MusculoskeletalInfectionsedited byJason H. CalhounJonT. MaderUniversityofTexasMedicalBranchGalveston,Texas,U.S.A.M A R C E LD E K K E R , I N C . N E W Y O R K B A S E LLibraryofCongressCataloging-in-PublicationDataAcatalogrecordforthisbookisavailablefromtheLibraryofCongress.ISBN:0-8247-0892-XThisbookisprintedonacid-freepaper.HeadquartersMarcelDekker,Inc.270MadisonAvenue,NewYork, NY10016tel:212-696-9000;fax:212-685-4540EasternHemisphereDistributionMarcelDekkerAGHutgasse4,Postfach812,CH-4001Basel, Switzerlandtel:41-61-260-6300;fax:41-61-260-6333WorldWideWebhttp://www.dekker.comThe publisher offers discounts on this book when ordered in bulk quantities. For more infor-mation, writetoSpecialSales=ProfessionalMarketingattheheadquartersaddressabove.Copyright # 2003byMarcelDekker,Inc.AllRightsReserved.Neither thisbooknoranypart maybereproducedortransmittedinanyformorbyanymeans,electronicormechanical,includingphotocopying,microlming,andrecording,orbyanyinformationstorageandretrieval system, without permissioninwritingfromthepublisher.Currentprinting(lastdigit):10 9 8 7 6 5 4 3 2 1PRINTEDINTHEUNITEDSTATESOF AMERICAInmemoryofJonT. MaderMarch21, 1944October25, 2002Dr. Jon T. Mader died while this book was in its nal stages of production.Whatwastobesimplyacompilationofwhatwevelearnedaboutmusculoske-letal infections over the last several decades has taken on a much greaterimportance. Thisbooknowservesasatributetoaremarkableman. Jonwasapioneerin theeldofmusculoskeletal infections;his work overthe years helpedtoincreaseour understandingof thetreatment of thesedifcult problems. Heworkedwithmanyof the authors inthis bookonnumerous projects, was afounding member of the Musculoskeletal Infections Society, and had many othercolleaguesintheareasofinfectiousdiseasesandhyperbaricmedicine.Ihadthepleasure of working closely with Jon for almost 20 years. His energy andcompassion were contagious. He loved his work and cared deeply for his patients.It is withgreat joyandsadness that I dedicatethis toJons memoryandhisremarkablecontributiontotheeldofmusculoskeletalinfections.Formywife, Karen, andchildren, Zoey, Ken, andSadie.JHCTomywife, Donna, andmychildren, SamandJonHenry, whohaveprovidedmealovingsanctuarythatgavemetheenergyandcouragetocompletethisproject.JTMPrefaceThe treatment of musculoskeletal infections continues to evolve. We have gleanedourknowledgefrompast andcurrent mastersandour patients. Ourknowledgewill be expanded and passed on to future generations. We are but a bridgebetweenthepastandfuture.JonT. MaderThe United Nations has declared the years 20002010 the Bone and JointDecade.Oneofthemajorcomponentsofboneandjointdisordersaremusculo-skeletal infections. We have spent the past 20 years treating patients withmusculoskeletalinfectionsandconductingresearchonthesedifcultinfections.When werststartedthisendeavorthere wasnot muchthatcouldbedonefor such patients, especially those with long bone osteomyelitis and severediabeticfootinfections.Inaddition,therewasnotmuchinterestinmusculoske-letal infections. These infections were considered more of a nuisance than a topicfor serious clinical and basic science research. At that time, the gold standard wasdebridementor amputationand sixweeksofparenteralantibiotics.Overthepasttwodecades, treatment of these infections has changed. We nowhave betterstagingsystems, surgeries, antibiotics, andadjunctivetherapies. Theinterest inthis topichas alsogrown. WehaveaMusculoskeletal Infections Society, andmoreclinical andbasicresearchisbeingdoneintheeld. Thepurposeofthisbook is to outline the current concepts for the treatment of musculoskeletalinfectionsandtoexploreemergingtechniquesfortreatment.viiThe rst chapter, onthe basic science of these infections, explores thestatement, The environment is everything and the bacteria are nothing.Physicians must understand the location of the infection (environment), theinfectingorganism(s),andthedefensesof thehostin ordertotreatanderadicatetheinfection.Thetriadofinfectionisdescribedindetailincludingtheinfectingpathogen, thepropertiesof thehost,and thesourceand locationoftheinfection.Inordertounderstandandtreat musculoskeletal infectionswemust havetreatment templates. Classicationof musculoskeletal infections helps providethistemplate. Chapter2describesastagingsystemforosteomyelitisthat helpsstratifytheinfectionandguidetherapy.Musculoskeletal infections are more difcult to treat in compromised hosts.The reasons for the difculty are carefully explored in the chapter on systemicallyandlocallyimmunocompromisedhosts. Physicians must developasystematicapproachforpatientswithcompromisedimmunesystemswhopresentwithsofttissue, joint, andboneinfections.It is better toprevent musculoskeletal infections rather thantreat them.Chapter4exploresthehistoryandcurrent rationalefortheuseofprophylacticantibioticsin musculoskeletal infections.Thetimingof antibioticadministration,roleofinteroperative dosing,duration of use,andchoiceofantibiotic(s)arealsodiscussed.Specic musculoskeletal infections andthe state-of-the-art treatment aredescribed in the chapters on open fractures, long bone osteomyelitis, septicarthritis, handinfections, totalhipinfections, totalkneeinfections, diabeticfootinfections, and vertebral osteomyelitis. Vertebral osteomyelitis is further stratiedinto hematogenous pyogenic infection of the spine, postoperative infections of thespine,andgranulomatousinfectionsofthespine.The treatment of osteomyelitis in neonates, infants, and children is differentfromthatinadults.Neonatesandinfantsarecompromisedhosts,whilechildrenare superhosts and are easier to treat than adults. Because of these differences, wehaveincludedachapteronpediatricosteomyelitis.The mainstays of musculoskeletal infections are surgery and culture-directedantibiotictherapy.Specicantibioticsthatareusefulinmusculoskeletalinfections andtheir potential toxicities are describedindetail inChapter 16.Includedinthatchapterisasectiononresistantorganismsandtheirtreatment.Surgeryisusuallymandatoryfor thetreatment of musculoskeletal infec-tions. The standardsurgeries have beendescribedinthe sections onspecicinfections. Muscleapsareuseful inalmost all theinfections, especiallywhenthere is soft-tissuecompromise. While the rationale forthe use of muscle aps isdescribedinthespecicchapters,thetechnicalaspectsanddetailsarepresentedinChapter17.The management of musculoskeletal infections includes antibiotics,surgery, and adjunctive therapy. The chapter on adjunctive therapy discussesviii Prefacetherolesandeffectivenessofsuchtherapiesinthemanagementofmusculoske-letal infection, including bioimplants, electrical stimulation, and hyperbaricoxygentherapy. TheuseofbioimplantsincludesPMMA, collagenhydroxyapa-tite, syntheticpolymers, andbrinsealant. Bioimplantsarebeingusedfor thedeliveryof antibioticsandbonemorphogenicprotein, asscaffolding, andasavehiclefor osteogenic cell delivery. The useof bioimplants is undergoinganexpansion and has the potential to change how we treat musculoskeletalinfections.The nal chapter discusses the state of the art in gene therapy inmusculoskeletal repair. Management of nonhealing wounds andbone defectshas beenaidedbythe development of newagents, includinggrowthfactors,cytokines, and bone morphogenicproteins. Away to get these agents to the rightlocationisthroughgenetherapy. Themost attractivefeatureofgenetherapyisthat therapeutic proteins can be delivered locally to the appropriate site inrelativelyhighconcentrationsandinasustainedfashion. Current goalsincludetheimprovementoftransfectionefcacyandspecicity,optimizinginducibleorcell-typespecicpromoters, andimprovinglocal applicationtechniques. Genetherapywillchangehowwetreatmusculoskeletalinfectionsandtheircomplica-tionsinthefuture.Musculoskeletalinfectionsarecommonproblemsthatinvolveavarietyofspecialties, includingorthopaedics, infectiousdiseases, plasticsurgery, vascularsurgery,internalmedicine, pediatrics,familymedicine, podiatry,andothers.Wefelt it was important to write a comprehensive book where all of these specialtiescould nd common ground. There are numerous articles and book chaptersspreadacross different specialties but it is difcult tondthe important andpertinentinformationinoneplace. Wehopethatthisbookwillllthatvoid.Wewouldliketothankourpatientswhohaveprovidedguidance. WeareindebtedtoKristi Overgaard, whowasthedrivingforcebehindthisbook. Ms.Overgaard organized, edited, and shepherded our book to the publishers.Acknowledgment must also be made to others who directly and heavilycontributed to this publication; for this we gratefully thank Drs. Joe Wang,LucaLazzarini,MarkShirtliff,JackLeFrock,andShaziaAmina.We wouldalsoliketothankthemanycontributorswhosededicationtotheirpatientsandtothetreatment of musculoskeletal infections contributed not only to this book, but alsototheentireeldofmusculoskeletalinfections.JasonH. CalhounJonT. MaderPreface ixContentsPreface vContributors vii1. TheBasicScienceofMusculoskeletalInfections 1MarkE. Shirtliff, JeffG. Leid, andJ. WilliamCosterton2. StagingandStagingApplicationinOsteomyelitis 63JonT. MaderandJasonH. Calhoun3. MusculoskeletalInfectioninSystemicallyandLocallyImmunocompromisedHosts 79JueWang, JasonH. Calhoun, LucaLazzarini, andJonT. Mader4. ProphylaxisofMusculoskeletalInfection 115BrentB. Wiesel andJohnL. Esterhai, Jr.5. OpenFractures:CurrentConceptsofManagement 131MichaelJ. Patzakis, CharalamposG. Zalavras, andPaulD. Holtom6. AdultLongBoneOsteomyelitis 149JonT. Mader, LucaLazzarini, andJasonH. Calhounxi7. SepticArthritis 183MarkE. ShirtliffandJackLeFrock8. HandInfections 211StephenB. Schnall9. TotalHipInfections 241KevinL. GarvinandJoshuaA. Urban10. PeriprostheticTotalKneeInfection 293EdwardJ. McPherson11. DiabeticFootInfection 325LucaLazzarini, JonT. Mader, andJasonH. Calhoun12. HematogenousPyogenicInfectionoftheSpine 341AlexanderG. Hadjipavlou, IoannisGaitanis, JamesW. Simmons, Jr.,andAnthonyMuffoletto13. PostoperativeInfectionsoftheSpineandTreatmentOptions 379AlexanderG. Hadjipavlou, IoannisGaitanis, PavlosKatonis,andJamesW. Simmons, Jr.14. GranulomatousInfectionsoftheSpine 421AlexanderG. Hadjipavlou, MichaelN. Tzermiadianos,IoannisGaitanis, andJeffNecessary15. PediatricOsteomyelitis 473LaurieO. HughesandJonT. Mader16. AntibioticActivitiesandToxicities 495JonT. Mader, JackLeFrock, HaissamS. El-Zaim,andJasonH. Calhoun17. MuscleFlaps 529RandySherman18. TheRolesandEffectivenessofAdjunctiveTherapyintheManagementofMusculoskeletalInfections 555JueWang, JasonH. Calhoun, JackLeFrock, andJonT. Maderxii Contents19. GeneTherapyinMusculoskeletalRepair:StateoftheArt 587JueWang, JasonH. Calhoun, andJonT. MaderIndex 613Contents xiiiContributorsJasonH. Calhoun, M.D.,F.A.C.S. DepartmentofOrthopaedicsandRehabilitation,UniversityofTexasMedicalBranch, Galveston, Texas,U.S.A.J.WilliamCosterton, M.D. CenterforBiolmEngineering, MontanaStateUniversity, Bozeman, Montana, U.S.A.HaissamS. El-Zaim, M.D.,Ph.D. DepartmentofOrthopaedicsandRehabilitation,UniversityofTexasMedicalBranch, Galveston, Texas,U.S.A.JohnL. Esterhai, Jr., M.D. DepartmentofOrthopaedicSurgery, HospitaloftheUniversityofPennsylvania, Philadelphia, Pennsylvania, U.S.A.KevinL. Garvin, M.D. DepartmentofOrthopaedicSurgeryandRehabilita-tion, UniversityofNebraskaMedicalCenter,Omaha, Nebraska, U.S.A.IoannisGaitanis, M.D. DepartmentofOrthopaedicSurgeryandTrauma-tology,SchoolofHealthSciences,University ofCrete,Heraklion,Crete,GreeceAlexanderG. Hadjipavlou, M.D. DepartmentofOrthopaedicSurgeryandTraumatology,SchoolofHealthSciences,UniversityofCrete,Heraklion,Crete,GreecePaulD. Holtom, M.D. DepartmentofOrthopaedicSurgery,KeckSchoolofMedicine, UniversityofSouthernCalifornia, LosAngeles, California, U.S.A.xvLaurie O. Hughes, M.D. The University of Arkansas for Medical Sciences andArkansasChildrensHospital, LittleRock, Arkansas, U.S.A.Pavlos Katonis,M.D. Department of Orthopaedic Surgery and Traumatology,SchoolofHealthSciences, UniversityofCrete, Heraklion, Crete, GreeceLucaLazzarini, M.D. InfectiousDiseasesandTropicalMedicineUnit, SanBortoloHospital, Vicenza, ItalyJackLeFrock, M.D. Sarasota, Florida, U.S.A.Jeff G. Leid, Ph.D. Center for Biolm Engineering, Montana State University,Bozeman, Montana, U.S.A.JonT. Mader, M.D.