Treating periprosthetic joint infections as biofilms: key diagnosis and management strategies

    Treating Periprosthetic Joint Infections as Biofilms: Key Diagnosis and Management Strategies Alice Tzeng, Tony H. Tzeng, Sonia Vasdev, Kyle Korth, Travis Healey, Javad Parvizi, Khaled J. Saleh PII: DOI: Reference:

S0732-8893(14)00447-7 doi: 10.1016/j.diagmicrobio.2014.08.018 DMB 13713

To appear in:

Diagnostic Microbiology and Infectious Disease

Received date: Revised date: Accepted date:

22 May 2014 19 August 2014 22 August 2014

Please cite this article as: Tzeng Alice, Tzeng Tony H., Vasdev Sonia, Korth Kyle, Healey Travis, Parvizi Javad, Saleh Khaled J., Treating Periprosthetic Joint Infections as Biofilms: Key Diagnosis and Management Strategies, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.08.018

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Treating Periprosthetic Joint Infections as Biofilms: Key Diagnosis and Management Strategies

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Tony H Tzeng, BSb

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Alice Tzeng, BAa

Sonia Vasdev, MD, MPH, MPAb

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Kyle Korth, BSc

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Travis Healey, BSb

Javad Parvizi, MD, FRCSd

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*Khaled J Saleh, BSc, MD, MSc, FRCS(C), MHCMb

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Academic Affiliation: a Koch Institute for Integrative Cancer Research Department of Biological Engineering Massachusetts Institute of Technology Cambridge, MA 02139, USA b

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Division of Orthopaedics and Rehabilitation Department of Surgery Southern Illinois University School of Medicine Springfield, IL 62794-9679, USA

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Rush University Medical College Chicago, IL 60612, USA

Department of Orthopaedics Rothman Institute Thomas Jefferson University Hospital Philadelphia, PA 19107, USA *Corresponding Author: Khaled J Saleh, BSc MD MSc FRCS(C) MHCM Division of Orthopedics and Rehabilitation, Chairman Director of Clinical and Translational Research Southern Illinois University School of Medicine P.O. Box 19679 Springfield, IL 62794-9679, USA Tel: (217) 545-8865 Fax: (217) 545-7901 E-mail: [email protected] Running Title: Treating PJI as Biofilms: Diagnosis and Management Word Counts: Abstract (150 words); Text (5,042 words)

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ACCEPTED MANUSCRIPT ABSTRACT Considerable evidence suggests that microbial biofilms play an important role in periprosthetic

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joint infection (PJI) pathogenesis. Compared to free-floating planktonic bacteria, biofilm bacteria

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are more difficult to culture and possess additional immune-evasive and antibiotic resistance mechanisms, making infections harder to detect and eradicate. This article reviews cutting-edge

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advances in biofilm-associated infection diagnosis and treatment in the context of current PJI

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guidelines and highlights emerging technologies that may improve the efficacy and reduce costs associated with PJI. Promising PJI diagnostic tools include culture-independent methods based

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on sequence comparisons of the bacterial 16S ribosomal RNA gene, which offer higher throughput and greater sensitivity than culture-based methods. For therapy, novel methods based

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on disrupting biofilm-specific properties include quorum quenchers, bacteriophages, and

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ultrasound/electrotherapy. Since biofilm infections are not easily detected or treated by conventional approaches, molecular diagnostic techniques and next-generation anti-biofilm

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treatments should be integrated into PJI clinical practice guidelines in the near future.

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ACCEPTED MANUSCRIPT KEYWORDS culture-negative, implant-associated infection, bacterial 16S rRNA, quantitative PCR, quorum

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sensing/quenching, bacteriophage

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ACCEPTED MANUSCRIPT 1. INTRODUCTION Although infection following primary total joint arthroplasty (TJA) remains relatively rare,

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occurring in ~1% of patients (Kurtz, et al., 2008), postoperative periprosthetic joint infections

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(PJI) nonetheless represent a serious concern for clinicians. These infections may place patients at greater risk for complications and often require costly treatments (Bozic & Ries, 2005).

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Further highlighting PJI’s growing significance, the American Association of Orthopaedic

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Surgeons (AAOS) and the Infectious Diseases Society of America (IDSA) have released independent sets of PJI clinical practice guidelines within the past few years (Della Valle, et al.,

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2010; Osmon, et al., 2013). Of high priority for the next iteration of PJI guidelines is consideration of the role of microbial biofilms in PJI pathogenesis and treatment, which has been

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supported by increasing evidence in recent years. In this review, we evaluate cutting-edge

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advances in biofilm-associated infection diagnosis and treatment in the context of the current periprosthetic infection guidelines and highlight emerging technologies that may improve the

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efficacy and reduce the costs of identifying and managing PJI.

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1.1 The Biofilm Paradigm

In most environments, including the human body, microbes preferentially exist as layers of aggregated sessile cells surrounded by extracellular biopolymer matrices, i.e. biofilms, rather than in free-floating planktonic form (Bjarnsholt, Ciofu, Molin, Givskov, & Hoiby, 2013). Biofilm formation occurs in several stages as motile cells gradually accrue on inorganic or organic surfaces. Importantly, cells within a biofilm become phenotypically different from their planktonic analogues (Fig. 1). In addition to reduced motility, bacteria in biofilms possess distinct gene transcription patterns and exhibit a spectrum of metabolic activity, with cells at the

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ACCEPTED MANUSCRIPT biofilm periphery growing more rapidly than the nutrient-deprived cells in the biofilm’s inner layers (Bjarnsholt, et al., 2013; P. Stoodley, et al., 2011).

