Total Synthesis and Microbiological Evaluation of Leopolic Acid A and Analogues.

New antimicrobial scaffolds are scarce, and there is a great need for the development of novel therapeutics. In this study, we report a convergent 9-step synthesis of leopolic acid A and a series of targeted analogues. The designed compounds allowed for incorporation of non-natural ureido dipeptide moieties and 4- and 5-position substituents around the 2,3-pyrrolidinedione of leopolic acid A. Leopolic acid A displayed modest antimicrobial activity (32 µg/mL) against MRSA, while the most active analogues displayed slightly improved activity (8-16 µg/mL). Additionally, several of the leopolic acid A analogues displayed promising antibiofilm activity, most notably having an MBEC:MIC ratio of ∼1. Overall, this work represents an initial SAR of the natural product and a framework for further optimization of these bioactive scaffolds within the context of bioactive pyrrolidinediones.

The Troublesome Ticks Research Protocol: Developing a Comprehensive, Multidiscipline Research Plan for Investigating Human Tick-Associated Disease in Australia.

In Australia, there is a paucity of data about the extent and impact of zoonotic tick-related illnesses. Even less is understood about a multifaceted illness referred to as Debilitating Symptom Complexes Attributed to Ticks (DSCATT). Here, we describe a research plan for investigating the aetiology, pathophysiology, and clinical outcomes of human tick-associated disease in Australia. Our approach focuses on the transmission of potential pathogens and the immunological responses of the patient after a tick bite. The protocol is strengthened by prospective data collection, the recruitment of two external matched control groups, and sophisticated integrative data analysis which, collectively, will allow the robust demonstration of associations between a tick bite and the development of clinical and pathological abnormalities. Various laboratory analyses are performed including metagenomics to investigate the potential transmission of bacteria, protozoa and/or viruses during tick bite. In addition, multi-omics technology is applied to investigate links between host immune responses and potential infectious and non-infectious disease causations. Psychometric profiling is also used to investigate whether psychological attributes influence symptom development. This research will fill important knowledge gaps about tick-borne diseases. Ultimately, we hope the results will promote improved diagnostic outcomes, and inform the safe management and treatment of patients bitten by ticks in Australia.

Tick killing in situ before removal to prevent allergic and anaphylactic reactions in humans: a cross-sectional study.

BACKGROUND: Tick anaphylaxis is a potentially fatal outcome of improper tick removal and management. OBJECTIVE: To investigate whether killing ticks in-situ with ether-containing sprays or permethrin cream, before careful removal by the mouthparts could reduce this risk. METHODS: This was a prospective study at Mona Vale Hospital Emergency Department (ED) in Sydney, New South Wales, over a 6-month period during the peak tick season of 2016. Tick removal methods, allergic/anaphylactic reactions were recorded for patients presenting with ticks in situ or having already removed the ticks themselves. Primary endpoint was allergic/anaphylactic reaction after tick killing/removal. RESULTS: One hundred twenty-one patients met study inclusion criteria. Sixty-one patients (28 known tick-hypersensitive) had ticks killed with Wart-Off Freeze or Lyclear Scabies Cream (5% w/w permethrin) before removal with fine-tipped forceps or Tick Twister. Three patients (2 known tick-hypersensitive) had allergic reactions (5%), none anaphylactic. The 2 known hypersensitive patients suffered reactions during the killing process and the third patient had a particularly embedded tick meaning it could not be removed solely by mouthparts. Fifty patients presented to the ED posttick removal by various methods, none using either fine-tipped forceps or Tick Twister, of which 43 (86%) experienced allergic reactions - 2 anaphylactic. Five patients suffered allergic reactions before presentation despite no attempt at kill or removal, but ticks had likely been disturbed by some other method. Five patients had live ticks removed in ED - 3 refused killing and had no reaction despite 1 having known hypersensitivity; 2 had ticks on eyelids contraindicating killing, 1 with known hypersensitivity but both had allergic reactions post removal. CONCLUSION: Results support killing ticks in-situ before careful removal by mouthparts to reduce allergic/anaphylactic reactions although further research is still required.

Phylogenetic characterisation of two novel Anaplasmataceae from Australian Ixodes holocyclus ticks: 'Candidatus Neoehrlichia australis' and 'Candidatus Neoehrlichia arcana'.

Recently, two novel species of Anaplasmataceae were detected in the Australian paralysis tick, Ixodes holocyclus, by 16S rRNA gene metabarcoding. Analysis of these sequences suggested that these novel organisms are closely related to the genus 'Candidatus Neoehrlichia'. In this study, phylogenetic analysis of 16S rRNA (1264 bp), groESL (1047 bp) and gltA (561 bp) gene sequences, and concatenated (2872 bp) sequences, all concur that these novel species belong in the genus 'Candidatus Neoehrlichia' and are most closely related to, but distinct from the only other recognised members of this genus, 'Candidatus Neoehrlichia mikurensis' and 'Candidatus Neoehrlichia lotoris'. Based on their unique molecular signature, we propose to designate these species 'Candidatus Neoehrlichia australis' (reference strain HT41R) and 'Candidatus Neoehrlichia arcana' (reference strain HT94R). Identical 'Candidatus Neoehrlichia australis' 16S rRNA, groESL and gltA sequences were detected in 34/391 (8.7 %) individual Ixodes holocyclus ticks, and sequences were most similar to 'Candidatus Neoehrlichia lotoris' (96.2 %, 83.1 % and 67.2 %, respectively) and 'Candidatus Neoehrlichia mikurensis' (96.2 %, 84 % and 68.4 % respectively). Likewise, identical 'Candidatus Neoehrlichia arcana' 16S rRNA, groESL and gltA sequences were detected in 12/391 (3.1 %) Ixodes holocyclus ticks, and sequences were most similar to 'Candidatus Neoehrlichia lotoris' (98.5 %, 88.7 % and 79.3 %, respectively) and 'Candidatus Neoehrlichia mikurensis' (96.3 %, 84 % and 67.4 % respectively). These new species are the first Anaplasmataceae (except Wolbachia spp.) to be found to be endemic to Australia. The pathogenic consequences of these organisms are yet to be determined.