{DivisionofHyperbaricMedicine, DepartmentofOrthopaedics and Rehabilitation, University of Texas Medical Branch, Galveston,Texas,U.S.A.EdwardJ.McPherson, M.D. DepartmentofOrthopaedicSurgery, KeckSchoolofMedicine,UniversityofSouthernCalifornia,LosAngeles,California,U.S.A.AnthonyMuffoletto, M.D. DivisionofSpineSurgery,DepartmentofOrthopaedics and Rehabiliation, University of Texas Medical Branch, Galveston,Texas,U.S.A.JeffNecessary, PA-C, B.S. DivisionofSpineSurgery,DepartmentofOrthopaedics and Rehabilitation, University of Texas Medical Branch, Galveston,Texas,U.S.A.MichaelJ.Patzakis, M.D. DepartmentofOrthopaedicSurgery, KeckSchoolofMedicine,UniversityofSouthernCalifornia,LosAngeles,California,U.S.A.StephenB. Schnall,M.D. Department of Orthopedic Surgery, Keck SchoolofMedicine, UniversityofSouthernCalifornia, LosAngeles, California, U.S.A.RandySherman, M.D.,F.A.C.S. DivisionofPlasticandReconstructiveSurgery, KeckSchoolofMedicine, UniversityofSouthernCalifornia, LosAngeles, California, U.S.A.{DeceasedOctober2002xvi ContributorsMarkE. Shirtliff, Ph.D. CenterforBiolmEngineering, MontanaStateUniversity, Bozeman, Montana, U.S.A.JamesW. Simmons, Jr., M.D. DivisionofSpineSurgery, DepartmentofOrthopaedics and Rehabilitation, University of Texas Medical Branch, Galveston,Texas,U.S.A.C. MelindaStevens DivisionofHyperbaricMedicine, DepartmentofOrthopaedicSurgery,UniversityofTexasMedicalBranch, Galveston, Texas,U.S.A.SaulTrevino, M.D. DepartmentofOrthopaedicsandRehabilitation,UniversityofTexasMedical Branch, Galveston, TexasU.S.A.MichaelN. Tzermiadianos, M.D. DepartmentofOrthopaedicSurgeryandTraumatology,SchoolofHealthSciences,UniversityofCrete,Heraklion,Crete,GreeceJoshuaA. Urban, M.D. DepartmentofOrthopaedicSurgeryandRehabilitation, University of Nebraska Medical Center, Omaha, Nebraska, U.S.A.BrentB. Wiesel, M.D. DepartmentofOrthopaedicSurgery, HospitaloftheUniversityofPennsylvania, Philadelphia, Pennsylvania, U.S.A.JueWang, M.D. DivisionofHyperbaricMedicine, DepartmentofOrthopaedics and Rehabilitation, University of Texas Medical Branch, Galveston,Texas,U.S.A.CharalamposG.Zalavras,M.D.,Ph.D. DepartmentofOrthopaedicSurgery,KeckSchoolofMedicine, UniversityofSouthernCalifornia, LosAngeles,California, U.S.A.Contributors xvii1TheBasicScienceofMusculoskeletal InfectionsMarkE.Shirtliff,JeffG.Leid,andJ.WilliamCostertonMontanaStateUniversity,Bozeman,Montana,U.S.A.I. INTRODUCTIONThe occurrence, type, severity, and clinical prognosis of bone and joint infectionsdepend upon the interplay within a factor triad that includes the characteristics oftheinfectingpathogen,thepropertiesofthehost,andthesourceofinfection.Inorderto describe accurately whycertaintypesof microbescausecertaintypesofmusculoskeletalinfections(MSIs),therelativecontributionofeachoneofthesefactors must be taken into account (Fig. 1). Therefore, we rst discuss the variousspecies of microbes responsible for MSIs. The virulence factors of the pathogensresponsible for the majority of musculoskeletal infection cases are also addressedwithin this introductory chapter, with specic emphasis on Staphylococcusaureus, the most commonlyisolatedbacterial species inthese infections. Wealso discuss host factors, including normal properties such as blood supply and itsrelationtoinfection, aswell ashost defectssuchaslocal trauma, age, vascularinsufciency, immunocompromise, phagocyte defects, and implanted medicaldevices. The nal part of the infectiontriadtobe discussedis the source ofinfection, whether it be via hematogenous introduction, extension of a contiguousfocus of infection, or direct inoculation frompenetrating trauma, compoundfracture, bites, orsurgicalcontamination.1II. MICROBIALSPECIESRESPONSIBLEFORMUSCULOSKELETALINFECTIONSVirtuallyeverybacterial species has beenreportedtocause MSIs. Ina1999prospective review ofpatients(N = 164)sufferingfromlongboneosteomyelitisbetween1994and1996, Staphylococcus spp. represented53%of all isolatedbacteria(1).Specically,S.aureusconstitutednearly80%ofallStaphylococcusspp. isolated from patients with osteomyelitis. Staphylococcus spp. are capable ofcausingosteomyelitis inimmunocompetent hosts as well as inimmature andimmunocompromisedindividuals.Someotherpathogenicmicroorganismsasso-Figure1 Thetriadofcausalfactorsinmusculoskeletalinfectionsinwhichtherelativecontributions of the properties of the host, properties of the microbe, and source ofinfection are diagrammatically displayed for three types of musculoskeletal infections: (A)propertiesoftheinfectingmicrobialspeciesdominate(e.g., S. aureusosteomyelitis),(B)thesourceoftheinfectionisthedominatingfactorinthedevelopment ofthisinfection(e.g., handosteomyelitis byPasteurellamultocida), or (C) thehost defect isthemajordeterminantintheinfectiousprocess(e.g.,Bacteroidesspp.osteomyelitisofthefootinadiabeticpatient).2 Shirtliff et al.ciatedwithosteomyelitisareEnterococcusspp., Streptococcusspp., Pseudomo-nas aeruginosa, Enterobacter spp., Mycobacterium spp., as well as anaerobic andfungal species (specically Candida spp.). Each of these pathogenic speciesindividuallyrepresents a verysmall minorityof infections. The immature orcompromisedimmunestatusofthehostistheprimarycauseof initialinfectionanddevelopment intoapersistent andchronicosteomyelitisinfectionbytheseotherspecies.Vertebralosteomyelitisisusuallyhematogenousinoriginbutmayalsobesecondarytotrauma. Most hematogenousinfectionsaremonomicrobic. Inthenormalhost,S.aureusremainsthemostcommonlyisolatedorganism.However,aerobic gram-negative rods are found in 30% of cases. Usual sources ofinfectioninclude thegenitourinarytract, skinandsoft tissue, respiratorytract, infectedintravenous catheter sites, postoperativewoundinfections, endocarditis, dentalinfection,andunknownsources.However,theprimaryinfectionfocusisusuallyunknown. Intravenous drugabuse causes a highincidence of infectionbyP.aeruginosaandSerratiamarcescens(2).Mostinstancesof longbone hematogenousosteomyelitis occurin childrenafter a bacteremic event. A single pathogenic organismis almost alwaysrecoveredfromthebone(35). Themost commonboneisolatesareStaphylo-coccusspp., the most common gram-negative organism isP.aeruginosa, and themostcommonanaerobesarePeptostreptococcusspp.(Table1).However,intheimmunocompromised patient, other organisms must also be considered includingfungi and mycobacteria. In contrast to hematogenous osteomyelitis, in contiguousfocus osteomyelitis multiple organisms are usuallyisolatedfromthe bone. S.aureus and coagulase-negative Staphylococcus spp. account for 75%of thebacterialisolates(35).However,gram-negative bacilliandanaerobicorganismsare frequentlyisolated. Ahigher rate of nasal andskincolonizationwith S.aureus, defectsinhost immunity, andimpairedwoundhealingall playrolesinfoot infection, especiallyinaninmmunocompromisedhost suchas adiabeticpatient. Supercial fungal skin infections, which are common in diabetic patients,mayalsoallowbacterialentrythroughmaceratedorbrokenskin.Multipleorganismsarefoundinpatientswithosteomyelitisinvolvingthesmallbonesofthefeet, includingS. aureus,coagulase-negativeStaphylococcusspp., Streptococcus spp., Enterococcus spp., gram-negative bacilli, and anae-robes. Aerobicgram-negativebacilliareusuallyapartofmixedinfection(6).The virulence and tropismof the microorganisms combined with theresistance or susceptibility of the synovia to microbial invasion are majordeterminants of joint infection. S. aureus, Streptococcus spp., and Neisseriagonorrhoeae are examples of bacteria that have a tropismfor the synovia,probably related to adherence characteristics and toxin production. Aerobicgram-negativebacilli suchas Escherichiacoli rarelyinfect thesynoviaexceptin the presence of an underlying and compromising condition. Once the organismBasicScienceof Musculoskeletal Infections 3isinsidethejoint,thevirulenceoftheorganismvaries.Inrabbits,intra-articularinjectionof105S.aureusintothekneejointresultedinmajorjointdestruction,but identical injections of N. gonorrhoeae or S. epidermidis caused nojointinammation(7).Table1 Osteomyelitis:CommonlyIsolatedOrganismsLongbonehematogenousosteomyelitis(monomicrobicinfection)Infant Childhood Adults16YearsGroupBStreptococcus Staphylococcusaureus StaphylococcusaureusStaphylococcusaureus Streptococcuspyogenes StaphylococcusepidermidisEscherichiacoli Haemophilusinuenzae Gram-negativebacilliPseudomonasaeruginosaSerratiamarcescensEscherichiacoliContiguousfocusosteomyelitiswithoutvasculardisease(polymicrobicinfection)StaphylococcusaureusStaphylococcusepidermidisStreptococcuspyogenesEnterococcusspeciesGram-negativebacilliAnaerobesDiabeticfootosteomyelitis(polymicrobicinfection)StaphylococcusaureusStreptococcusspeciesEnterococcusspeciesProteusmirabilisStaphylococcusepidermidisPeptostreptococcusspeciesDiphtheroidsPseudomonasaeruginosaAnaerobes(e.g., Bacteroidesspp.)Vertebralosteomyelitis(monomicrobicinfection)StaphylococcusaureusStaphylococcusepidermidisPseudomonasaeruginosa4 Shirtliff et al.Themost commonetiological agent ofall septicarthritiscasesinEuropeand all nongonococcal cases in the United States is S. aureus (812). Therepresentation of S. aureus is more pronounced in patients with either rheumatoidarthritis or diabetes. After S. aureus, Streptococcus spp. are the next mostcommonlyisolatedbacteria fromadult patients sufferingfromseptic arthritis(1116). Whereas one study showed a high representation of Streptococcuspneumoniae(13), Streptococcuspyogenesisusuallythemost commonstrepto-coccal isolate, oftenassociatedwithautoimmunediseases, chronicskininfec-tions, and trauma (1114). Groups B, G, C, and F, in order of decreasingpreponderance, arealsoisolated, especiallyinpatientssufferingfromimmuno-deciency, diabetes mellitus, malignancy, and severe genitourinaryor gastro-intestinal infections (1114). Gram-negative bacilli account for approximately10%20% of cases (1014,16). Patients with a history ofintravenous drug abuse,extremesof age, or immunocompromisedisplayahigher prevalenceof gram-negativeinfection. Themost commongram-negativeorganismsarePseudomo-nasaeruginosaandE.coli.Anaerobesarealsoisolatedinasmallpercentageofcases, usually those of diabetic patients and patients with prosthetic joints.Approximately10%of patientswithnongonococcal septicarthritishavepoly-microbicinfections.Historically, Haemophilusinuenzae,S.aureus,andgroupAstreptococciwere