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An estimated 80% of human infections can be attributed to biofilms, and nearly three

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decades ago, Gristina and Costerton promulgated the idea that many periprosthetic joint infections involve microbial biofilms (1984), which has since been supported by direct and

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indirect observations. PJI often demonstrates key characteristics of biofilm-associated infections

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(Table 1). Moreover, the most common microbial causative agents of PJI, staphylococci, streptococci, and enterococci (Pandey, Berendt, & Athanasou, 2000), form biofilms in other

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types of infection, including jaw osteonecrosis and ureteric stent infections (Keane, Bonner, Johnston, Zafar, & Gorman, 1994; Sedghizadeh, et al., 2008). Additionally, a handful of studies

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have provided direct clinical evidence of biofilm formation on implants through electron or

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confocal laser scanning microscopy analysis of surgically removed infected prosthesis components (Neut, van Horn, van Kooten, van der Mei, & Busscher, 2003; Paul Stoodley, et al.,

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2008; Tunney, et al., 1998). Whether these studies represent outliers or the norm in PJI cases

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remains to be seen, but the collective evidence to date suggests that biofilms occur in at least a subset of PJI cases. Given that biofilm-forming microbes evade most conventional bacterial diagnosis and treatment strategies, it is surprising that biofilms do not factor more into the AAOS and IDSA guidelines for PJI.

2. BIOFILM-ASSOCIATED PJI DIAGNOSIS For initial assessment of patients with symptoms of possible PJI, such as wound drainage over the prosthesis and/or local and systemic inflammatory signs (Osmon, et al., 2013), both AAOS and IDSA strongly suggest erythrocyte sedimentation rate (ESR) measurement and C-reactive

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ACCEPTED MANUSCRIPT protein (CRP) testing (Fig. 2). If abnormal ESR or CRP is detected, then arthrocentesis is highly recommended, with the aspirated fluid sent for white blood cell count and differential as well as

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aerobic and anaerobic microbiologic culture to determine treatment options (Della Valle, et al.,

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2010; Osmon, et al., 2013). Although these criteria may readily identify patients with earlypostoperative PJI (occurring within a few months), patients with late-chronic PJI (occurring

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several months to years) may be difficult to distinguish from those with aseptic implant

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loosening, since both conditions may present simply as loose prosthesis accompanied by pain (Osmon, et al., 2013).

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Both the timing and the lack of systemic inflammation in chronic PJI may in fact indicate biofilm-associated infection (Table 1). Furthermore, given that most biofilm species escape

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detection by conventional culture-based methods, a large proportion of culture-negative

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infections may be misdiagnosed as aseptic loosening (Achermann, Vogt, Leunig, Wust, & Trampuz, 2010; Tunney, et al., 1998) and fail to receive appropriate treatment. Further

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complicating the issue is that many patients have previously received antibiotic therapy, which

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may eliminate the planktonic bacteria that is more easily detected by traditional approaches (Achermann, et al., 2010; Peel, Dowsey, Aboltins, Daffy, & Stanley, 2013). Hence, there is an urgent need for alternative, culture-independent methods of PJI detection. Of great relevance are strategies based on molecular diagnostic methods. In addition to offering the ability to reveal dormant or metabolically inactive organisms, molecular detection methods can be completed in much less time than culture-based methods (hours versus days) (Cazanave, et al., 2013; John William Costerton, et al., 2011; Krimmer, et al., 1999). These methods have great potential as PJI diagnostic tools (Table 2), especially in conjunction with improved culture methods such as implant sonication, which yield more sensitive culture results

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ACCEPTED MANUSCRIPT than tissue biopsy or synovial fluid sampling, prolonged culture incubation for up to 14 days to allow anaerobic microbial growth, and multi-sample collection from the infection site (Larsen,

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Lange, Xu, & Schønheyder, 2012; Trampuz, et al., 2007).

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2.1 Polymerase Chain Reaction (PCR)

PCR has routine clinical applications in genetic testing and in the detection of infectious agents

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including human immunodeficiency virus and Mycobacterium tuberculosis (Hamady & Knight,

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2009; Moure, et al., 2011). In PCR, target gene sequences undergo successive cycles of enzymatic amplification by DNA polymerase, as specified by a known pair of primers

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complementary to the sequence of interest. The 16S ribosomal RNA (rRNA) gene is the most common amplification target, as the gene comprises both highly conserved and hypervariable

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regions: the conserved regions serve as binding sites for universal bacteria primers, while the

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hypervariable regions can be probed by specific primers to identify bacterial taxa. Several studies have demonstrated that PCR of prosthesis sonicate fluid is at least as sensitive and specific as

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sonicate fluid culture (Cazanave, et al., 2013; Gomez, et al., 2012; Grif, et al., 2012; Lévy &

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Fenollar, 2012; Rampini, et al., 2011; Xu, et al., 2012) and may have a higher PJI detection rate than culture for patients recently treated with antibiotics (Cazanave, et al., 2013; Gomez, et al., 2012; Grif, et al., 2012). Concurrently, PCR can be used to assess the antibiotic resistance of PJI microbes through the use of primers that amplify specific resistance genes such as mecA; this strategy has been validated for methicillin-resistant staphylococci detection in PJI (DubouixBourandy, et al., 2011; Titecat, et al., 2012). The majority of studies employing PCR in PJI diagnosis have used the quantitative variant (qPCR), which enumerates the total abundances of bacterial taxa or antibiotic-resistant bacteria (depending on the primers) in patient samples (Bjerkan, et al., 2012; Cazanave, et al., 2013; Gomez, et al., 2012; Grif, et al., 2012; Xu, et al.,

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ACCEPTED MANUSCRIPT 2012). qPCR has the advantage over standard PCR in that the bacterial load of each species in polymicrobial infections can be evaluated.

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Overall, PCR offers many improvements for PJI detection over culture-based methods,

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including higher-throughput analysis in 96- or 384- well plate formats, reduced hindrance by prior antibiotic therapy, and more rapid output. Once patient samples have been collected, DNA

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extraction can be performed using commercial kits in just a few hours, with subsequent, largely

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automated PCR amplification of the extracted DNA requiring several additional hours (Cazanave, et al., 2013; John William Costerton, et al., 2011). One potential drawback of PCR is the risk of

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detecting false positives, namely DNA from dead or contaminating environmental bacteria or from primer cross-reactivity with human tissue (Bjerkan, et al., 2012; Cazanave, et al., 2013; Xu,

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et al., 2012). False positive incidence can be decreased by performing PCR on several

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independent samples from each patient and by using primers specific for pathogen virulence factor genes alongside the 16S rRNA gene primers, increasing specificity.

