Staphylococcus aureus is often found in orthopaedic infections and may be protected from commonly prescribed antibiotics by forming biofilms or growing intracellularly within osteoblasts. To investigate the effect of non-antibiotic compounds in conjunction with antibiotics to clear intracellular and biofilm forming S. aureus causing osteomyelitis. SAOS-2 osteoblast-like cell lines were infected with S. aureus BB1279. Antibiotics (vancomycin, VAN; and dicloxacillin, DICLOX), bacterial efflux pump inhibitors (piperine, PIP; carbonyl cyanide m-chlorophenyl hydrazone, CCCP), and bone morphogenetic protein (BMP-2) were evaluated individually and in combination to kill intracellular bacteria. We present direct evidence that after gentamicin killed extracellular planktonic bacteria and antibiotics had been stopped, seeding from the infected osteoblasts grew as biofilms. VAN was ineffective in treating the intracellular bacteria even at 10× MIC; however in presence of PIP or CCCP the intracellular S. aureus was significantly reduced. Bacterial efflux pump inhibitors (PIP and CCCP) were effective in enhancing permeability of antibiotics within the osteoblasts and facilitated killing of intracellular S. aureus. Confocal laser scanning microscopy (CLSM) showed increased uptake of propidium iodide within osteoblasts in presence of PIP and CCCP. BMP-2 had no effect on growth of S. aureus either alone or in combination with antibiotics. Combined application of antibiotics and natural agents could help in the treatment of osteoblast infected intracellular bacteria and biofilms associated with osteomyelitis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1086-1092, 2018.
Summary Parasites can have profound effects on host behaviour and species interactions, but the consequences of these impacts are inadequately understood. Three common crayfish in northern Wisconsin and Michigan (native Orconectes virilis, non‐native O. propinquus and non‐native and invasive O. rusticus) are intermediate hosts for trematode parasites, Microphallus spp. Some species in the genus Microphallus alter host behaviour, increasing their predation risk, but the effects of microphallids on crayfish are unknown. Orconectes propinquus replaces O. virilis in most lakes where they are introduced, and O. rusticus replaces both. These species replacements have major effects on macrophytes, macroinvertebrates and fish. Therefore, differential parasite impacts on crayfish could have community‐level effects if competitive outcomes are altered. We examined the shelter affinity of infected and uninfected individuals of all three species in laboratory experiments in the presence and absence of a conspecific. We also observed behaviour during agonistic interactions, and measured boldness by quantifying how quickly crayfish emerged from shelter with a predatory fish present. Infection with Microphallus substantially altered crayfish shelter affinity, shelter competition and boldness, though infection affected each species differently. Infection reduced shelter affinity in O. propinquus and the ability of O. virilis to compete for shelter against uninfected conspecifics. Infected crayfish were bolder in the presence of a predatory fish. Our results suggest that infection with Microphallus alters crayfish behaviour so that all three species are more vulnerable to predation. Orconectes propinquus is likely to suffer the greatest increase in predation when infected, due to a reduced affinity for shelter coupled with increased boldness. In lakes where crayfish species coexist, O. rusticus will probably be less affected by the parasite than either congener. Therefore, crayfish parasites could alter crayfish abundance and species composition in north temperate lakes via behavioural modifications.
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