the most common causes of infectious arthritis in children below2years of age. However, the overall incidence of H. inuenzae as a cause ofsepticarthritisisdecreasingbecauseof theH. inuenzaetypeb(Hib) vaccinenowgiventochildren(17). A1999studyof165casesofacutehematogenousosteomyelitis or septic arthritis treated in the years before and after theadvent of theHibvaccinedemonstratedthat musculoskeletal infectionduetothis bacterial species was reduced to nearly nonexistent levels (18). Therefore, thecoverageof H. inuenzaeas part of theempirical antibioticcoveragemaynolongerbeneededinthemanagement ofacutesepticarthritisinHibvaccinatedchildren. While H. inuenzae has lost its predominance as the most com-monlyidentiedgram-negative pathogeninpediatric populations, the normaloropharyngeal resident of youngchildren, Kingellakingae, mayhavetakenitsplace,specically in patientslessthan24 months of age (1922). In fact,a 1995study found that the nearly half of the clinical isolates fromacute septicarthritis patients less than 2 years old were K. kingae (21). However, theseresults haveyet toberepeatedinother centers. Clinical datasuggest that theorganismmay gain access to the bloodstreamin the course of an upperrespiratory infection or stomatitis (23). In children above the age of 2, S.aureus, streptococci, H. inuenzae, and N. gonorrhoeae have usually beenisolated(2426), althoughH. inuenzaemayhavealsolost its predominanceinthispatientagegroup(19).BasicScienceof Musculoskeletal Infections 5Microbiological associationsexist withconcomitant diseasestates. Septicarthritis that follows cases ofinfectious diarrhea may be caused byShigella spp.,Salmonellaspp., Campylobacterspp., or Yersiniaspp. (27,28). However, thesecases may reect a formof reactive arthritis. A rare formof migratingpolyarthritismaybecausedbyStreptobacillusmoniliformis.Inhumanimmuno-deciency virus(HIV)-infected patients, S. aureus continues to be the mostcommon isolate (approximately 30%) (29). However, there is an increasednumber of opportunisticpathogensisolatedfromthis patient subset, includingS. pneumoniae, mycobacterialspecies, andfungal species(29,30).RelativelyrareinWesternEurope, thediplococcusgram-negative bacterialspecies N. gonorrhoeae is the most common cause of septic arthritis in the UnitedStates(11,12,31).Thenumberofcasesofgonorrheadecreasedby72%between1975 and 1997 and this decrease was correlated with a reduction in disseminatedgonococcal infectionandarthritis(32). However, thereportedrateincreasedby9.2%between1997and1999andin2000stoodat133.2casesper100,000peryear(32). Specically,therateofgonococcalinfectionamonghomosexualmenhas demonstrated an alarming increase. These increased incidence rates may alsocausehighernumbersofobservedgonococcalarthritiscases.A. PropertiesoftheMicrobesintheDevelopmentofMusculoskeletalInfectionsSinceS.aureushasbeenextensivelystudiedwithregardtoitsroleinMSIsandcausesthemajorityoftheinfectiouscases, weusethisbacterial speciesasthetypical pathogen in our discussion of bone and joint infections. Otherrepresentative species, including N. gonorrhoeae and P. aeruginosa, are alsodescribedintheirrelationtoMSIs.1. StaphylococcusaureusSince Staphylococcus aureus spp. demonstrate a wide diversityof infections(tropical pyomyositis, lowerrespiratorytract infections[pneumonia], supercialskininfections[boils,sties,carbuncles],localizedabscesses,endocarditis,osteo-myelitis, toxic shock syndrome, serious skin infections [pyodermatitis], foodpoisoning,bacteremia,empyema,pyopneumothorax,andexfoliative diseases),itis not surprising that S. aureus has evolved a wide variety of virulence products tocause disease. The pathogenesis of staphylococcal infections is multifactorial, andit is difcult to determine the precise role of any given factor in infection. Most ofthevirulencefactors seemtobespecicallyadaptedtosurvival andinfectionwithinthehost. Staphylococcal products that havearoleininfectionmaybeclassiedasvirulencefactorsresponsibleforadherence, direct host damage, orimmunoavoidance. There are also a number of enzymes and extracellular proteins6 Shirtliff et al.that may have a role invirulence. These factors have a specic role in thecolonization and infection process in bone and joint infections, and theirexpressionis coordinated throughout the various stages of infection(Figs. 2and3). Therefore, thedifferential regulationof thesevirulencefactors duetostaphylococci population levels and environmental factors is extremely importantinthedevelopmentof infection.a.) Regulation. Staphylococcus aureus produces a large number ofextracellular andcell-associatedproducts that maycontributetovirulenceanddevelopmentofpersistentinfections.Mostofthesevirulencefactorsseemtobespecicallyadaptedtosurvivalandinfectionwithinthehost.Duringearlyexponential growthwhencell densityis low, proteins thatpromote adherence and colonization (such asbronectin binding protein, proteinA,staphylokinase,andcoagulase)areexpressed.Whencellgrowthreacheshighdensities, theproduction of theadherenceand colonizationfactorsissuppressed,whilesecretedtoxinsandenzymesareexpressed(suchasenterotoxinsB,CandD, epidermolytic[exfoliative]toxinA, a-, b-, and d-hemolysin, serineprotease,nuclease, type5capsularpolysaccharide, clumpingfactor,leukocidin, phospha-tidyl-specicphospholipaseC,fattyacidmodifyingenzyme,lipase,hyaluronatelyase[hyaluronidase], andtoxicshocksyndrometoxin1). Manyofthesepost-exponential phase proteins are involved in damaging the host, obtaining nutrientsfromthehost for pathogengrowth, anddisseminatingafter theorganismhasadequatelycolonizedandincreasedinnumbertopromoteanactiveinfection.The expression of most of these staphylococcal products is underpartial orcompletecontrol ofthestaphylococcal accessoryregulator(sar)andtheacces-sorygene regulator (agr) system. Duringearlylogarithmic growth, a proteinencoded by repressor of toxins (rot) inhibits the expression of agr-activatedvirulencefactors(33). Onceactivationoftheagrandsarregulatorylocioccursduringthelateexponential phase, thereisanincreasedtranscriptionof anagrregulatoryRNAmoleculeknownasRNAIII (34). RNAIII immediatelyblockstranscriptionof surfaceproteingenes and, withahypothesizedtimingsignal,upregulates transcription of extracellular pathogenicity factors (such as exo-toxins). The primary regulatory function of RNAIII is at the level of transcriptionby an undetermined mechanism that may involve one or more regulatory proteins(35).ThisregulatoryRNAmoleculeisalsocapableofcontrollingproductionofatleasttwo virulencefactors, a-hemolysin(hla)andproteinA(spa),atthelevelof translation. At the beginning of exponential phase growth, the expression of a-hemolysinisnormallyinhibitedthroughintramolecularbasepairingthat blockstheribosomalbinding site (35,36).Laterin exponentialphase growth,RNAIIIisexpressed and folds into a stable but inactive regulatory molecule. After asignicantlag,thesecondarystructureofRNAIIIchangesthroughanunknownagent, and the 5/region of RNAIII is then able to hybridize with a complementaryBasicScienceof Musculoskeletal Infections 78 Shirtliff et al.5/untranslatedregionof a-hemolysinmesengerRNA(mRNA),therebymakingthetranscriptsaccessiblefortranslationinitiation(36).Conversely,the3/regionofRNAIIIcontainssequencescomplementarytotheleadersequenceofspaandhybridization is believed to inhibit translation of protein A. In addition, SarA (theprimaryproduct of sar) has beenshowntohave aninhibitoryeffect ontheexpression of a number of genes, including cna, sea, sar, and the agr operon (37).Therefore, sarAexpressionmaybeautoregulated, but theinteractionswiththiscomplexsystemarestillbeingelucidated.IthasbeenhypothesizedthattheactivityofRNAIIIisregulatedthroughapopulation-sensing autocrine system (quorum sensing) that involves the productsof theagr locus (3841). This locus consists of two divergent transcription units,driven bypromotersP2andP3.TheP2operoncontainsfour genes,agrB,agrD,agrC, andagrA, andtheP3operoncodesforRNAIII(seepreviousdiscussion)(42).An octapeptide with a uniquethioesterringstructure (referred to as theagrautoinducingpeptide[AIP])isgeneratedfromitsprecursor,AgrD,andsecretedout of the cell through the action of the AgrB membrane protein (Fig. 4) (43). Asthe concentrationof AIPincreases inthe extracellular microenvironment, theinteractionbetweenAIPandthehistidinekinasereceptor protein, AgrC, alsoincreases. This interaction possibly acylates AgrC and enables it to phosphorylateand thereby activate an intracellular agr-encoded protein (AgrA) (35,44).AgrAP then increases transcription at the P2 promoter. With SarA(themajor transcript of the sar operon), AgrAPalsoincreases thetranscriptionat the P3promoter, resulting in elevated intracellular levels of RNAIII (45).Therefore, as AIPconcentrates in the extracellular environment, the level ofRNAIIIincreases, allowingthegrowthphasedependentreductioninadherencefactor productionandincreaseinextracellular pathogenicityfactor production.AIPnotonlyiscapableofactivatingtheagrreguloninselfstrains,butcanalsoinhibit theagr activation of otherS.aureus strains. SarA (the primary productofsar) has been found to be autoregulatory, and it mediates virulence factorexpressionthroughagr transcriptionbybindingtothe promoter regionas aFigure2 DiagrammaticrepresentationofS. aureusattachment totissuesduetohigh-level expression of adherence factors to host extracellular matrix proteins. After attachmentand localized multiplication, the constitutive secretion of the quorum sensing autoinducingpeptide(AIP) allowstheconcentrationof thisquorumsensingsignal. Asthemicrobialpopulationincreases, individual cells withinthepopulationbegintocompetewithoneanother for nutrients. At this time the AIP is at high enough levels to activate theproduction of a wide variety of staphylococcal virulence factors that enable the microbe todamage the host andobtainmore nutrients or explore newniches. At the same time,capsule production increases while adherence factor production is reduced, therebyenablingtheS. aureustobecomeabletoresistphagocytosis.BasicScienceof Musculoskeletal Infections 910 Shirtliff et al.dimer (46). SarAhas alsobeenshowntointeract directlywiththe promoterregionsof anumber of genes, includingthecodingregionsfor theP2andP3promoter of agr, protein A, bronectin binding protein, and a-hemolysin (37,47).However, theinteractions withthis complexsystemarestill beingelucidated.SincesarandRNAIIIhomologshavebeenidentiedinanumberofcoagulase-negative Staphylococcus spp., including S. lugdunensis (48), S. epidermidis(49,50), S. simulans, andS. warneri (50), this regulationsystemis alsousedbytheothermembersofthestaphylococcalgenus.Another regulatorylocus, termedS. aureus exoproteinexpression(sae),wasdiscoveredin1997(51). Thissystemiscomposedoftwogenes, saeRandsaeS, encodingaresponseregulatorandahistidineproteinkinase, respectively,Figure4 Structuresofsomecommonmicrobialsignalingmolecules.Figure3 Scanningelectronmicroscope(SEM) andtransmissionelectronmicroscope(TEM) images of the progression of S. aureus osteomyelitis from colonization, to localizedmultiplication, to embedding within a dense glycocalyx to form a biolm that is resistant toremovalbythehost