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The other major drawback of PCR is that pathogen identification may not be

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straightforward: if universal primers are used to amplify the 16S rRNA genes of all bacteria present, the resulting PCR amplicons must be sequenced and compared to known sequences, a lengthy and costly process that requires quality databases. Yet if taxon-specific 16S rRNA gene primers are used, microbes not belonging to the surveyed taxa will not be detected. Fortunately, refinements have developed for both approaches, and comprehensive databases of humanassociated microbes have become widely available (Human Microbiome Project, 2012). The next-generation technique of pyrosequencing allows for massively parallel, rapid identification of bacteria at a much lower cost per base than traditional Sanger sequencing. The greater breadth and depth of pyrosequencing—hundreds of thousands of sequences can be generated in a single

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ACCEPTED MANUSCRIPT run—means that low abundance species have a higher chance of being detected (Hamady & Knight, 2009). An alternative to sequencing is the Ibis platform, which relies on electrospray

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ionization mass spectrometry to determine the masses and therefore base pair compositions and

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abundances of PCR amplicons. Bacteria can then be identified by algorithmic comparison with an extensive database of microbial signature masses (John William Costerton, et al., 2011; Ecker,

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et al., 2008). A related method using matrix-associated laser desorption/ionization time-of-flight

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(MALDI-TOF) mass spectrometry in conjunction with the Biotyper database to distinguish microbes directly by their distinct native protein peaks, bypassing the need for PCR

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amplification, has also been employed to identify bacterial isolates from clinical infections (Harris, et al., 2010; Seng, et al., 2009). Additionally, the increasing availability of sequence and

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mass databases for antibiotic resistance genes has allowed analogous pyrosequencing and mass

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spectrometry approaches to be used for characterizing microbial antibiotic sensitivity (Kristiansson, et al., 2011; Seng, et al., 2009). Of course, both pyrosequencing and mass

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spectrometry-based methods require specialized equipment that may not be readily available at

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many hospitals, though technological advances should eventually increase the accessibility of these instruments. Alternatively, panels of primers specific for the bacteria genera most commonly responsible for PJI may be used to analyze patient samples, optimizing chances of detection without the need for subsequent sequencing or equipment aside from a conventional thermocycler; this method has been used to diagnose culture-negative PJI due to Staphylococcus, Streptococcus, Enterococcus, and several other bacteria groups (Cazanave, et al., 2013). 2.2 Fluorescence In Situ Hybridization (FISH) FISH provides spatial resolution of bacterial groups on excised tissue or implant sections. In this method, samples are fixed and permeabilized to allow short, fluorescent DNA or peptide nucleic

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ACCEPTED MANUSCRIPT acid probes to hybridize with their targets. Analogous to PCR primers, these probes are complementary to regions of the 16S rRNA or antibiotic resistance genes. The samples can then

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be examined using a fluorescence microscope to check for fluorescently labeled bacteria. Using

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FISH, the physical structure of biofilms can be visualized, particularly if confocal microscopy is employed (John William Costerton, et al., 2011; Krimmer, et al., 1999; Nistico, et al., 2009).

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Though there is a dearth of direct comparisons between culture-based methods and FISH for PJI

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diagnosis, FISH has been shown to be at least as sensitive and specific as culture in detecting pathogens in clinical blood samples (Gescher, et al., 2008; Kempf, Trebesius, & Autenrieth,

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2000). Additionally, FISH can identify bacteria in culture-negative infections including PJI, even if the bacteria are encapsulated within biofilms (John William Costerton, et al., 2011; Krimmer,

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et al., 1999; Nistico, et al., 2009; P. Stoodley, et al., 2011; Xu, et al., 2012).

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In contrast to PCR, FISH remains relatively low throughput, yet like PCR, only the surveyed bacterial taxa or antibiotic resistance genes are detected. However, FISH readily

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circumvents several weaknesses of PCR, making it an ideal complementary diagnostic strategy.

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PCR may measure dead as well as live bacteria, but with FISH, dead bacteria can be excluded by co-administration of a viability stain (Savichtcheva, Okayama, Ito, & Okabe, 2005). Furthermore, unlike PCR, FISH allows facile identification of environmental bacteria contamination and probe cross-reactivity with human tissue, as false positive samples fail to appear integrated with the host surface, lack characteristic biofilm structure, or indicate cells much too large to be prokaryotic (Ehrlich, et al., 2012). 2.3 DNA Microarrays Microarrays are composed of thousands of microscopic DNA probes organized in a grid. For microbial analysis, these probes are generally complementary to taxon-specific fragments of the

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ACCEPTED MANUSCRIPT 16S rRNA gene or portions of antibiotic resistance genes. Clinical sample DNA is fluorescently labeled and then allowed to hybridize with microarray probes, with relative abundances of each

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taxon and antibiotic-resistant bacteria determined by fluorescent intensity of the corresponding

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microarray spot (Hamady & Knight, 2009). Existing microarrays such as PhyloChip (Wilson, et al., 2002) and HuGChip (Tottey, et al., 2013) cover large portions of the human microbiome and

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could be assayed for PJI detection; custom microarrays encompassing common PJI-associated

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pathogens and their associated virulence factors could also be fabricated. The microarray approach is potentially cheaper and more rapid for identifying microbial causative than

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sequencing-based methods, though detection is limited to the probes printed on the microarray (DeSantis, et al., 2007; Hamady & Knight, 2009). In a direct comparison of a 16S rRNA gene

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microarray with pyrosequencing for oral microbiome characterization, both methods exceled at

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high-throughput characterization of common microbial taxa; for less common genera, pyrosequencing provided broader and more sensitive detection (Ahn, et al., 2011). Improved

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microarray designs could circumvent these drawbacks, and meanwhile, the current designs

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present a compromise between cost/time and diagnostic sensitivity.

