Biology subjects

Gunn, N. J.

Publications and source records attributed to Gunn, N. J..

4 recordsLinked to original sources

bioRxiv2025

Chronic Staphylococcus aureus infection of osteocytes promotes an intracellular viable-but-non-culturable phenotype

Osteomyelitis associated with periprosthetic joint infection (PJI) is a serious and growing complication of orthopaedic joint replacement surgery, with a failure to cure rate within 2-years of between 15-46%. PJI is a severe chronic infectious disease with a 20% recurrence rate and culture-negativity in 7-39% of cases, of which 14-27% are PCR-positive, findings that remain poorly explained. Osteocytes, the most numerous bone cell type, have been identified as potential long-term intracellular reservoirs for Staphylococcus aureus, shielding it from antimicrobial treatment. Here we demonstrate that in chronic intra-osteocytic infections, S. aureus consistently adopts a viable-but-non-culturable (VBNC) state, capable of spontaneous host cell escape and reversion to active growth. Whole-genome sequencing identified single nucleotide polymorphisms (SNPs) in gene pairs between clones. Two revertant clones exhibited SNPs in srrB and murA2, and five exhibited SNPs in garR and upstream of tatB. While the functional importance of these changes is uncertain, they suggest that diverse genetic mechanisms underly entrance into and exit from the intracellular VBNC state. These findings provide a compelling explanation for culture-negative, PCR-positive osteomyelitis and have direct implications for the diagnosis and treatment of PJI.

microbiology↗

bioRxiv2023

Antibiotics at Clinical Concentrations Show Limited Effectivity Against Acute and Chronic Intracellular S. aureus Infections in Osteocytes

ObjectivesCase numbers of osteomyelitis are rising and chronic infections remain difficult to cure. While it is known that the major pathogen Staphylococcus aureus can persist intracellularly in osteocytes, the effectivity of antibiotics against this condition remains largely unknown. We sought to determine if current clinically utilised antibiotics were capable of clearing an intracellular osteocyte S. aureus infection. MethodsRifampicin, vancomycin, levofloxacin, ofloxacin, amoxicillin, oxacillin, doxycycline, linezolid, gentamicin and tigecycline were assessed for their MIC and minimum bactericidal concentrations (MBC) against 11 S. aureus clinical isolates and the reference strain ATCC 25923, at pH 5.0 and 7.2 to mimic lysosomal and cytoplasmic environments, respectively. Those antibiotics whose bone achievable concentration was commonly above their respective MICs for the strains tested were further assayed in a human osteocyte infection model under either acute or chronic conditions. Osteocyte-like cells were treated at 1, 4 and 10x the MIC for 1 and 7 days following infection (acute model), or after 14 days of infection (chronic model). The intracellular effectivity of each antibiotic was measured in terms of colony forming unit (CFU) reduction, small colony variant (SCV) formation and bacterial mRNA expression change. ResultsOnly rifampicin, levofloxacin and linezolid reduced intracellular CFU numbers significantly in the acute model. The effect was larger after 7 days compared to 1 day of treatment. However, no treatment reduced the quantity of bacterial mRNA, nor prevented non-culturable bacteria from returning to a culturable state. DiscussionThese findings indicate that S. aureus adapts phenotypically during intracellular infection of osteocytes, adopting a reversible quiescent state which is protected against antibiotics, even at 10x their MIC. Thus, new therapeutic approaches are necessary to cure S. aureus intracellular infections in osteocytes.

microbiology↗

bioRxiv2023

Beyond the Colony-Forming-Unit: Rapid Bacterial Evaluation in Osteomyelitis

Examination of bacteria/host cell interactions is important for understanding the aetiology of many infectious diseases. The colony-forming-unit (CFU) has been the standard for quantifying bacterial burden for the past century, however, this suffers from low sensitivity and is dependent on bacterial culturability in vitro. Our data demonstrate the discrepancy between the CFU and bacterial genome copy number in an osteomyelitis-relevant co-culture system and we confirm diagnosis and quantify bacterial load in clinical bone specimens. This study provides an improved workflow for the quantification of bacterial burden in such cases.

microbiology↗

bioRxiv2023

Failure to clear intra-osteocytic Staphylococcus aureus is not the result of antibiotic-mediated autophagy dysregulation

BackgroundStaphylococcus aureus is a major causative pathogen of osteomyelitis. The intracellular infection of osteocytes and related bone cells can persist despite application of gold-standard clinical interventions. The mechanisms by which intracellular S. aureus persists during antibiotic therapy are unknown. In this study, we apply S. aureus to an in vitro model of differentiated osteocytes to investigate whether antibiotic-mediated dysregulation of autophagy contributes to this phenomenon. MethodsHuman osteocyte-like cells were exposed to combinations of rifampicin, vancomycin and modulators of autophagy, in the presence or absence of S. aureus. Intracellular bacterial growth characteristics were assessed through CFU analysis, viable bacterial DNA abundance and the rate of escape into antibiotic-free medium, in parallel with measures of host cell autophagic flux. ResultsRifampicin, alone or in combination with vancomycin, caused a rapid decrease in the culturability of the intracellular bacterial community, concomitant with stable or increased absolute bacterial DNA levels. Both antibiotics significantly inhibited autophagic flux. However, while the modulation of autophagic flux affected bacterial culturability, this modulation did not affect viable bacterial DNA levels. ConclusionsAutophagy was shown to be a factor in the host-pathogen relationship in this model, as its modulation affected the growth state of intracellular S. aureus with respect to both their culturability and propensity to escape the intracellular niche. Whilst rifampicin and vancomycin treatments moderately suppressed autophagic flux acutely, this did not explain the paradoxical response of antibiotic treatment in decreasing S. aureus culturability while failing to clear bacterial DNA and hence intracellular bacterial load. Thus, whilst rifampicin and vancomycin exhibited off-target effects that modulated autophagy in osteocyte-like cells, this could not explain the persistent infection observed for S. aureus.

microbiology↗