immunesystemandantimicrobialtherapy. (PhotographscourtesyofMaryElizabethPowers, Dissertation, 1990.UniversityofCalgary.)BasicScienceof Musculoskeletal Infections 11withsignicant homologytootherbacterial two-component regulatorysystems(52). Itscontrol ismediatedat thetranscriptionlevel, andmutationof thesaelocus loweredinvivovirulencebydrasticallydiminishinglevelsofa- andb-hemolysins and coagulase and slightly reducing levels of protein A(53,54).Several environmental signalshavealsobeenimplicatedinthesar=agr-depen-dent or -independent regulation of virulence factors. These signals includenonmaintained pH(55), osmolarity (56,57), glucose (58), deoxyribonucleicacid(DNA) topology(56), NaCl, sucrose (59), temperature (60), aminoacidavailability(61), andpresenceof O2andCO2. Also, ahomologtotheferricuptake regulator (Fur) of gramnegatives was isolated in2000andfoundtoregulate thestaphylococcal ironregulated (sir) operon,which hasbeenproposedto constitute a siderophore-transport systemin S. aureus (62). Many of thereactions of S. aureus to these environmental cues are classied as stressresponses, andtheyarebelievedtoberegulatedbysigmafactors that controlgene expression. Inthis species, there are twovarieties of sigma factors, thehousekeepingsigmafactor (sA) andthealternativesigmafactor (sB), that areexpressedandactivateresponsegenesuponentryintostationaryphaseandinresponse to environmental stress. sBIs believed to be regulated by the product ofthe gene directly upstream of sBcoding region, rsbW (63). This protein is able tobind to the alternative sigma factor, thereby posttranslationally inhibiting itsactivityuntilastressresponseisneeded.b.)AdherenceFactors. Asstatedearlier,thepathogenmustcolonizethetarget tissuethrough adherence in orderto initiateinfection.Staphylococcusspp.haveavarietyof receptorsfor host proteins, termedmicrobial surfacecompo-nents, that mediateadherencetotheextracellular matrixinbonesandjointsorimplantedmedical devices byrecognizingadhesivematrixmolecules (6466).Some of the host matrix proteins are bronectin and laminin (adherence proteins),elastin (whichimparts elastic properties), collagen (whichprovides structuralsupport), and hyaluronic acid (a glycosaminoglycan that is abundant in thejointsand the matrix and provides cushioning through hydration ofits polysaccharides).There arealso anumberof bone-or joint-specicmatrixproteins.These includeosteopontin (a soluble phosphoprotein that acts as a cytokine and osteoclastattachment proteinandisneededfor boneinjuryrepair andremodeling), bonesialoprotein(whichinteractswithosteoblastsandactsasanucleatorforcalciumhydroxyapatiteformation), andvitronectin(anadhesiveglycoproteinthat regu-latesadhesionandthecoagulation, brinolytic, andcomplement cascadesandalso allows bone resorption when bound to osteoclasts). Eight adhesin genes havebeen determined: genes encoding brinogen binding proteins ( b, cA, and fbpA)(6769), bronectinbindingprotein( fnbAandfnbB) (70), acollagenreceptor(cna) (71), an elastin binding protein (ebpS) (72), and a broad specicity adhesin(map)thatmediateslow-levelbindingofseveralproteinsincludingosteopontin,12 Shirtliff et al.collagen, bonesialoprotein, vitronectin, bronectin, andbrinogen(73). Also,thismicroorganismhasbeenshowntopossessanumberofotherhost proteinbinding receptors in which the genes have not yet been determined. These includea laminin- (52 kd) (74), a lactoferrin- (450 kd) (75), and a transferrin- (42 kd) (76)bindingprotein. Thestaphylococcal receptorthat bindslamininmaybeusedinextravasation (77). These receptors were found in S. aureus but were absent fromthe noninvasive pathogenS. epidermidis (77). The lactoferrinandtransferrinreceptorsbindtothesehost ironbindingproteinsandmaybeusedasadhesinsand=orasironacquisitionmechanisms.Inaddition,S.aureusexpressesa42-kdprotein,proteinA,whichisboundcovalentlytotheouterpeptidoglycanlayer oftheircell walls.Thisadherenceproteinisable tobindtothehostplateletgC1qR(amultifunctional,ubiquitouslydistributedcellularprotein,initiallydescribedasa bindingsite for theglobular heads of the complement complexC1q) (78).Therefore,proteinAmaybeabletopromoteadhesiontositesofvascularinjuryandthrombosis andhas beenimplicatedas animportant colonizationfactor.ProteinAproductionisrepressedbythesarlocusviabothRNAIII-dependentand-independent mechanismsduringpostexponential phasegrowth(45). Thisprotein is also associated with S. aureus immunoavoidance (discussed later).Many of these and other staphylococcal cell wall proteins must be exported out ofthe bacterial cell inorder tointeract withthe extracellular environment. Thisexportcanbeeitheratargetingprocess(theproteinisexportedandhasbindingdomains for cell wall secondarypolymerssuchas teichoicacids) or asortingprocess(aC-terminal conservedaminoacidsequence, LPXTG, that directstheexportandcovalentattachmenttothepeptidoglycan)(79).Increasing evidence supports the importance of staphylococcal surfacecomponents as virulence determinants by allowing initial colonization. In anumber of studies, mutants inthese receptors stronglyreducedthe abilityofstaphylococci to produce infection. In addition, there was signicant binding of S.aureustobonesialoprotein,bronectin, andcollagentype1inamousemodel,indicatingthat adherenceremains akeyphaseintheearlystages of infection(80).Expressionofadhesinspermitstheattachmentofthepathogentocartilage.In a murine septic arthritis model, inoculation of mice with mutants of thecollagen adhesin gene showed that septic arthritis occurred 43% less often than inthecorrespondingwildtype(81). Collagenadhesinpositivestrains werealsoassociatedwiththeproductionof highlevelsof immunoglobulinG(IgG) andinterleukin-6 (IL-6) (81). Also, vaccination with a recombinant fragment of the S.aureuscollagenadhesinwasabletoreducethesepsis-inducedmortalityrateto13%, compared with 87% in the control group (82). However, the role of collagenadhesionofS. aureusasamajorvirulencefactorwasquestionedin1999sinceapproximately30%60%ofclinical isolatesdonot displaycollagenbindinginvitro or the cna-encoded collagen adhesin (83). Staphylococcal bronectin-bindingproteins (FbpAandFbpB) mayhaveamajor roleinthecolonizationBasicScienceof Musculoskeletal Infections 13and virulence of MSIs. In a 2000 study, all of the tested clinical isolates(N = 163) contained one or both of the coding regions for these binding proteinsand95%ofthesestrainshadacomparablebronectinbindingcapacitytothatseeninastaphylococcal referencestrainknowntobindbronectinefciently(84). Inaddition, aninvivostudyof endocarditis inarat model showedthatmutantsdecient forbronectin-bindingproteinwere250-foldlessadherent totraumatizedheartvalves(85).Also,S.aureusadherencetominiplatesfromiliacbonesofguineapigswasthreetimeshigherthanthatofthebronectin-bindingproteindefective mutantstrain(86).Itislikelybronectin-bindingproteinsplayanimportant roleinboneandjoint infections, especiallythoseassociatedwithinitial trauma or implanted medical devices (87). These receptors play anadditionalroleinanintracellularimmunoavoidancestrategy(discussedlater).c.) Factors Causing Damage tothe Host. Duringacute infection, theinnate immune system responds to the peptidoglycan wall (via N-formylmethionineproteinsandteichoicacids) ofS.aureus toproduceproinammatorycytokines (such as interleukin-1b[IL-1b], IL-6, and tumor necrosis factor-a[TNF-a])andC-reactive protein. BacterialDNA(specicallyunmethylatedCpGmotifs)hasalsobeenshowntoelicit anintenseinammatoryresponse(88,89).When bacterial DNA fromS. aureus, E. coli, or synthetic, unmethylatedoligonucleotides containing CpGmotifs was injected into the knee joint ofmice, thedevelopment of arthritis occurredquicklyandlastedupto14days,whereasmethylatedDNAhadnosignicanteffect.Also,theaffectedtissuewascharacterized by monocyte and macrophage inux with the release of theirassociatedcytokinesandchemokinesandtheabsenceofTcells.S. aureus also secretes a number of enterotoxins (A, B, C13, D, E, G, H, I,andJ)andtoxicshocksyndrometoxin(TSST-1). TheenterotoxinsandTSST-1havebeenshowntoactivatetheimmunesystemprofoundlywhenevaluatedinanimal models, increasing mortality rates and exacerbating host inammatory cellinvasion, cytokine release, and tissue degradation in the acute phase of theinfection(80,90). Theyact assuperantigensbybindingtotheconservedlateralregions of the host major histocompatibility complex class II molecule and T cellreceptor. Althoughonlyapproximately1in10,000Tcellsis activatedduringnormal presentation of a nonself antigen, 2%20% of all T cells may be activatedbya superantigen(90). These activatedTcells are thenable toincrease therelease of a number of cytokines, such as the interleukins (90), interferon-g (IFN-g), and TNF-a (91). This upregulated production of cytokines causes a signicantsystemic toxicity, suppresses the adaptive immune responses, and inhibits plasmacell differentiation. Also, the stimulated Tcells proliferate and then rapidlydisappear,apparentlybecauseofapoptosis(92).Therefore,immunesuppressionmay be due to generalized immunosuppression and T cell deletion. Since14 Shirtliff et al.superantigentoxinsareusuallyproducedduringthepostexponentialphaseofanestablishedinfection, theymayalsoaidinlocal immune deciencyandhostdamageseeninboneandjointinfections.Theimportanceof thesesuperantigentoxins insepticarthritis has beendemonstratedinanimal models of septicarthritis. Most animals infectedwithstrainsofS. aureusisogenicforTSST-1orentertoxins(AD)hadfrequent andsevere joint infection (80). However, 80% of animals infected with strains devoidof thesetoxinshadnosymptoms, andthoseremaininganimalsdemonstratingsymptomshadonlymildortransientarthritisinfections(80).Vaccinationwitharecombinant formof staphylococcal enterotoxinAdevoidof superantigenicitywas able to demonstrate signicant protection from S. aureus sepsis in mice (93).TheseenterotoxinsandTSST-1alsosubvert thecellular andhumoral immunesystemandmaytherebypromoteasustainedandmorefulminantacuteinfectionorenablealocalizedosteomyelitistodevelopintoachronicinfection.However,sinceastudyin2000demonstratedthatexpressionofTSST isnegligibleatlowoxygenpartial pressures(94), theimportanceofthistoxinintherelativelyO2-deprivedenvironmentofaninfectedbone, whencomparedtothatinsupercialabscessesorcasesofsepticarthritis, isstillasourceofdebate.Staphylococcalhemolysinexpressionisincreasedduringpostexponentialphase growth.Amongotherstimulatorysignals,thesar=agrregulon plays aroleinthispostexponential expression. a-Hemolysinissecretedasamonomer thatattachestohostmembranesandpolymerizesintoahexamericringchannel(95)andhasbeenfoundtobeasignicantmediatorofvirulence(96).Althoughthishemolysinonlybindstohumanerythrocytesinanonspecicmanner,itcanstillmediatesignicant host cell lysiswhenproducedinhighconcentrationsintheinfection environment (97). Also, a-hemolysin promotes signicant bloodcoagulationbymediatingneutrophil adhesion(98), platelet aggregation(viaabrinogen-dependent mechanism) (99), and its nonlytic attack on human platelets(100). Inaddition, this hemolysincanformchannels innucleatedcells (e.g.,endothelial cells) through which calcium ions freely pass (101,102). The calciuminuxisresponsiblefor thevasoregulatoryprocessandinammatoryresponsedisturbances seen in severe infection(103). Finally, a-hemolysin