3. BIOFILM-ASSOCIATED PJI TREATMENT Preventative measures such as skin preparation, body exhaust suits, and laminar flow should be taken pre-, intra-, and post- operatively to minimize chances of PJI; these measures have been extensively reviewed (Illingworth, et al., 2013; Legout & Senneville, 2013). If PJI nonetheless occurs, IDSA provides extensive management strategy guidelines, which range from debridement and retention to prosthesis exchange or in serious cases, resection arthroplasty (Fig. 2). Several courses of antimicrobial therapy are recommended in conjunction with all procedures.

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ACCEPTED MANUSCRIPT For staphylococcal PJI, the treatment course consists of oral rifampin accompanied by intravenous pathogen-specific antibiotics, followed by rifampin with a companion oral drug. In

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some instances, indefinite oral antimicrobial suppression may be initiated after the initial therapy.

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For non-staphylococcal PJI, intravenous or highly bioavailable oral pathogen-specific antibiotics are prescribed, with chronic oral antimicrobial suppression following as needed (Osmon, et al.,

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2013). Unfortunately, bacteria within biofilms possess extreme resistance to conventional

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antimicrobials, due largely to slower growth rates and the ease of resistance gene transfer in close quarters. The physical structure of biofilms results in creation of an antibiotic gradient,

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with inner cells receiving less antibiotic and thus greater opportunity to develop resistance. Furthermore, biofilm bacteria mutate more rapidly than their planktonic counterparts, a

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phenomenon linked to increased oxidative stress in the biofilm environment both endogenously

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and induced in response to antibiotics (Bjarnsholt, et al., 2013). Biofilms possess physical, physiological, and/or adaptive resistance mechanisms against

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common microbicide classes such as aminoglycosides, antimicrobial pepties, β-lactams, and

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quinolones (Bjarnsholt, et al., 2013). Indeed, frequently used PJI antimicrobial agents (rifampicin, vancomycin, tigecycline, clindamycin, trimethoprim/sulfamethoxazole, ciprofloxacin, cloxacillin, daptomycin, fosfomycin) proved largely unable to eradicate in vitro biofilms cultured from a panel of clinical Staphylococcus aureus and S. epidermidis samples (Molina-Manso, et al., 2013). Antibiotic resistance is not limited to staphylococci: several clinical clones of Enterococcus faecalis, another common PJI pathogen, also showed strong biofilm production and significant antibiotic multi-resistance (Arciola, et al., 2008). Of the antimicrobial therapies recommended by IDSA, only the transcriptional inhibitor rifampin and the cell wall biosynthesis inhibitor meropenem have shown consistent anti-biofilm activity. Rifampicin is well documented to be

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ACCEPTED MANUSCRIPT effective for the eradication of staphylococcal biofilms (John, et al., 2009; Kadurugamuwa, et al., 2003; Raad, et al., 2007; Saginur, et al., 2006) and may be efficacious against Propionibacterium

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acnes and other biofilms as well (Furustrand Tafin, Corvec, Betrisey, Zimmerli, & Trampuz,

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2012). Rifampin may decrease microbial adherence to surfaces, resulting in the release of planktonic bacteria that are more susceptible to antimicrobial agents (Saginur, et al., 2006). As

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the IDSA guidelines state, rifampin should not be used as a single agent due to the high

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likelihood of resistance development (Osmon, et al., 2013); it works synergistically with numerous other drugs, including quinolones and vancomycin, to achieve cure rates between 83%

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and 100% (Lynch & Robertson, 2008; Zimmerli & Moser, 2012). Meropenem is often used to treat Pseudomonas aeruginosa biofilms, as it is β-lactamase stable and hence eludes a common

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antibiotic resistance mechanism (Hill, et al., 2005; Moskowitz, et al., 2011), and may be

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effective against other Gram-negative bacterial biofilms. Like rifampin, meropenem works best in combination with other microbicides such as fluoroquinolones or aminoglycosides

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(Rodríguez-Pardo, et al., 2014). In general, efficacious anti-biofilm agents should exhibit both

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high activity within biofilms and adequate penetration of infected soft tissue and bone; appropriate treatment regimens have been reviewed by Zimmerli and Moser (2012). Given that current-generation antimicrobial therapies tend to suppress rather than eradicate biofilms, with limited drugs possessing true anti-biofilm activity, there is an urgent need for alternate biofilm-associated PJI management options. One possible solution, which has been briefly explored, is combination therapy of high-dose antibiotics with known anti-biofilm agents including azithromycin, colistin, meropenem, and rifampin. Combination therapy with drugs against known biofilm antibiotic resistance mechanisms such as efflux pumps and oxidative stress could also bolster efficacy (Bjarnsholt, et al., 2013). Ultimately, antimicrobial

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ACCEPTED MANUSCRIPT combination therapy requires careful optimization of timing and dosages to maximize efficacy while minimizing toxicity and is limited by the many mechanisms of biofilm resistance to

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conventional microbicides. Novel methods based on disrupting biofilm-specific properties alone

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or in combination with existing therapeutics are thus highly appealing (Table 3). 3.1 Quorum Quenching

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One promising next-generation treatment is the inhibition of quorum sensing, a form of inter-

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and intra- species cell-to-cell bacterial communication used by many pathogenic bacteria including S. aureus and P. aeruginosa (Estrela, Heck, & Abraham, 2009; Hirakawa & Tomita,

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2013; Tay & Yew, 2013; Zhu & Kaufmann, 2013). Such strategies, referred to as quorum quenching, aim to impede bacterial communication, preventing both the expression of

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extracellular signaling molecules (autoinducers) and the resulting coordination of bacterial group

