gastrointestinal tract. This report provides a platform for further investigations into SBE and for understanding the genetic relationships that exist between C. pecorum strains detected in association with other infectious diseases in livestock.

Determination of the effect of JO146, a CtHtrA inhibitor, on the chlamydial developmental cycle and persistence

The role of Chlamydia trachomatis HtrA (CtHtrA) for the growth and pathogenesis of this organism is presented. Inhibition of CtHtrA led to loss of infectious progeny in Chlamydia, particularly during heat stress or recovery from penicillin persistence. Isolation of CtHtrA inhibitor resistant mutants identified positive selection for mutants in fatty acid related pathways. Importantly, HtrA inhibition was effective against clinical isolates of C. trachomatis. Thus the findings combined indicate that CtHtrA is an important chlamydial growth factor that has the potential to be targeted for the development of anti-chlamydial drugs in the future.

Comparative analysis of the uropathogenic Escherichia coli surface proteome by tandem mass-spectrometry of artificially induced outer membrane vesicles

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections. For successful colonisation of the urinary tract, UPEC employ multiple surface-exposed or secreted virulence factors, including adhesins and iron uptake systems. Whilst individual UPEC strains and their virulence factors have been the focus of extensive research, there have been no outer membrane (OM) proteomic studies based on large clinical UPEC collections, primarily due to limitations of traditional methods. In this study, a high-throughput method based on tandem mass-spectrometry of EDTA heat-induced outer membrane vesicles (OMVs) was developed for the characterisation of the UPEC surface-associated proteome. The method was applied to compare the OM proteome of fifty-four UPEC isolates, resulting in the identification of 8789 proteins, consisting of 619 unique proteins, which were subsequently interrogated for their subcellular origin, prevalence and homology to characterised virulence factors. Multiple distinct virulence-associated proteins were identified, including two novel putative iron uptake proteins, an uncharacterised type of chaperone-usher fimbriae and various highly prevalent hypothetical proteins. Our results give fundamental insight into the physiology of UPEC and provide a framework for understanding the composition of the UPEC OM proteome.

Wavelet-based multiresolution analysis of Wivenhoe Dam water temperatures

Water temperature measurements from Wivenhoe Dam offer a unique opportunity for studying fluctuations of temperatures in a subtropical dam as a function of time and depth. Cursory examination of the data indicate a complicated structure across both time and depth. We propose simplifying the task of describing these data by breaking the time series at each depth into physically meaningful components that individually capture daily, subannual, and annual (DSA) variations. Precise definitions for each component are formulated in terms of a wavelet-based multiresolution analysis. The DSA components are approximately pairwise uncorrelated within a given depth and between different depths. They also satisfy an additive property in that their sum is exactly equal to the original time series. Each component is based upon a set of coefficients that decomposes the sample variance of each time series exactly across time and that can be used to study both time-varying variances of water temperature at each depth and time-varying correlations between temperatures at different depths. Each DSA component is amenable for studying a certain aspect of the relationship between the series at different depths. The daily component in general is weakly correlated between depths, including those that are adjacent to one another. The subannual component quantifies seasonal effects and in particular isolates phenomena associated with the thermocline, thus simplifying its study across time. The annual component can be used for a trend analysis. The descriptive analysis provided by the DSA decomposition is a useful precursor to a more formal statistical analysis.

Phylogenetic lineage and pilus protein Spb1/SAN1518 affect opsonin-independent phagocytosis and intracellular survival of Group B Streptococcus