has been showntointerferewithlymphocyteDNAreplication(98). Thesemultipleeffectsof a-hemolysinonthehost contributetothevasculardisturbancesandimmunode-ciencyseeninstaphylococcalinfections,therebycontributingtoacuteinfectionsand the development of chronic MSIs. The pathogenic properties of a-hemolysinwere found in 1999 to occur only when another staphylococcal toxin, theleukocyte-specic g-toxin (discussed later), was also present in the infectingstrain(104).Another type of hemolysin, sphingomyelinase (b-hemolysin), has onlyweak cytocidal effects on human granulocytes, broblasts, lymphocytes, anderythrocytes (105). Instead, this hemolysinspecicallyattacks andkills thoseBasicScienceof Musculoskeletal Infections 15cellswithmembranesrichinsphingomyelin, suchasmonocytes. Thedeathofmonocytesreducestheeffectivenessof theimmuneresponseandsponsorstherelease of cytokines (IL-1b, IL-6, andsoluble CD14). These cytokine-relatedeventsmaybeimportantintheinfectiousprocess.d-Hemolysin, the translation product of RNAIII of the agr regulon,specicallybinds tomonocytes andneutrophils (106). d-Hemolysinpromotestheproductionandreleaseoftumornecrosisfactor-ainmonocytes(106).Also,this toxin upregulates the expression of neutrophil complement receptor 3.Althoughthis toxinwas unabletoprimeneutrophils directlyfor anenhancedresponse, it enhanced neutrophil priming by lipopolysaccharide or tumor necrosisfactor. Therefore, thesimultaneouspresenceof monocytes, neutrophils, andd-hemolysin-producing staphylococci may overactivate host inammatoryresponse, resulting in host tissue damage in the microenvironment of boneinfection. However, the exact role of this toxin in infection remains to beelucidatedadequately.Leukocidin (LukSF-PV) and g-hemolysin (HlgAB and HlgCB) specicallylyseleukocytes. Eachof thesetoxins is composedof aninterchangeabletwo-componentsystem.TheactivetoxinisformedbytakingoneproteinfromtheScomponent family(LukS-PV, HlgA, andHlgC)andonefromtheFcomponentfamily (LukF-PVand HlgB) (107,108). The S component is most likelyresponsible for the specic cytopathic effect of each of the toxins; the Fcomponent isresponsibleforthecommonleukocytebindingactivity. AlthoughLukFand HlgAproteins showvery strong similarity, they are encoded ondifferent gene loci (109). Since these cytotoxins specicallyinteract andlyseleukocytes, theycontributetotheinhibitionof infectionclearancebythehostimmunesystem, therebyenablingstaphylococcal speciestopersist.The role of exfoliative (epidermolytic) toxins A and B is well demonstratedincasesofexfoliativedermatitis(i.e., staphylococcalscaldedskinsyndrome)inwhich the epidermis separates fromthe stratumgranulosum. However, thesetoxinshavenotbeenstudiedinrelationtotheireffectonS. aureusvirulenceinMSIs.Also,theyhavebeenshownnottobebacterialsuperantigens(110).Theymaystill contributetovirulencesincetheywereclassiedin2000aspossibleserineproteases(110).Inadditiontothislargearrayoftoxins,a2000studyhasidentied a novel gene locus that encodes ve exotoxin-like proteins. The in vivorelevanceoftheseproposedtoxinsmuststillbedemonstrated(111).d.) Immunoavoidance Factors. The ability of the bacteria to evadeclearance by the host immune response and to promote a sustained acute infection(e.g., septic arthritis) or a persistent, chronic infection (e.g., osteomyelitis) residesin a number of staphylococcal defense mechanisms, including, but not limited to,IgGinactivation(viaproteinAattachment), antiphagocyticcapsuleproduction,biolmformation, andinvasionandsurvivalinmammaliancells.16 Shirtliff et al.ProteinAisboundcovalentlytotheouterpeptidoglycanlayeroftheircellwalls. This receptor binds to the Fc portion of IgG and presents the Fab fragmentoftheantibodytotheexternalenvironment.Therefore, theFcportionisunableeither tobindcomplement or tosignal polymorphonuclear leukocytes, therebyinterferingwithstaphylococcalopsonizationandphagocytosis.Thisinterferencehas been demonstrated in vitro and in animal models with subcutaneousabscessesandperitonitis. ProteinAalsocoatsthestaphylococcal cellwallwithhost Fab fragments, and the ability of the immune systemto recognize thepathogen as nonselfishindered. The importanceof proteinA inS.aureus septicarthritiswas demonstratedina1997studyinwhichstrains that obtainedthisvirulencefactorcausedgreaterinammationandcartilagedestruction(96).Capsular polysaccharide may interfere with opsonization and phagocytosis.Amongthe11reportedserotypes, capsuletypes5and8(microcapsuleprodu-cers)constitutethevastmajority(75%94%)ofclinicalisolates(112114).Thecapsule of these two serotypes was found to be muchsmallerthanthe capsule ofotherserotypesofS.aureus(suchascapsuletype1)orpathogenicspeciessuchas Streptococcus pneumoniae. Unencapsulated and microencapsulated strainsdemonstrated a high rate of serum clearance when compared to fully encapsulatedstrains. Therefore, the role of capsular polysaccharide in opsonization andphagocytosiswasquestioned(112).However,thethincapsulemaybenecessaryin early infection stages in order to allowthe interaction of staphylococcaladhesionfactorswithhostproteins(suchasbrinandbronectin).Inonestudy,it was shownthat asmall capsulewas necessaryfor broblast attachment byprotein A of S. aureus, and a fully encapsulated strain reduced binding efciency(115). In another study, the thin capsule was shown to be necessary for binding tobonecollagentype1, sincehighcapsularexpressionactuallyinhibitedbinding(116). Once these microorganisms adhere to solid surfaces (such as bone,implants, or joint tissue), both in vitro and in vivo, staphylococci producelarger quantities of cell-associatedcapsulethanthosegrowninliquidcultures(117). Specically, type 5 and type 8 capsule production was shown to bestrongly upregulated during postexponential growth (i.e., after adhesion andcolonization) by agrregulation and perhapsotherregulatorysystems (118).Thisupregulated capsule production makes themmore resistant to antimicrobialtreatmentandhostimmuneclearance.Therefore,oncestaphylococcaladherenceproteins establish the infection, the pathogen enters postexponential growth phaseand begins producing a thicker capsule that covers and hides the highlyimmunogenicadherenceproteins. This thicker type5andtype8capsulehasbeen found to be serumresistant through inhibition of phagocytosis andopsonization(112,119). Theeffect ofthisstaphylococcalpolysaccharidemicro-capsule in murine arthritis was explored in a 1997 study in which strainsexpressingtype 5capsulewere showntoproduce a higher rate of mortality,higher frequency of arthritis, and more severe form of the disease when comparedBasicScienceof Musculoskeletal Infections 17tocapsulemutants(119).Inaclinicaltrial,a vaccine(StaphVax)thatconsistsofisolatedtype5andtype8capsularpolysaccharideswasabletoreduceinfectionratessignicantly, by57%inahigh-riskpopulationfor aslongas10months(120).Staphylococcus spp. can also produce a multilayered biolm. A biolm is amodular community of microbes embedded within a host- and=or microbe-derivedhydratedmatrix(usuallyexopolysaccharide) that exists at a phase ordensityinterface.Thisinterfacemaybebetweenasolidsupport(e.g.,softtissueor bone) anda liquidmedium(e.g., extracellular uid, blood, mucin) (121).Biolmthickness canvaryfromasinglelayer toathickcommunityof cellsembeddedwithinathickpolymericmatrix. Structural analysesin1995demon-stratedthatbiolmspossessasophisticatedarchitectureinwhichmicrocoloniesexist in discrete pillar or mushroom-shaped structures (122). Between thesestructures, anintricatechannel networkprovidesaccesstoenvironmental nutri-ents. It has beenhypothesizedthat the development andmaintenance of thisphenotypemaybemediatedthroughtheactionof quorumsensingsystemsinbiolm-producingmicrobes(123126).ThemultilayeredS. aureusbiolmisembeddedintoaglycocalyx(127).The glycocalyx develops on devitalized bone (such as the involucrum) ormedicallyimplanteddevices(128). Thepresenceof implantsisapredisposingfactorinthedevelopment of infectionsincetheyarecoatedwithhost proteinssoonafterimplantation, andthisprovidesanexcellent sourceofattachment forany bacteria remaining after debridement surgery (64,69,129,130). Onceattached, the bacteriacanformtheglycocalyx, or slime layer, whichprotectsthebacteriafromnormalhostdefensesandsystemicantibiotics(131134).Thispathogen usually grows in coherent microcolonies in the adherent biolm, whichis oftensoextensive that the underlyinginfectedbone or implant surface isobscured. This layer prevents the inward diffusion of a number of antimicrobials,allowing bacterial escape fromthe bactericidal and bacteriostatic effects ofantimicrobial therapy(131134). Furthermore, thosebacteriathat surviveanti-biotic clearance often develop resistance to the impregnated antibiotic andregrow. This resistance has been clinically demonstrated by the isolation ofsmall colonyvariantsof S. aureusresistant togentamicinfromthewoundsofpatientstreatedwithgentamicin-impregnatedpolymethylmethacrylate(PMMA)beads (135). Also, the glycocalyx displays antiphagocytic properties, therebyallowing the bacteria to evade clearance by the hosts immune system (136138).Theglycocalyxismainlycomposedofteichoicacids(80%)andstaphylococcaland host proteins (139). Host proteins such as brin are derived fromtheconversionofbrinogenbythestaphylococcalcoagulaseprothrombincomplex(discussedlater)(140).ItwasfoundthatthebiolmproducedbyS.epidermidisalso contains the capsular polysaccharide=adhesin (PS=A) that mediates celladherencetobiomaterialsandapolysaccharideintercellular adhesin(PIA) that18 Shirtliff et al.may mediate bacterial accumulation into cellular aggregates (141,142). PS=A is ahigh-molecular-mass(>250-kd)moleculethat iscomposedofacid-stablepoly-mers of b1,6-linked glucosamine. PIAis a polymer of b1,6-linked N-acetylglucosamine residues with a molecular mass of less than 30 kd that is synthesizedthrough genes present on the intercellular adhesion locus (ica) (63,141). S. aureusand other Staphylococcus spp. also contain an ica locus, and its deletion results inthe loss of biolm-forming ability (63). The presence of glycocalyx was noted in76%ofS.aureus,57%ofStaphylococcusepidermidis,75%ofEscherichiacoli,and50%ofPseudomonasaeruginosaclinicalosteomyelitisisolates(143).Clinical strainsofStaphylococcusspp. areabletopersist byanumberofglycocalyxproperties. First, thislayerhasbeenshowntoprotect theembeddedpathogens from the action of antimicrobial agents and the host immune system byforming a mechanical barrier (144). Second, local immune deciency oftenoccurs throughfrustratedphagocytosis since the normal phagocyticprocessesare devoted to the removal of the glycocalyx and the implant, if present.Therefore, theenergyandresourcesoftheimmunesystemthatwouldnormallybe used to ght infection are subverted. Third, the glycocalyx may activatemonocyte production of prostaglandin E2 to inhibit T cell proliferation indirectly(145). Finally, this glycocalyx has been shown to inhibit polymorphonuclearleukocytesdirectly(136).S. aureus has also been shown to survive intracellularly after internalizationbyculturedosteoblasts (146). Type5capsuleproductionof invivogrownS.aureus (i.e. internalized in cultured osteoblasts) was shown in 1998 to beupregulatedwhencomparedtoS. aureus growninvitro(147). Therefore, thecapsule