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behaviors (Estrela, et al., 2009; Hirakawa & Tomita, 2013; Zhu & Kaufmann, 2013). Since this communication system is pivotal to the expression of virulence and biofilm formation genes,

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quorum sensing inhibition can potentially be an effective and specific anti-biofilm approach

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(Hirakawa & Tomita, 2013; Zhu & Kaufmann, 2013). Additionally, several studies have suggested that because quorum sensing does not directly affect fitness and viability, selection pressures for resistance to quorum quenching compounds may be less strong than for antibiotics (Tay & Yew, 2013; Zhu & Kaufmann, 2013). Key quorum quenching strategies include 1) inhibition of autoinducer synthases; 2) enzymatic degradation of autoinducers; and 3) ligand mimicry to inhibit signaling receptors (Estrela, et al., 2009; Hirakawa & Tomita, 2013; Tay & Yew, 2013; Zhu & Kaufmann, 2013). Expansive natural and synthetic chemical compound libraries have been screened for quorum quenching efficacy, facilitating the development of ligand analogues (Estrela, et al., 2009; Zhu &

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ACCEPTED MANUSCRIPT Kaufmann, 2013). Recent studies have investigated derivatives of naturally produced quorum sensing inhibitors such as furanones, which are produced by certain alga, and their sulfur

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analogues, thiophenones, along with ajoene, extracted from garlic (Jakobsen, et al., 2012; Lonn-

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Stensrud, Landin, Benneche, Petersen, & Scheie, 2009; Lonn-Stensrud, Naemi, Benneche, Petersen, & Scheie, 2012). Other potential avenues for quorum quenching include inhibition of

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signaling molecule secretion, interference with downstream signaling pathway factors,

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inactivation of autoinducers using antibodies or other compounds like cyclodextrin, and activation of regulatory quorum sensing suppressors or biofilm autolysis mechanisms (Estrela, et

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al., 2009; Hirakawa & Tomita, 2013; Zhu & Kaufmann, 2013). Although an array of quorum quenching compounds have undergone testing in vitro and

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in animal models, none have yet reached clinical trials (Hirakawa & Tomita, 2013; Zhu &

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Kaufmann, 2013). Current challenges include potential cytotoxic effects on eukaryotic cells, inability of ligand analogues to outcompete natural ligands for signal inhibition, and the high

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specificity of autoinducers for individual pathogen quorum sensing systems (Estrela, et al., 2009;

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Zhu & Kaufmann, 2013). Nonetheless, it is probable that these challenges can be surmounted using protein engineering approaches (Tay & Yew, 2013). Since quorum quenching alone may be insufficient to completely resolve biofilm-associated infections, combination therapy with other treatment modalities such as antibiotics will likely also be necessary (Zhu & Kaufmann, 2013). Overall, better understanding of the roles of various autoinducers and quorum sensing systems in biofilm formation, maintenance, and virulence is required to develop more effective therapeutic quorum quenching compounds. 3.2 Bacteriophages

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ACCEPTED MANUSCRIPT Another promising strategy for treating biofilm-associated PJI is bacteriophage therapy, which has been employed to treat dysentery, wound infections, and osteomyelitis in patients. Though

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their popularity has declined in the current era of widespread antibiotic usage, several phage

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therapies have recently passed or are undergoing phase I and II clinical trials (Donlan, 2009; Lu & Koeris, 2011). Phages are viruses that infect and lyse bacteria with specificity ranging from

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strain- to species- or multi-species levels (Azeredo & Sutherland, 2008; Donlan, 2009). This high

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specificity has attractive clinical implications: since phages can only replicate within their host bacteria species, therapeutic pathogen-specific phages would become concentrated at infection

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sites (Azeredo & Sutherland, 2008). Unlike antibiotics, which require a higher concentration to eradicate older biofilms, phage efficacy does not appear to be affected by biofilm age. Using a

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variety of enzymes such as polysaccharide depolymerases, phages are also able to break down

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biofilm extracellular matrices, which often hamper antibiotic diffusion (Donlan, 2009). Additionally, phages remain effective in low-nutrient and low-temperature environments, and

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phage production is fast and relatively inexpensive (Azeredo & Sutherland, 2008; Donlan, 2009).

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Several bacteriophages have been identified that specifically target a variety of biofilmforming bacteria, including Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli (Carson, Gorman, & Gilmore, 2010; Donlan, 2009; Pires, Sillankorva, Faustino, & Azeredo, 2011; Yele, Thawal, Sahu, & Chopade, 2012). However, the species-specificity of phages presents a challenge, since it requires accurate identification of the biofilm organism; a further complication is that biofilms often comprise multiple species (Azeredo & Sutherland, 2008). Potential solutions include engineering phages with broader host specificity, utilizing a cocktail of phages, and developing more efficient biofilm bacteria identification assays (Donlan, 2009; Lu & Koeris, 2011). Other hurdles to phage therapy include

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ACCEPTED MANUSCRIPT phage inactivation by the immune system, safety considerations such as endotoxin release or horizontal gene transfer (HGT) of virulence factors, and systemic immune activation (Donlan,

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2009; Lu & Koeris, 2011). These difficulties may be surmounted by using phage-derived

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proteins rather than whole phages as therapy, decreasing immunogenic and HGT potential. In particular, lysins, a class of hydrolytic enzymes, can perforate the cell walls of specific bacteria

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and may synergize with antibiotic therapy (Azeredo & Sutherland, 2008; Fenton, et al., 2012;

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Pastagia, Schuch, Fischetti, & Huang, 2013). Furthermore, no studies have yet reported the development of lysin resistance after exposure (Fenton, et al., 2012; Pastagia, et al., 2013).