Bacterial Profiling Reveals Novel "Ca. Neoehrlichia", Ehrlichia, and Anaplasma Species in Australian Human-Biting Ticks.

In Australia, a conclusive aetiology of Lyme disease-like illness in human patients remains elusive, despite growing numbers of people presenting with symptoms attributed to tick bites. In the present study, we surveyed the microbial communities harboured by human-biting ticks from across Australia to identify bacteria that may contribute to this syndrome. Universal PCR primers were used to amplify the V1-2 hyper-variable region of bacterial 16S rRNA genes in DNA samples from individual Ixodes holocyclus (n = 279), Amblyomma triguttatum (n = 167), Haemaphysalis bancrofti (n = 7), and H. longicornis (n = 7) ticks. The 16S amplicons were sequenced on the Illumina MiSeq platform and analysed in USEARCH, QIIME, and BLAST to assign genus and species-level taxonomies. Nested PCR and Sanger sequencing were used to confirm the NGS data and further analyse novel findings. All 460 ticks were negative for Borrelia spp. by both NGS and nested PCR analysis. Two novel "Candidatus Neoehrlichia" spp. were identified in 12.9% of I. holocyclus ticks. A novel Anaplasma sp. was identified in 1.8% of A. triguttatum ticks, and a novel Ehrlichia sp. was identified in both A. triguttatum (1.2%) ticks and a single I. holocyclus (0.6%) tick. Further phylogenetic analysis of novel "Ca. Neoehrlichia", Anaplasma and Ehrlichia based on 1,265 bp 16S rRNA gene sequences suggests that these are new species. Determining whether these newly discovered organisms cause disease in humans and animals, like closely related bacteria do abroad, is of public health importance and requires further investigation.

Tick bite anaphylaxis: incidence and management in an Australian emergency department.

OBJECTIVES: Ticks are endemic to the eastern coastline of Australia. The aim of the present study is to describe the incidence of tick bites in such an area, the seasonal and geographical distribution, the incidence of anaphylaxis due to tick bite and its management. METHODS: We retrospectively analysed emergency presentations of patients with tick bites to Mona Vale Hospital on Sydney's Northern Beaches over a 2 year period from 1 January 2007 to 1 January 2009. We recorded the geographical and seasonal distribution of tick bites as well as the symptoms from tick bite and its emergency management. RESULTS: We report over 500 cases of tick bites presenting to a single New South Wales hospital over a 2 year period, of which 34 resulted in anaphylaxis. Cutaneous symptoms were the most common feature associated with anaphylaxis (32/34, 94%). Forty per cent (13/34) of patients with tick bite anaphylaxis had a history of allergy or previous anaphylaxis. Seventy-six per cent (26/34) of patients were administered adrenaline either prior to presenting or in the ED, while 97% (33/34) were treated with steroids. Fifty-three per cent were referred to an immunologist and only one-quarter were discharged with an adrenaline auto-injector. CONCLUSION: We report 34 cases of tick bite anaphylaxis over a 2 year period at a single hospital in a tick endemic area. The variation in the presenting symptoms and signs, as well as in management highlights the need for increased awareness for tick bite management in tick endemic areas.

Incidence of adverse events during prehospital rapid sequence intubation: a review of one year on the London Helicopter Emergency Medical Service.

BACKGROUND: To establish the incidence of hypoxemia and hypotension during prehospital rapid sequence intubation (RSI) in trauma patients attended by the London Helicopter Emergency Medical Service (HEMS) and to compare it with historical control data from published studies of both hospital and prehospital RSI. METHODS: A retrospective observational study during a 12-month period of London HEMS. All mission reports from the period March 1, 2003 to February 28, 2004 were reviewed and all intubations involving the use of drugs were included in the analysis. Measurements of oxygen saturation (SpO2) and systolic blood pressure (SBP) were obtained from the printed record produced by the portable monitor. RESULTS: During the 12-month period 244 RSIs were performed. Completed SpO2 data were available on 175 patients (71.7%), and of those 32 (18.3%) experienced hypoxemia (SpO2 <90%, or >10% fall if initial SpO2 <90%). Completed SBP data were available for 192 patients (79.1%), and of those 25 (13%) experienced hypotension (SBP <90 mm Hg or >10 mm Hg fall if initial SBP <90 mm Hg). No patients developed both hypoxemia and hypotension. CONCLUSIONS: Rates of hypoxemia and hypotension during prehospital RSI performed by London HEMS are relatively low. They are less than that found in previous studies of prehospital RSI and are similar to those reported in studies of in-hospital emergency RSI undertaken in the emergency department or ward setting. We therefore conclude that prehospital RSI has an acceptably low complication rate when performed by appropriately trained personnel.