not only may resist phagocytosis and opsonization, but may alsocontribute to intracellular survival. In addition to osteoblasts, staphylococcihavedemonstratedinternalizationintootherculturedmammaliancells, suchasbovinemammaryglandepithelial cells, humanumbilical veinendothelial cells,andpulmonaryepithelial cellsisolatedfromacysticbrosispatient (148150).Specically, initial adherencetoglandularepithelial cellshasbeenshowntobemediated by bronectin receptors on this pathogen (148), possibly usingbronectinas a bridge between the host cell andbacterial receptors for thishost factor. After adherence, bacteriamaybeinternalizedbyhost mechanismsinvolving membrane pseudopod formation (seen in established bovine mammaryepithelial cell lines) or through receptor-mediated endocytosis via clathrin-coatedpits(seeninmouseosteoblastsandepithelialcells)(148,151).Ineithercase,thedependenceupontheactionofhostcytoskeletalrearrangementsthroughmicro-lamentsisevident.After internalization, staphylococci may induce apoptosis (via a hostcaspasedependent mechanism) or survive intracellularly (148,149,152,153).Induced apoptosis may further the host cell damage seen in MSIs withoutcausingfuther inammation. Also, staphylococci mayescapeclearancebytheBasicScienceof Musculoskeletal Infections 19immunesystemandantimicrobial therapybypersistingwithinthesehost cells.Thissurvival wasdemonstratedin vivo in2000whenS.aureuscellswerefoundinthecytoplasmof embryonicchickosteoblastsandosteocytesinmineralizedbonematrix(154). Inanother study, S. aureuswasfoundwithinpolymorpho-nuclearneutrophilsin an in vivo infection model (155).These infectedhost cellswereabletoestablishinfectioninna veanimals. Therefore, thispathogenmayutilizeinvasionasanimmunoavoidancetechniqueduringthehostinammatoryresponse. AfterthedownregulationoftheadaptiveimmuneresponsethroughTcellapoptosis(mediatedbysuperantigens,othertoxins,andinvasion),fulminantand=orpersistentinfectionmayresult.e.) Other Staphylococcal Enzymes. Staphylokinase binds to plasminogen,and this complex activates plasminlike proteolytic activity that causes dissolutionofbrinclots.Althoughtheactivityofthisenzymehasnotbeendirectlyrelatedto virulence,theimportanceofescapingbrinclotstoinvadesurroundingtissueisapparent. Anotherenzyme, fattyacidmodifyingenzyme(FAME), allowsthemodicationofhostderivedantibacteriallipidsinabscesses(156).Therefore,inthe intramedullary abscesses associated with osteomyelitis (Brodies abscess), thisenzyme may be responsible forprolonged bacterial survival and the developmentof osteomyelitis. Coagulase(cna), althoughnot anenzyme, isanextracellularprotein thatformsacomplex calledstaphylothrombinbybindinghostprothrom-bin(157). Thiscomplexisabletoconvert host brinogentobrininorder topromotelocalizedclotting. This proteinis mainlyexpressedduringtheexpo-nentialgrowthphasebutisthoughttobedownregulatedduringlateexponentialphase by direct binding and inhibition by SarA(158). Coagulase also hasbrinogen-binding capacity in the absence of thrombin (159). Although nodifferencesinvirulencewereobservedbetweenwild-typestrainsandcoagulasemutants in several infection models, the collagen binding adhesin was found to beimportantinthepathogenesisofcornealinfectionintherabbit(160162).Also,one can speculate as to the benets derived from locally impeding blood ow andpromotingabrin-richareaforaugmentedcolonizationbyStaphylococcusspp.Thisgenusalsopossessesanumberofotherenzymes(lipase, nucleases, serineprotease, andmetalloprotease) that areusedtoacquirehost nutrients suchaslipids, nucleotides, and amino acids, respectively. Staphylococcal iron acquisitionismediatedthroughsiderophores, suchasthe42-kdtransferrinbindingproteinand the 450-kd lactoferrin binding protein (composed of multimers of 62- and 67-kdsubunits).AlthoughthepathogeniceffectsofureasehavenotbeenevaluatedinMSIs, it isacritical virulencedeterminant forcolonization, urolithiasis, andsevereacutepyelonephritis. It isanenzymethat convertsureatoammoniaandcarbon dioxide, thereby surrounding the bacteriumin a protective layer ofammonia. The ammonia is also toxic to host cells, and urease has a directinammatory activity on epithelial tissue. Therefore, urease aids in penetration of20 Shirtliff et al.thebacteriumintotissues andblood. Theregulationmechanismis still beingdetermined.In summary,S.aureus infectsand elicits astrongnative immuneresponse,cytokine release, and high T cell activation. This pathogen is able to use a numberofimmunoavoidancestrategieswhilethehostimmunesystemcausesdamagetoself tissues and blood vessels in the area ofinfection. Damage may cause localcirculatory and immune compromise. The high T cell activation eventually resultsin apoptosis and a weakened immune system, enabling this pathogen to produce asustainedanddestructiveinfectioneffectively. Althoughthebacterial productsdiscussedhavebeenshowntoincreaseboneandjoint damageinacutesepticarthritis, many more S. aureus virulence factors have not yet been tested.Therefore, futurestudieswill undoubtedlyidentifyotherfactorsthat playaroleinMSIs.2. NiesseriagonorrhoeaeAsmentionedpreviously, N. gonorrhoeaeisthemost commoncauseof septicarthritisintheUnitedStates(11,12,31).Thisdiplococcuspossessesanumberofcell surface structures that have been implicated in virulence. Initial attachment tohost epitheliumis mediated by long, hairlike protein projections called pili.Whetherthismembranestructureisassembled(Pil)ornot(Pil),alsoknownas phase variation, is determined by posttranslational proteolytic cleavage,variations inhomologous recombination, andslippedstrandDNAreplicationresulting in frameshift mutations (163). In addition, the antigenic character of thepili is altered by homologous recombination between coding regions for thevariouspilinsubunits.ProteinIisthemainproteinontheouter membrane. It isaporinthat isexpressed in two different forms, a protein IAvariant that is almost alwaysassociatedwithdisseminatedinfectionandproteinIBthat is associatedwithstrain causing localized infections. Those strains that are able to cause adisseminatedinfectioninhosts witha normal immune systemdisplayserumresistance(164). ProteinIAallows stableserumresistancebybindingtohostfactorH.ThisbacteriallyboundhostfactorefcientlyinactivatesC3b(acentralfactorinboththeclassicalandalternative complementcascade)intoiC3b(165),thereby reducing the efcacy of the host complement system. This porin may alsobe responsible for the prevention of phagolysosomal fusion in polymorpho-nuclear leukocytes and a reduced oxidative burst, thereby allowing survivalwithin these cells. Another extracellular gonococcal protein is protein II, which isalsocalledOpasince coloniesexpressing protein II on theirsurfacehave a moreopaqueappearance. Thisproteinisbelievedtocooperateinthemoreintimateattachmentafterinitialpiliinteraction. Inaddition,proteinIIisabletoattachtothe lipooligosaccharide (LOS) of other N. gonorrhoeae, thereby enabling the cellsBasicScienceof Musculoskeletal Infections 21to bind to one other and form microcolonies. These microcolonies may also aid intheinitiationof mucosal surfaceattachment. ProteinII is capableof avoidingclearancebythehost immunesystembyphaseandantigenicvariation(166).Phase variation occurs through slipped strand synthesis that produces a frameshiftmutation and produces a prematurely terminated form of the protein. In addition,multiple variants of the protein II gene exist, and, therefore, the antigeniccharacter of proteinII canbechangedbyhomologousrecombinationof thesevariants.Althoughthisproteinisimportantformucosalinfections,mostisolatesfrom DGI patients are missing protein II fromtheiroutermembraneand grow toformtransparent colonies. ProteinIII is another porinthat is prevalent onthebacterialsurface.TheantibodiesdirectedagainstproteinIIIarenotbactericidal,and they sterically inhibit antibody binding to protein I and unsialylated LOS thatwould likely result in bactericidal action (167). Therefore, the generation of theseblockingantibodiesmaypreventserumbactericidalaction.LOSislikethelipopolysaccharideofothergram-negativebacteriaexceptthat its carbohydrate portion does not have the complex structure of the repeatingOside chain. LOShas endotoxin activity and is largely responsible for thesynovial damage in gonococcal arthritis (168,169). Although stable serumresistanceisduetoproteinI.A., unstableresistanceismediatedbytheabilityof some gonococcal strains to attach covalently activated forms of host sialic acidto the galactose residues on LOS(170). This covalent attachment coats thebacterial cell inhost proteins andavoids complement activation. Inaddition,opsonizationbycomplementcomponentsandformationofthemembraneattackcomplexofthecomplementsystemareinhibited.N.gonorrhoeaealsoproducesanIgAprotease that mayaidincolonization. However, the relevance of thispotentialvirulencefactoringonococcalpathogenesiswillneedfurtherstudy.3. PseudomonasaeruginosaP.aeruginosaisagram-negative,ubiquitous,free-livingbacterialspeciesthatisabletosurvive ina wide variety ofenvironmentalextremes.Ithasapredilectionfor moist environments and can infect plants, insects, lower animals, and humans(171). It has been described as the quintessential opportunist in human infectionsandiscapableofcausingfatal systemicdiseaseincertainconditions. Someofthese conditions arisewhennormal cutaneous or mucosal barriers have beenbreached or bypassed (e.g., penetrating trauma, surgery, or intravenous drugabuse);whenimmunologicaldefensemechanismshave beencompromised(e.g.,by chemotherapy-induced neutropenia, hypogammaglobulinemia, extremes ofage, diabetes mellitus, cystic brosis, cancer, or acquired immunodeciencysyndrome [AIDS]); when the protective function of the normal bacterial ora hasbeen disrupted by broad-spectrum antibiotic therapy; and=or when the patient has22 Shirtliff et al.beenexposedtoreservoirsassociatedwithahospitalenvironment(172).There-fore, this pathogenic bacterial species is able to gain access to the musculoskeletalsystembyanumberofdifferentroutes.a.)VirulenceFactors. Onceintroducedintoasusceptiblelocationwithinahost, aP. aeruginosabiolmmaydevelopondevitalizedtissueor medicallyimplanteddevicestoproduceaninfection. Whereaspseudomonal adherenceismediatedbytype IVpili, initial colonizationis augmentedbythe activityofagella, whichallowsmotilityandplacesthebacteriumcloseenoughtosolidstructures for the pili to adhere (173176). Also, this organismproducesneuraminidasethat enhancespili bindingbyremovingsialicacidresiduesfromhost glycoproteinGM1, makingit abetter receptorfor thepili (177,178). Onceattached,thisorganismcanformafullymaturebiolmstructurecomposedofacomplexchannelsystemthatprovidesevendeeplyembeddedbacteriaaccesstonutrients inthis modular community. It is believedthat thechannel systemismaintainedbycell-to-cellsignaling(179,180).High-molecular-weight alginatepolymersareusedtoretainP. aeruginosacellsefcientlywithinthebiolmmatrix.However,largeconglomeratesofcellsoftendetach,diffuseawayfromtheparentalbiolm,andreattachtothesurface,thereby allowing biolm spread in the mature biolm form. This detachment maybemediatedbystress duetohydrodynamicowand=or bythepseudomonalenzyme alginate lyase encoded by algL. This enzyme is capable of