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As with other anti-microbial strategies, phages may work best in combination therapy settings, which minimize the development of resistance mechanisms (Donlan, 2009; Lu & Koeris,

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2011). Phages may be combined with traditional antibiotics, phage cocktails may be employed,

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or anti-biofilm phages may be co-administered with engineered phages that specifically target phage resistance mechanisms (Lu & Koeris, 2011). A recent study validated the in vitro efficacy

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of phage therapy and rifampicin treatment for eradication of S. aureus biofilms (Rahman, Kim,

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Kim, Seol, & Kim, 2011). Synergy between phage therapy and antibiotics has also been demonstrated using a rat model of implant infection: combination therapy significantly decreased MRSA or P. aeruginosa bacterial loads compared to either treatment alone (Yilmaz, et al., 2013). Although one study employed a known bacteriophage as well as modified derivatives to eliminate S. aureus biofilms with no incidences of phage resistance (Kelly, McAuliffe, Ross, & Coffey, 2012), another group noted the in vitro development of phage resistance (Moons, Faster, & Aertsen, 2013), further underscoring the importance of employing phage therapy as a combination therapy rather than as a monotherapy 3.3 Ultrasound and Electrotherapy

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ACCEPTED MANUSCRIPT Ultrasound treatment in combination with antibiotics has significantly reduced or even completely eradicated S. epidermis, S. aureus, P. aeruginosa, and E. coli biofilms in vitro

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(Wanner, et al., 2011; Yu, Chen, & Cao, 2012). Moreover, this combination was significantly

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more effective than monotherapy for eradicating biofilms from implanted polyethylene disks in rabbit models (Carmen, et al., 2005; Rediske, et al., 2000). Although most studies to date have

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applied high-energy ultrasound, high-energy ultrasound treatments present the risk of micro-

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fractures and hematoma formation; a better alternative may be to use low-energy ultrasound, which may actually enhance tissue regeneration and bone healing (Wanner, et al., 2011). Limited

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evidence indicates that lower frequencies can be similarly efficacious against biofilms: one in vivo study demonstrated that pulsed ultrasound treatment at the same peak intensity resulted in

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the same effect as continuous treatment, but at a lower total power and without damaging tissue

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(Rediske, et al., 2000; Yu, et al., 2012).

Additionally, some data suggest a similar combinatory response to electricity, a

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“bioelectric effect” in which electric currents enhance antibiotic effectiveness against biofilms.

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Various antibiotic classes, including aminoglycosides and quinolones, have shown increased effectiveness in the presence of electric current (J. W. Costerton, Ellis, Lam, Johnson, & Khoury, 1994; Jass & Lappin-Scott, 1996; Wellman, Fortun, & McLeod, 1996), and in particular, combination with electric current significantly improved the in vitro efficacy of vancomyocin against MRSA biofilms (del Pozo, Rouse, Mandrekar, et al., 2009). Bioelectric effects aside, electrotherapy alone has also exhibited dose- and time- dependent “electricidal effects”, resulting in significant biofilm reduction in vitro (del Pozo, Rouse, Mandrekar, et al., 2009; Vergidis & Patel, 2012). Furthermore, in a rabbit infection model, electrotherapy alone proved significantly

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ACCEPTED MANUSCRIPT more effective in reducing S epidermis chronic foreign-body osteomyelitis compared to doxycycline-treated and control animals (Del Pozo, Rouse, Euba, et al., 2009).

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Ultimately, further research is required to elucidate the mechanisms underlying treatment

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efficacy as well as to translate preclinical studies for clinical application (Carmen, et al., 2005; Rediske, et al., 2000; Vergidis & Patel, 2012; Yu, et al., 2012). For instance, effective and safe

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thresholds for frequency, peak intensity, and power in ultrasound therapy should be established

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(Yu, et al., 2012). In addition, the generalizability of these approaches to various biofilm

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organisms and antibiotic classes should be explored.

4. FUNGAL BIOFILM DIAGNOSIS AND TREATMENT

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Bacteria are not the only biofilm-forming microorganisms: fungal biofilms can also be clinically

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challenging to diagnose and treat (Lynch & Robertson, 2008). Specifically, Candida species are often implicated in PJI and other medical device infections (Lynch & Robertson, 2008). Candida

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albicans, the most ubiquitous PJI-associated fungal pathogen, is noted for its capacity to form

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biofilms (Chandra, et al., 2005). Unfortunately, certain diagnosis tools for bacterial biofilms, such as Gram staining, are not effective for diagnosing fungal biofilms, and furthermore, serological values and synovial fluid cell counts cannot identify fungal infections if the patient presents with both fungal and bacterial infections (Azzam, et al., 2009). IDSA treatment recommendations include debridement and resection arthroplasty, though debridement may not be sufficient to resolve fungal infections (Azzam, et al., 2009). Particularly troubling is that the most prevalent approach to treating fungal biofilm infections, antifungal compound treatment and a two-stage exchange, has an overall success rate of around 50% (Legout & Senneville, 2013).

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ACCEPTED MANUSCRIPT 4.1 Fungal Biofilm Diagnosis Current techniques for diagnosing fungal biofilm species include cultures, serological analyses,

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PCR, DNA microarrays, and 26S rRNA sequencing, all of which are analogous to methods used

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for bacterial biofilm identification (Azzam, et al., 2009; Bu, et al., 2005). MALDI-TOF mass spectrometry has also been successfully used for species identification based on fungal proteins

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detected in the sample (Bader, et al., 2011; Bille, et al., 2012; Marklein, et al., 2009).

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Additionally, a recent study presented preliminary results for a species-specific assay based on antibodies to gliotoxin produced by A. fumigatus (Bugli, et al., 2014). Nonetheless, definitive

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standardized, evidence-based clinical guidelines for fungal biofilm diagnosis should be

4.2 Fungal Biofilm Treatments

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established in the near future (S. X. Zhang, 2013).