Opsonin-independent phagocytosis of Group B Streptococcus (GBS) is important in defense against neonatal GBS infections. A recent study indicated a role for GBS pilus in macrophage phagocytosis (Maisey et al Faseb J 22 2008 1715-24). We studied 163 isolates from different phylogenetic backgrounds and those possessing or lacking the gene encoding the pilus backbone protein, Spb1 (SAN1518, PI-2b) and spb1-deficient mutants of wild-type (WT) serotype III-3 GBS 874391 in non-opsonic phagocytosis assays using J774A.1 macrophages. Numbers of GBS phagocytosed differed up to 23-fold depending on phylogenetic background; isolates possessing spb1 were phagocytosed more than isolates lacking spb1. Comparing WT GBS and isogenic spb1-deficient mutants showed WT was phagocytosed better compared to mutants; Spb1 also enhanced intracellular survival as mutants were killed more efficiently. Complementation of mutants restored phagocytosis and resistance to killing in J774A.1 macrophages. Spb1 antiserum revealed surface expression in WT GBS and spatial distribution relative to capsular polysaccharide. spb1 did not affect macrophage nitric oxide and TNF-alpha responses; differences in phagocytosis did not correlate with N-acetyl d-glucosamine (from GBS cell-wall) according to enzyme-linked lectin-sorbent assay. Together, these findings support a role for phylogenetic lineage and Spb1 in opsonin-independent phagocytosis and intracellular survival of GBS in J774A.1 macrophages.

Molecular characterisation of the Vacuolating Autotransporter Toxin in Uropathogenic Escherichia coli

The vacuolating autotransporter (AT) toxin (Vat) contributes to Uropathogenic Escherichia coli (UPEC) fitness during systemic infection. Here we characterised Vat and investigated its regulation in UPEC. We assessed the prevalence of vat in a collection of 45 UPEC urosepsis strains and showed that it was present in 31 (68%) of the isolates. The isolates containing the vat gene corresponded to three major E. coli sequence types (ST12, 73 and 95) and these strains secreted the Vat protein. Further analysis of the vat genomic locus identified a conserved gene located directly downstream of vat that encodes a putative MarR-like transcriptional regulator, which we termed vatX. The vat-vatX genes were present in the UPEC reference strain CFT073 and RT-PCR revealed both genes are co-transcribed. Over-expression of vatX in CFT073 led to a 3-fold increase in vat gene transcription. The vat promoter region contained three putative nucleation sites for the global transcriptional regulator H-NS; thus the hns gene was mutated in CFT073 (to generate CFT073hns). Western blot analysis using a Vat-specific antibody revealed a significant increase in Vat expression in CFT073hns compared to wild-type CFT073. Direct H-NS binding to the vat promoter region was demonstrated using purified H-NS in combination with electrophoresis mobility shift assays. Finally, Vat-specific antibodies were detected in plasma samples from urosepsis patients infected by vat-containing UPEC strains, demonstrating Vat is expressed during infection. Overall, this study has demonstrated that Vat is a highly prevalent and tightly regulated immunogenic SPATE secreted by UPEC during infection.

Biofilm formation by multidrug resistant Escherichia coli ST131 is dependent on type 1 fimbriae and assay conditions.

Escherichia coli sequence type 131 (ST131) have emerged as a pandemic lineage of important multidrug resistant pathogens worldwide. Despite many studies examining the epidemiology of ST131, only a few studies to date have investigated the capacity of ST131 strains to form biofilms. Some of these studies have reported contrasting findings, with no specific ST131 biofilm-promoting factors identified. Here we examined a diverse collection of ST131 isolates for in vitro biofilm formation in different media and assay conditions, including urine from healthy adult women. We found significant differences among strains and assay conditions, which offers an explanation for the contrasting findings reported by previous studies using a single condition. Importantly, we showed that expression of type 1 fimbriae is a critical determinant for biofilm formation by ST131 strains and that inhibition of the FimH adhesin significantly reduces biofilm formation. We also offer direct genetic evidence for the contribution of type 1 fimbriae in biofilm formation by the reference ST131 strain EC958, a representative of the clinically dominant H30-Rx ST131 subgroup. This is the first study of ST131 biofilm formation in biologically relevant conditions and paves the way for the application of FimH inhibitors in treating drug resistant ST131 biofilm infections.

Complete genome sequences of seven Helicoverpa armigera SNPV-AC53-derived strains

Wild-type baculovirus isolates typically consist of multiple strains. We report the full genome sequences of seven alphabaculovirus strains derived by passage through tissue culture from Helicoverpa armigera SNPV-AC53 (KJ909666).