alginatedegradationandthereforecaninducebiolmsloughinganddispersion(181).Thebacterial biolmphenotypehasbeenshowntoprotect theembeddedbacteria fromnormal host defenses andsystemic antibiotics. Specically, thebiolmprotectstheorganismfromdirect antibody- andcomplement-mediatedbactericidal mechanisms andfromopsonophagocytosis (172). Also, themajorconstituent of pseudomonal biolms, thebacteriallyderivedextracellular poly-saccharide matrix, displays antiphagocytic properties, thereby allowing thebacteria to evade clearance bythe hosts immune system(182). The biolmalso acts as a mechanical barrier and prevents the inward diffusion of a number ofantimicrobials,therebyincreasingminimalbactericidalconcentrationsbygreaterthan100timesandallowingbacterialescapefromthebactericidalandbacterio-static effects of antimicrobial therapy (183185). Furthermore, those bacteria thatsurvive antibiotic clearance often develop or increase their resistance to theantibiotic and regrow upon treatment cessation. Meanwhile, the organismproducesanumber of extracellular enzymes, includingalkalineprotease(apr),elastase(lasB),serineprotease(lasA),andhemolyticphospholipaseC(plcS).Inaddition, the PA-IL and PA-IIL lectins (lecA and lecB) are produced and appear tofunction as adhesins as well as cytotoxins for respiratory epithelial cells. P.aeruginosaalsoproduces thevirulence factors exotoxinAandexoenzyme S(toxAand exoS). Exotoxin Aadenosine diphosphate(ADP)-ribosylates hostBasicScienceof Musculoskeletal Infections 23elongationfactor-2, causinghost translationinhibition(186,187). Thistoxinissimilartodiphtheriatoxinandcontributestotissuedamageanddiminishestheactivityofphagocytes. ExotoxinShasADP-ribosylatingactivitysimilartothatseen in many exotoxins and has been found to ribosylate and inactivate Gproteins like the pertussis and cholera toxins (188). In order to achieve fullenzymaticactivity, exoenzymeSmust beactivatedbya hostcellprotein,termedfactor-activatingExoS(FAS)(189).ExoenzymeS isextremelyimportantfortheabilityofP. aeruginosatocausediseasesincedisruptingthegeneincreasedtheLD50byafactorof104inburnedmousemodels(188). Thebreakdownofhosttissuesbytheseextracellular bacterial productscreatesconditionsthat enhancebacterial proliferation, invasion, and tissue injury. These activities may culminateinbloodstreaminvasionanddissemination.b.) Regulation. P. aeruginosa regulates these virulence factors in acomplex, but coordinated, wayina microbial cell densitydependent mannerthrough quorumsensing and response to environmental cues. The quorumsensing ability in P. aeruginosa is very different fromthat in the S. aureussystemandisdependentupontwodistinctbutinterrelatedsystems, lasandrhl.There is a denite hierarchy of these systems, with the las systemtakingprecedence. These two systems work in concert to upregulate a number ofpseudomonal factors that enable this pathogen to survive in highly diverseenvironments. Inthelasquorumsensingsystem, thelasI geneproduct directsthe formation of the extracellular autoinducing signal N-(3-oxododecanoyl)homoserinelactone(3-oxo-C12-HSL) (Fig. 4), whosesecretiontotheextracel-lularenvironmentisaidedbyP.aeruginosaefuxpumpsencodedbythemexA-mexB-ompRoperon(190). As3-oxo-C12-HSLconcentratesintheextracellularenvironment, it istakenupbyP. aeruginosacellsandinteractswiththeLasRtranscriptional activator. This LasR-3-oxo-C12-HSL complex is then able toactivatetheexpressionofanumberofgenes, includinglasB(elastase), lasA(aserineproteasethat nicks elastinandworks synergisticallywithelastase), apr(alkalineprotease), toxA(exotoxinA), bothxcpoperons (xcpPQandxcpR-Z,encoding the type II secretion apparatus), rhlR, and lasI itself (191). Theformation of the LasR-3-oxo-C12-HSL complex may be aided by GroESLchaperonins as seenintheluxsystemof V. scheri (192). Thesechaperoninsare upregulated in response to heat shock and the resultant protein misfolding viatheRpoHandAlgUalternativesigmafactors. Also, thetranscriptionoflasRisinduced in response to glucose limitation, and this induction is mediated throughthe virulence factor regulatorcyclic adenosine monophosphate (vfr-cAMP)complex(discussedlater)(193).The second P. aeruginosa quorum sensing system consists of the regulatoryproteinRhlRandthediffusibleautoinducerN-butyryl homoserinelactone(C4-HSL) (Figs. 4 and 5) synthesized by the product of rhlI. In contrast to 3-oxo-C12-24 Shirtliff et al.Figure5Modelofthelas=rhlquorumsensingsysteminP.aeruginosa.Seetextforgenedescriptions.(AdaptedfromRef.179.)BasicScienceof Musculoskeletal Infections 25HSL, C4-HSLis freely permeable. Apparently the length and=or degree ofsubstitution of the N-acyl side chain determines whether an autoinducer isfreely diffusible or is subject to active efux by P. aeruginosa. As in thehomologous las system, once the diffusible autoinducer C4-HSL attains adequatelevels, it binds and activates the RhlR transcriptional regulator. RhlR-C4-HSL hasbeen shown to regulate the rhamnolipid biosynthesis operon rhlAB, alkalineprotease, pyocyanin, PA-ILandPA-IILlectins, lasB-encodedelastase, andrhlIitself(191).Thehierarchy ofthelas=rhlsystemisaidedbytheinhibitoryactionof the unbound las autoinducer, 3-oxo-C12-HSL, on the binding of C4-HSL to theRhlRtranscriptional activator. The upregulationof lasRtranscriptionandtheresultingelevatedconcentrationsoftheLasR-3-oxo-C12-HSLcomplexallowtherhl system to be subsequently activated. The rhl quorum sensing system was alsoshownin2000tobeinhibitedbythealternativesigmafactor, RpoS(194), andactivatedbythegactwo-component regulatorysystemthat respondstogrowthphase (Figs. 4 and 5) (187). In addition, the formation of the RhlI-C4-HSLcomplexhasbeenshowntobeaidedbyGroESLchaperoninsasseenintheluxsystemofV. scheri andpossiblyintheformationoftheLasR-3-oxo-C12-HSLcomplex(192).Another factor inthis quorumsensingsystemis the negativeregulator,RsaL(195). InP. aeruginosa,LasRand3-oxo-C12-HSLgloballyregulatemanyproductsassociatedwithvirulence, aswellasthesecondP. aeruginosaquorumsensing system. It has been theorizedthat at lowcell density, RsaLinhibitstranscriptionof lasI bybindingtothe lasI operator region, therebyblockingactivationbyLasR-3-oxo-C12-HSL(195). Asthecelldensityincreases, sodoestheintracellularconcentrationof3-oxo-C12-HSL, whichallowssufcientLasR-3-oxo-C12-HSLformationtoinhibit RsaLcompetitivelyfor bindingtothelasIoperator. Thus, it appears that during the early stages of growth, RsaL blocks thequorumsensingcascadebyinhibitingthetranscriptionof lasI. Finally, it wasfoundthatthisorganismproducesanotherintercellularsignal,thePseudomonassp. quinolone signal (PQS), that was identied as a 2-heptyl-3-hydroxy-4-quinolone(196,197). PQSis producedmaximallyat latestationaryphaseandworks by activating transcription of the rhlI gene (and to a lesser degree lasR andrhlR).Itisnotknownwhat activatestheproductionofPQS,butthismoleculeisprobablynotinvolvedinsensingcelldensity.The3-oxo-C12-HSLandtheC4-HSLof thelasandrhl systems, respec-tively, have been well studied; other homoserine lactones may have beenidentied in P. aeruginosa by using a combination of reporters, thin layerchromatography, and comparison to HSL standards (198). In P. aeruginosaisolatesderived from cysticbrosisrelated chronic lung infections, investigatorswere able to detect up to ve additional HSLs: N-hexanoyl-L-homoserine lactone(C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), N-(3-oxodecanoyl)-L-26 Shirtliff et al.homoserine lactone(3-oxo-C10-HSL), and N-(3-oxotetradecanoyl)-L-homoserinelactone (3-oxo-C14-HSL). This studyalsonotedthat the productionof HSLsshowedpronouncedreductionwhenthepatient was subsequentlycocolonizedwith Burkholderia cepacia. Although only one cocolonized patient was evaluated,thepropertiesof multispeciesinfectionsandpotential interspeciescommunica-tionmaybeimportantincysticbrosisandinbiolms.Althoughthequorumsensingsystemisanextremelyimportant determi-nantofvirulencefactorexpressioninP.aeruginosa,thesystemmustbeabletorespondadaptivelytoenvironmental stimuli. Stimuli that havebeenshowntoaffect this system(either directlyor indirectly) include heat shock, iron andglucoseavailability, RpoS-mediatedinhibition(possiblyduetospecicaminoacidstarvation), andentryintostationarygrowthphasemediatedbyunknownstimulatorsofthegactwo-componentsystem.Theregulationof biolmformationandvirulencefactor expressionis acomplex interaction among a number of regulatory cascades in P. aeruginosa. Forexample, exotoxinA, thediphtherialiketoxinresponsiblefor proteinsynthesisdisruptionineukaryoticcells, is regulatedbyanumber of environmental andquorumsensing signals (Fig. 6). Some of the environmental signals that P.aeruginosa responds to are the presence of glucose, iron and nitrogen availability,oxygen levels, temperature variations, pH, osmolarity, amino acid starvation, andultravioletdamage.TheP. aeruginosaresponsetoglucoselimitationismediatedthroughthevirulence factor regulator (Vfr) that demonstrates signicant homologytothecAMPreceptorprotein(CRP)ofE.coli(193).Whenglucoseisinshortsupply,theintracellular concentrationof cAMPisupregulatedinmost microbes. InP.aeruginosa, twocAMPmoleculesbindtheinactiveVfrdimer. ThiscAMP-Vfrcomplexis thenable tobinda consensus dyadsymmetrical sequence inthepromoterregion of a number of operons, including the quorum sensingregulator(lasR) and genesrequiredfortheutilizationof various carbonsources, through ahelix-turn-helixbindingmotif (193). Uponbinding, thecomplexpromotesthelocalization of the RNA polymeraseholoenzyme (RNAP)to the promoterregionthrough interaction between the b-subunit of the RNAPand the cAMP-Vfrcomplex. As previously mentioned, the cAMP-Vfr complex is able to activate thetranscriptionof lasR, therebyactivatingthequorumsensingcascadewhentheautoinducer, 3-oxo-C12-HSL, ispresent at signicant levels. It isinterestingtonotethat thetranscriptionof thevfrgeneitself hasrecentlybeenshowntobeactivated by both the las and rhl autoinducertranscriptional regulator complexes.Another carbonmetabolite regulator, termedthe catabolite repressioncontrol(Crc) protein, has been recently found. This protein is able to sense carbon sourceavailabilityandaffectsexpressionofthetypeIVpili structural subunit PilAtopromote microcolony formation on biolms. As demonstrated in mutationstudies, P. aeruginosa Crc mutants are only capable of formingthin biolmBasicScienceof Musculoskeletal Infections 2728 Shirtliff et al.monolayersinsteadofconglomeratingintoanumber of microcoloniesthroughtype-IVdependenttwitchingmotility(199).Therefore,Crcmay representa linkbetweencarbonavailabilityandthedecisiononwhether or not toenter intoabiolmmodeofgrowth.SinceP. aeruginosaprefersaerobicmetabolismthat utilizesanumber ofiron-containing enzymes, this microbial species has evolved a number ofstrategies to obtain iron fromits environment (200). In order to conserveenergyandresources, P. aeruginosatightlyregulatestheexpressionof itsironacquisitionsystemstolimit their activityiniron-richenvironments. Thisiron-dependentregulationcentersontheactivityofarecentlyisolatedhomologueoftheferricuptakeregulator(Fur)inEscherichiacoli (200,201). Whenironisinamplesupply, FurbindsFe2andisabletoattachtoapalindromicconsensussequence (termed the Fur box) in the promoter regions ofiron-regulated genes,thereby