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Currently used antifungal drugs include azoles, echinocandins, and polyenes (Ramage, Robertson, & Williams, 2014). However, key drawbacks of these compounds include limited

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spectrum of utility and serious side effects, as well as increasing drug resistance (Wong, et al.,

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2014). Azoles, in particular, have demonstrated lack of efficacy against various Candida biofilm species (Ramage, et al., 2014). Echinocandins have proven more effective but do not thoroughly eliminate biofilms and possess slower kinetics than amphotericin B, a polyene. In contrast, polyenes exhibit dose-dependent renal toxicity (Ramage, et al., 2014). Development of novel treatment modalities is thus essential. Antifungal quorum sensing inhibitors aim to disrupt auto-regulation mechanisms that govern fungal pathogenicity and biofilm formation. One recent study characterized the novel quorum quenching compound thiazolidinedione-8, finding specific anti-biofilm and antiadherence activity against C. albicans (Feldman, Al-Quntar, Polacheck, Friedman, & Steinberg,

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ACCEPTED MANUSCRIPT 2014). Another study showed that 2-dodecenoic acid isomers, which are structurally similar to the signaling factor cis-11-methyl-2-dodecenoic acid, disrupted biofilm structure and growth in

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vitro (Y. Q. Zhang, Cai, Yang, Weng, & Wang, 2011). Since neither of these compounds

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completely eradicated fungal biofilms, a combinatory therapy may prove more efficacious. Another promising antifungal biofilm agent is chitosan, derived from the natural

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crustacean polymer chitin. Proposed mechanisms for its antifungal activity are cell wall

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interference, fungal transcription or translation inhibition, and microbial growth inhibitory metalchelation (Cobrado, et al., 2012). Chitosan has shown significant anti-biofilm activity against C.

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albicans both in vitro and in vivo (Cobrado, et al., 2012; Martinez, et al., 2010). Ultimately, further research needs to be performed to identify optimal strategies for employing quorum

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quenchers and chitosan, alone and in combination with current antifungal agents, against fungal

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5. CONCLUSIONS

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biofilm PJI in order to maximize efficacy while minimizing toxicity.

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The biofilm paradigm for understanding bacterial colonization has revolutionized environmental microbiology and has powerful implications for clinical infections such as PJI. Further work is needed to quantify the prevalence of biofilm-associated PJI cases, but based on bacterial growth habits in other environments and infection sites, the majority of chronic PJI cases are likely linked to biofilms. Since biofilm-associated infections are not easily detected by culture-based methods and often resist management by conventional antimicrobial therapy, molecular diagnostic techniques and next-generation anti-biofilm treatments should be integrated into PJI clinical practice guidelines as soon as possible (Fig. 2).

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ACCEPTED MANUSCRIPT 6. ACKNOWLEDGEMENTS We would like to thank Dr. Noreen J. Hickok for the generous contribution of scanning electron

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microscopy images to this manuscript.

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Rahman, M, Kim, S, Kim, SM, Seol, SY, & Kim, J (2011) Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin. Biofouling, 27: 1087-1093. Ramage, G, Robertson, SN, & Williams, C (2014) Strength in numbers: antifungal strategies against fungal biofilms. International Journal of Antimicrobial Agents, 43: 114-120. Rampini, SK, Bloemberg, GV, Keller, PM, Büchler, AC, Dollenmaier, G, Speck, RF, & Böttger, EC (2011) Broad-range 16S rRNA gene polymerase chain reaction for diagnosis of culturenegative bacterial infections. Clin Infect Dis, 53: 1245-1251. Rediske, AM, Roeder, BL, Nelson, JL, Robison, RL, Schaalje, GB, Robison, RA, & Pitt, WG (2000) Pulsed ultrasound enhances the killing of Escherichia coli biofilms by aminoglycoside antibiotics in vivo. Antimicrob Agents Chemother, 44: 771-772. Rodríguez-Pardo, D, Pigrau, C, Lora-Tamayo, J, Soriano, A, del Toro, MD, Cobo, J, Palomino, J, Euba, G, Riera, M, Sánchez-Somolinos, M, Benito, N, Fernández-Sampedro, M, Sorli, L, Guio, L, Iribarren, JA, Baraia-Etxaburu, JM, Ramos, A, Bahamonde, A, Flores-Sánchez, X, Corona, PS, Ariza, J, & the, RGftSoPI (2014) Gram-negative prosthetic joint infection: outcome of a debridement, antibiotics and implant retention approach. A large multicentre study. Clinical Microbiology and Infection: n/a-n/a. Saginur, R, Stdenis, M, Ferris, W, Aaron, SD, Chan, F, Lee, C, & Ramotar, K (2006) Multiple combination bactericidal testing of staphylococcal biofilms from implant-associated infections. Antimicrob Agents Chemother, 50: 55-61. Savichtcheva, O, Okayama, N, Ito, T, & Okabe, S (2005) Application of a direct fluorescencebased live/dead staining combined with fluorescence in situ hybridization for assessment of survival rate of Bacteroides spp. in drinking water. Biotechnology and bioengineering, 92: 356-363. Sedghizadeh, PP, Kumar, SKS, Gorur, A, Schaudinn, C, Shuler, CF, & Costerton, JW (2008) Identification of microbial biofilms in osteonecrosis of the jaws secondary to bisphosphonate therapy. J Oral Maxil Surg, 66: 767-775. Seng, P, Drancourt, M, Gouriet, F, La Scola, B, Fournier, P-E, Rolain, JM, & Raoult, D (2009) Ongoing revolution in bacteriology: Routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis, 49: 543-551. Stoodley, P, Ehrlich, GD, Sedghizadeh, PP, Hall-Stoodley, L, Baratz, ME, Altman, DT, Sotereanos, NG, Costerton, JW, & Demeo, P (2011) Orthopaedic biofilm infections. Current orthopaedic practice, 22: 558-563. Stoodley, P, Nistico, L, Johnson, S, Lasko, L-A, Baratz, M, Gahlot, V, Ehrlich, GD, & Kathju, S (2008) Direct demonstration of viable Staphylococcus aureus biofilms in an infected total joint arthroplasty: A case report. The Journal of Bone & Joint Surgery, 90: 1751-1758. Tay, SB, & Yew, WS (2013) Development of quorum-based anti-virulence therapeutics targeting Gram-negative bacterial pathogens. International journal of molecular sciences, 14: 1657016599. Titecat, M, Loiez, C, Senneville, E, Wallet, F, Dezeque, H, Legout, L, Migaud, H, & Courcol, RJ (2012) Evaluation of rapid mecA gene detection versus standard culture in staphylococcal chronic prosthetic joint infections. Diagnostic microbiology and infectious disease, 73: 318321. Tottey, W, Denonfoux, J, Jaziri, F, Parisot, N, Missaoui, M, Hill, D, Borrel, G, Peyretaillade, E, Alric, M, Harris, HM, Jeffery, IB, Claesson, MJ, O'Toole, PW, Peyret, P, & Brugere, JF

