Larvae of the greater wax moth (Galleria mellonella) are susceptible to infection with C. burnetii, an obligate intracellular bacterial pathogen. We show that bacteria are found in hemocytes after infection, and occupy vacuoles which are morphologically similar to Coxiella-containing vacuoles seen in infected mammalian phagocytes. We characterized the infection by transcriptome profiling of bacteria isolated from the hemocytes of infected larvae and identified 46 highly upregulated genes. The encoded proteins are predicted to be involved in translation, LPS biosynthesis, biotin synthesis, scavenging of reactive oxygen species, and included a T4SS effector and 30 hypothetical proteins. Some of these genes had previously been shown to be upregulated in buffalo green monkey (BGM) cells or in mice, whilst others appear to be regulated in a hostspecific manner. Altogether, our results demonstrate the value of the G. mellonella model to study intracellular growth and identify potential virulence factors of C. burnetii.

Staphylococcus pseudintermedius causes opportunistic infections in dogs. It also has significant zoonotic potential, with the emergence of multidrug-resistance leading to difficulty treating both animal and human infections. Manuka honey has previously been reported to inhibit many bacterial pathogens including methicillin resistant Staphylococcus aureus and is successfully utilised in both clinical and veterinary practice. Here we evaluated the ability of manuka honey to inhibit strains of S. pseudintermedius growth alone and in combination with antibiotics, and its capacity to modulate virulence within multiple S. pseudintermedius. All 18 of the genetically diverse S. pseudintermedius strains sequenced and tested were inhibited by ≤ 12% (w/v) medical grade manuka honey, although tolerance to five clinically relevant antibiotics was observed. The susceptibility of the isolates to four of these antibiotics was significantly increased (p ≤0.05) when combined with sub lethal concentrations of honey, although sensitivity to oxacillin was decreased. Virulence (DNase, protease and haemolysin) activity was also significantly reduced (p ≤ 0.05) in over half of isolates when cultured with sub lethal concentrations of honey (13, 9 and 10 isolates respectively). These findings highlight the potential for manuka honey to be utilised against S. pseudintermedius infections. Importance Staphylococcus pseudintermedius is an important member of the skin microbial community in animals and can cause opportunistic infections in both pets and their owners. The high incidence of antimicrobial resistance in S. pseudintermedius highlights that this opportunistic zoonotic pathogen can cause infections which require prolonged and intensive treatment to resolve. Manuka honey has proven efficacy against many bacterial pathogens and is an accepted topical treatment for infections in both veterinary and clinical practice so is a particularly appropriate antimicrobial for use with zoonotic pathogens such as S. pseudintermedius. Here we demonstrate that manuka honey is not only highly potent against novel multi-drug resistant S. pseudintermedius isolates, but also acts synergistically with clinically relevant antibiotics. In addition, manuka honey modulates S. pseudintermedius virulence activity, even at subinhibitory concentrations. In a clinical setting these attributes may assist in controlling infection, allowing a more rapid resolution and reducing antibiotic use.

Coxiella burnetii is an intracellular pathogen responsible for causing Q fever in humans, a disease with varied presentations ranging from a mild flu-like sickness to a debilitating illness that can result in endocarditis. The intracellular lifestyle of C. burnetii is unique, residing in an acidic phagolysosome-like compartment within host cells. An understanding of the core molecular biology of C. burnetii will greatly increase our understanding of C. burnetii growth, survival and pathogenesis. We used transposon-directed insertion site sequencing (TraDIS) to reveal C. burnetii Nine Mile Phase II genes fundamental for growth and in vitro survival. Screening a transposon library containing >10 000 unique transposon mutants revealed 512 predicted essential genes. Essential routes of synthesis were identified for the mevalonate pathway, as well as peptidoglycan and biotin synthesis. Some essential genes identified (e.g. predicted type IV secretion system effector genes) are typically considered to be associated with C. burnetii virulence, a caveat concerning the axenic media used in the study. Investigation into the conservation of the essential genes identified revealed that 78 % are conserved across all C. burnetii strains sequenced to date, which probably play critical functions. This is the first report of a whole genome transposon screen in C. burnetii that has been undertaken for the identification of essential genes.