repressing their expression. When P. aeruginosa is grown in iron-limitingconditions, Fe2 dissociates from the complex, causing Fur release and repressionremoval.GenescontrolledeitherdirectlyorindirectlybytheFursystemincludethosethatcodeforotherregulatoryproteins(e.g., thesigmafactorPvdS), iron-scavengingproteins(e.g., pyochelinandpyoverdinsiderophores), proteasesthatdegrade the iron-binding host proteins, the cytotoxin exotoxin A that enables ironreleasefromsusceptiblehostcellstooccur,proteinsinvolvedinbasicmetabolicprocesses (e.g., Krebs cycle), and proteins responsible for oxidative stresssurvival (e.g., superoxidedismutase) (202204). Alterationsinironconcentra-tionshavebeenshowntoaffect thequorumsensingsystem; suchinteractionsmay be mediated indirectly through the vfr regulation system, as seen in V.scheri(205).In reference to respiration, P. aeruginosa can utilize inorganic electronacceptors(otherthanoxygen)forgrowth. However, thisspeciesisincapableoffermentativemetabolismandgenerallygrows morefastidiouslyinoxygenatedenvironmentssinceitprefersaerobicmetabolism. Therefore, itisnotsurprisingthat thisorganismaltersitsgeneexpressioninresponsetooxygenlevels. Thiscontrolismediatedthroughtheoxygen-sensingtranscriptionalregulatorprotein,anaerobicnitraterespiration(ANR)protein,whichishomologoustotheFNRinE. coli (206). This protein forms a [4Fe-4S]2cluster under conditions of low O2.Thisclusterformationhasbeenshowntopromotedimerizationandbindingtopromoterregionsofgeneswhosefunctionsfacilitateadaptationtogrowthunderanaerobic conditions (e.g., denitrication enzymes and=or their regulators) (207).Therearenodatatosupport thedirect roleof ANRinthe regulationof theFigure6 Thecomplexinteractionof theP. aeruginosaquorumsensingsystemandenvironmental stimuli intheregulationof toxA(exotoxinA) transcription. Seetext forgenedescriptions.BasicScienceof Musculoskeletal Infections 29quorumsensingsystem. However, O2levelsmust betakenintoaccount whenevaluatingcell-to-cellsignalingexperimentaldatasinceANRhasbeenshowntoactivate the transcription of a large number of enzymes associated with anaerobicmetabolismwhile repressingthe expressionof those enzymes responsibleforaerobic metabolism. By taking into account the effect of oxygen-dependentregulation, one may prevent the incorrect assumption of causal relationshipsbetweengeneexpressionandquorumsensingsystemactivity.Thisorganismhasbeenshowntoadapttoaminoacidstarvationthroughacomplexseriesofregulatoryeventstermedthestringent responsethathasbeendescribedasaglobal regulationmechanism. Briey, thisresponse(well eluci-datedinE. coli) is mediatedthroughthe accumulationof unchargedcognatetranscriptional RNA (tRNA). When the ratio of aminoacyl-charged to -unchargedtRNAfallsbelowacriticalthreshold,occupationofthevacantmRNAcodonattheribosomal AsitebyunchargedcognatetRNAleads tostallingof peptidechainelongation. Also, thesynthesisof thepseudomonal nucleotide(p)ppGppfrom guanosine triphosphate and adenosine triphosphate (GTP) (ATP) is inducedinaribosomal-dependentidlingreaction(208,209).IthasbeendemonstratedinE.colithatthe(p)ppGppinhibitsRNApolymerase,causingthedownregulationof a wide range of energetically demanding cellularprocesses (e.g., the synthesisof stable RNA), stimulation of certain amino acid synthesis pathways (e.g.,isoleucine), andinductionofstationaryphase-specicgenesthroughtheeffectsof the stationary-phase=stress-specic sigma factor (a.k.a. ssor RpoS) (208,210).Whereas the s70(a.k.a. sD) factor is responsible for the transcription ofconstitutive-expressedandhousekeepinggenes, sshas beenimplicatedinthetranscription regulation of over 50 genesinE.coli in response to not only aminoacidstarvation, but alsoosmoticstress, acidshock, heat shock, oxidativeDNAdamage, andtransitiontostationaryphase. Transcriptional regulationof rpoSexpressionhasbeendemonstratedtobeunderpositivecontrolby(p)ppGppandnegativecontrol bythecAMPreceptor protein. Also, translational control hasbeenascribedtoanumber of other factors includinganRNA-bindingprotein(Hfq),anucleoidhistonelikeprotein(H-NS),andasmallregulatoryRNA(dsrARNA)that destabilizesthesecondarystructureinrpoSmRNAtoallowtransla-tional initiation. However, proteolysis of RpoS by the ClpPX protease (due to theremovalofprotectionofRpoSbythechaperoneprotein,DnaK)seemstobethemain regulation mechanism (211). This sigma factor was shown in 2000 to inhibitrhlI transcription, thereby reducing the level of C4-HSL- and RhlR-RhlI-regulatedgenetranscription(194).P. aeruginosa also mediates changes in gene expression through a complexarray of other alternative RNA polymerase sigma factors in response to a numberof environmental stressors. Heat shock is one example of an environmental stress,andthepseudomonal responseismediatedthroughthecombinedeffect of theextracytoplasmicstress factor, sE(a.k.a. AlgU), andsH(a.k.a. RpoHors32)(212).AlgUandRpoHrespondtotheaccumulationofmisfoldedproteinsinthe30 Shirtliff et al.periplasmic and cytosolic bacterial compartments, respectively. Specically, AlgUis able to upregulate its own expression as well as the expression of genes codingfor RpoHand the enzymes of the alginate biosynthetic pathway (212). Theantisigma factor products of mucA and mucB normally inhibit the activity of AlgU(213,214). However, inpatientssufferingfromcysticbrosis, theseantisigmafactor coding regions are often mutated, resulting in the conversion of nonmucoidstrainsinto the mucoid variety by allowing fortheconstitutive overproductionofalginate(214,215).ThisexcessofalginateproductioninP.aeruginosaresultsinthe formation of biolm microcolonies consisting of exopolysaccharide-embeddedcells. Thesebiolmmicrocoloniesdemonstratehigh-level resistancetohost orantimicrobial clearancestrategies. InreferencetoRpoH, manyof itsregulatory effects can be linked to its activation of GroESL proteins (192). Theseproteins act as chaperonins that sequentially promote correct folding of a numberofproteinsandaidin theformationofproteincomplexes,possiblyincludingtheLasRquorumsensingregulatorwiththeLasIproduct, 3-oxo-C12-HSL, andtheformationof the RhlR-C4-HSLcomplex(191,192). Twoother environmentalstressorsthat regulategeneexpressionthroughalternativesigmafactorsaretheneed for agella (mediated by sF, a.k.a. s28or RpoF coded by iA) and nitrogendepletion(sNa.k.a. s54orRpoN)(216,217).Allofthesigmafactorsdiscussedhave been well studied in E. coli, but their role in pseudomonal stress response andbiolmformationisstillunclear.GacAand GacS are highly conserved among Pseudomonas spp. anddemonstrate upregulationof expressionuponentryintostationaryphase as aresultofanunknownsignal(187). gacSEncodesthecognatesensorkinasethatactivates the response regulator coded for by gacA by phosphorylation (218). ThisGacS=GacAsystemstrictly controls the expression of extracellular products(antibiotics,exoenzymes,andhydrogencyanide)whencellsareinthetransitionfrom exponential to stationary phase. This system has also been found to increasethe production of the C4-HSLautoinducer of the pseudomonal rhl quorumsensingsystem(187). It washypothesizedthat activatedGacA, byvirtueof itstypical C-terminal helix-turn-helixDNAbindingmotif, regulatedthetranscrip-tion of target genes. However, 1999 evidence points to posttranscriptional controlbyinteractingwiththemRNAribosomalbindingsiteofGacA-controlledgenes(219).Theimportanceofthisregulatorysystemcanbedemonstratedbyastudyinwhicha gacAmutant of P. aeruginosahas attenuatedvirulence inanimalmodels(218).III. PROPERTIESOFTHEHOSTAnumber of virulence factors of pathogenic microorganisms allowpersistentinfections, andmanyhost factorsassumeasignicant role. ThelocalizationofmanyMSIswithinspecicsitesofthehostisduetothe vasculararchitectureofBasicScienceof Musculoskeletal Infections 31these sites. In addition, any systemic or local factor of the host that affectsimmune surveillance, metabolism, and local vascularity reduces the ability of thehost to resolve the infection and may result in the development of MSIs (Table 2).Anumberofthesefactorsarediscussedinthesectionsthatfollow.A. TheNormalHostVasculatureIncases of longboneosteomyelitis, themetaphyses of thelongbones (tibia,femur) aremost frequentlyinvolved(1). Theanatomical characteristics of themetaphysealregionseemtoexplainthisclinicallocalization(220). Theafferentartery ends in the metaphyses as narrow capillaries that make sharp loops near thegrowthplateandenterasystemoflargevenoussinusoidswherethebloodowbecomes slowand turbulent. These capillary loops are essentially the end-artery branches ofthenutrientartery. Thisstructureleadsto a slowing ofbloodow in the area and presumably allows bacteria to settle and initiate aninammatoryresponse. The histological features of the regionmayalsobe acontributingfactor.Themetaphysealcapillarieslackphagocyticliningcellsandthe sinusoidal veins contain functionallyinactive phagocytic cells (221); thisstructure further allows growth of microorganisms. Any end-capillary obstructioncouldleadtoanareaofavascularnecrosis. Minortraumaprobablypredisposesthe infant or child to infection by producing a small hematoma, vascularobstruction, andsubsequent bone necrosis that are susceptible to inoculationfromatransient bacteremia(222). Vertebral hematogenous osteomyelitis loca-Table 2 SystemicorLocalFactorsThatAffectImmuneSurveillance,Metabolism,andLocalVascularitySystemic(Bs) Local(BI)Diabetismellitus MajorvesselcompromiseRenal, hepaticfailure SmallandmediumvesseldiseaseMalnutrition ExtensivescarringChronichypoxia ArteritisImmunosuppressionor Radiationbrosisimmunedeciency ChroniclymphedemaMalignancy VenousstasisImmunedisease NeuropathyExtremesofage Tobaccoabuse[_2packsperday]Chronicgranulomatousdisease Presenceof implantsLocalizedtrauma32 Shirtliff et al.lizes within the cancellous bones, particularly the lumbar and thoracic areas of thespinebecauseoftheirrichbloodsupplyandreducedshear,therebyallowingforcolonizationandinfection.In cases of septic arthritis, the architecture of the normal joint space allowsfortheeasyhematogenousentryofbacteriasincethewell-vascularizedsynovialmembrane has no limiting basement membrane. Bacteria may also gain entry intothejointbydirectintroductionorextensionfromacontiguoussiteof infection.Once bacteria are seeded within the closed joint space, the lowuid shearconditionsallowbacterialadherenceandinfection.The virulenceandtropismofthe microorganisms combinedwiththeresistance or susceptibility of the synoviatomicrobialinvasionaremajordeterminantsofjointinfection.B. GeneralizedVascularInsufciencyMostpatientswhohaveMSIsandgeneralizedvascularinsufciencysufferfromdiabetes(6).Thediminishedarterialbloodsupplyhastraditionallybeenconsid-eredtobe the major predisposingfactor inthe initiationof infectionanditsprogressiontoachronicstate(6).Observationsin1999suggestthatneuropathymaybeanequallyimportantfactor(223).Identiable neuropathyasacomplica-tionof diabetesmellitusispresent inapproximately80%of patientswithfootdisease(223). Neuropathymaycausefoot infectionthroughthreemechanisms.First, patients with decreasedsensation suffer mechanical or thermal injurieswithout awareness, leading to skin ulcerations. Second, motor neuropathyaffecting the intrinsic muscles of the foot predisposes the patient to gaitdisturbancesan