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1. Scanning electron micrograph of Staphylococcus aureus (ATCC25923) biofilm growth

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on a Nunc coverslip in trypticase soy broth after 18 hours at 37°C.

Figure 2. Updated algorithm for the treatment and management of periprosthetic joint infections.

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Cutting-edge technologies for biofilm detection and eradication can be considered

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complementary to and synergistic with current clinical standards.

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Figure 1

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Figure 2

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ACCEPTED MANUSCRIPT Table 1 Biofilm-like characteristics of chronic PJI Paradigmatic biofilm explanation

References

Infection local to device rather than systemic

Biofilm stationary on prosthesis surface

(Khoury, Lam, Ellis, & Costerton, 1992; Stoodley, et al., 2011)

Infection resolves after device removal

Biofilm removed along with prosthesis (substrate to which it is adhered)

Infection occurs months to years postoperatively

Biofilm formed in a multistage process over time

Minimal inflammatory symptoms present

Biofilm evades host immunity, resulting in dampened inflammation

Culture-negative infection indicated

Biofilm species not readily dislodged for sampling, do not grow under laboratory conditions

(Berbari, et al., 2007; HallStoodley, et al., 2012; Trampuz, et al., 2007)

Biofilm highly antibiotic resistant through multiple mechanisms

(Bjarnsholt, Ciofu, Molin, Givskov, & Hoiby, 2013; Molina-Manso, et al., 2013; Nishimura, Tsurumoto, Yonekura, Adachi, & Shindo, 2006)

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Common characteristic of chronic PJI

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Infection resists antibiotic therapy

(Osmon, et al., 2013; Stoodley, et al., 2011) (Khoury, et al., 1992; Osmon, et al., 2013) (Jensen, Kharazmi, Lam, Costerton, & Hoiby, 1990; Khoury, et al., 1992; Osmon, et al., 2013)

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ACCEPTED MANUSCRIPT Table 2 Comparison of PJI diagnosis strategies Advantages

Disadvantages

Sensitivity/ Specificity*

Aerobic/ anaerobic culture

Inexpensive; readily available

<1% of known microbes culturable; slow; culture bias; lowthroughput; sensitive to prior antibiotic therapy

56‒85% / 94‒100%

PCR

Rapid; can be quantitative; highthroughput

Limited by primer selection; higher false positive risk

Pyrosequencing

Rapid; quantitative; very high-throughput; extremely sensitive; quality database available

Requires specialized equipment; higher false positive risk

Ibis/MALDITOF

Rapid; quantitative; high-throughput; sequencing not required

FISH

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Method

(Jordan, Smith, Saithna, Sprowson, & Foguet, 2014; Legout & Senneville, 2013; Rak, BarlicMaganja, Kavcic, Trebse, & Cor, 2013) (Achermann, Vogt, Leunig, Wust, & Trampuz, 2010; Gomez, et al., 2012; Rak, et al., 2013; Titecat, et al., 2012)

60‒97% / 70‒100%

(Bravo, et al., 2009; Gadsby, et al., 2011)

Requires specialized equipment and comprehensive databases; higher false positive risk

78‒81% / ~95%

(GreenwoodQuaintance, et al., 2013; Melendez, et al., 2014; Seng, et al., 2009)

Rapid; spatial resolution; false positives easily identified

Limited by probe selection; lowthroughput

N/A

Rapid; semiquantitative; very high-throughput; inexpensive

Limited by probe selection; higher false positive risk

85‒91% / 88‒94%

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70‒100% / 94‒100%

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References

(Uchida, et al., 2009; Witney, et al., 2005)

*Results can vary depending on factors such as sample size, sample collection technique, previous antibiotic therapy, culture incubation duration, and culture media used. Future efforts should focus on optimizing protocols for each of these methods.

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ACCEPTED MANUSCRIPT Table 3 Comparison of PJI treatment strategies Advantages

Disadvantages

Conventional antibiotics

Well-documented efficacies and side effects; readily available

Multiple antibiotic resistance mechanisms; complex dose management needed for combo therapy

Quorum quenching compounds

Biofilm-specific; lower risk of acquired resistance

Still in experimental development; second therapeutic modality may be required for complete bacteria eradication

Bacteriophages

Biofilm-specific; unaffected by biofilm age or extracellular matrix; rapid and inexpensive to produce

Ultrasound/ Electrotherapy

Lower risk of acquired resistance (physical biofilm disruption); lowenergy ultrasound regimens may enhance healing

Still in experimental development; combination therapy needed to circumvent resistance; narrow species-specificity of current phages necessities accurate pathogen identification Still in experimental development; incomplete knowledge about mechanisms or safety/efficacy thresholds

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Method

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ACCEPTED MANUSCRIPT Highlights

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Periprosthetic joint infections (PJI) often show key features of biofilm infections Biofilm infections evade culture-based diagnosis and resist standard antibiotics 16S rRNA gene-based methods allow high-throughput, sensitive PJI detection Antimicrobials work best as cocktails or combined with biofilm-targeting therapies Biofilm diagnosis and management should be integrated into PJI clinical guidelines

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