research article hyaluronidase in clinical isolates of

Research ArticleHyaluronidase in Clinical Isolates of Propionibacterium acnes

Harmony Tyner1 and Robin Patel12

1Division of Infectious Diseases Department of Medicine Mayo Clinic Rochester MN USA2Division of Clinical Microbiology Department of Laboratory Medicine and Pathology Mayo Clinic 200 First Street SWRochester MN 55905 USA

Correspondence should be addressed to Robin Patel patelrobinmayoedu

Received 17 September 2014 Revised 14 January 2015 Accepted 2 February 2015

Academic Editor James Dooley

Copyright copy 2015 H Tyner and R Patel This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objectives We sought to describe the prevalence of a hyaluronidase gene and hyaluronidase production in 197 clinical isolates of Pacnes we assessed kinetics of hyaluronidase production in a subset of three isolatesMethods The hyaluronidase gene was detectedusing polymerase chain reaction Hyaluronidase production was detected by growing isolates on BHI agar containing 400120583gmLhyaluronic acid and 1 albumin and flooding plates with 2N glacial acetic acid to precipitate unbound hyaluronic acid with a zoneof clearing representing a positive phenotype Hyaluronidase production kinetics were measured as a function of hyaluronic aciddigestion over time in a liquid medium Results A hyaluronidase gene and hyaluronidase production were detected in 100 and 97of P acnes isolates respectively Hyaluronidase production in liquid medium was detectable after 96 hours of growth ConclusionsHyaluronidase production is nearly universal among P acnes isolatesThree days appear to be required for significant hyaluronidaseproduction in a liquid medium Detection of hyaluronidase in tissue specimens may be a strategy to differentiate P acnes infectionfrom colonization when P acnes is isolated from a clinical specimen

1 Introduction

A slow-growing member of the normal microbiota of seba-ceous glands which may be beyond the reach of topicalantiseptics Propionibacterium acnes has long been recog-nized as a contaminant in clinical cultures In specific clinicalscenarios however P acnes is increasingly recognized asa pathogen Notable examples include P acnes shoulderarthroplasty- and spinal instrumentation-associated infec-tion Recognition of the pathogenicity of P acnes in suchcases is dependent on its recovery from multiple separatelycollected specimens from a site expected to be sterile inassociation with clinical symptoms Because P acnes hastraditionally been considered a contaminant its recovery hasnot been historically emphasized in clinical microbiologylaboratories And despite its recognition as a pathogen it isalso frequently isolated as a contaminant Recent studies haveinformed strategies for ideal recovery of pathogenic P acnesfrom clinical specimens [1 2] but this issue remains a topicof controversy

The main symptom associated with P acnes orthopedicforeign body infection is pain Other findings classicallyassociated with infection including fever and elevated whiteblood count (WBC) sedimentation rate (ESR) and C-reactive protein (CRP) are uncommon The absence ofabnormal inflammatory markers and other typical signs ofbacterial infection may lead to P acnes infection being disre-garded because despite repeated isolation of this organismother findings pointing to a diagnosis of implant-associatedinfection may be lacking Current guidelines for prostheticjoint infection (PJI) diagnosis [3] rely on features that may beabsent in P acnes PJI

The reason for the paucity of acute inflammatory mark-ers associated with P acnes infection remains incompletelydefined P acnes secretes a number of proteins [4] the P acnessecretome includes 20 proteins The identity and function ofonly a few of these proteins have been described and charac-terized Among the proteins in the secretome are the enzymeshyaluronidase chondroitin sulfatase and gelatinase [5 6]Hyaluronidases are enzymes that degrade hyaluronic acid

Hindawi Publishing CorporationInternational Journal of BacteriologyVolume 2015 Article ID 218918 6 pageshttpdxdoiorg1011552015218918

2 International Journal of Bacteriology

a component of the extracellular matrix Hyaluronic acid ispresent in tissues throughout the body including bones andjoints contributing to the viscosity of synovial fluid and thelubrication of joints It is also a part of the extracellular struc-ture of chondrocytes involved in tissue healing The ability ofbacteria to degrade hyaluronic acidmay be a virulence factorenabling penetration of hyaluronidase-producing organismsinto tissues rich in hyaluronic acid including joints creatingan advantage for establishing growth in articular spaces Inaddition to P acnes other Gram-positive pathogens such asStreptococcus pneumoniae Streptococcus intermedius Strepto-coccus constellatus Streptococcus dysgalactiae Staphylococcusaureus Peptostreptococcus species Clostridium perfringensandClostridium septicum as well as other species of Propioni-bacterium including P granulosum produce hyaluronidase[7]

Herein we defined the prevalence of a hyaluronidase geneand of hyaluronidase production in a collection of clinicalisolates of P acnes We also defined the time course of in vitrohyaluronidase production by P acnes using a subset of threeclinical isolates

2 Methods

21 P acnes Isolate Collection 197 clinical isolates of P acneswere studied including isolates associated with clinicallysignificant monomicrobial infections such as PJI spineinfections and endocarditis as well as isolates from clinicalspecimens with polymicrobial growth and isolates represent-ing contamination (Table 1) Of the 197 isolates 71 were asso-ciated with clinically significant growth defined as growthof ge20 colony forming units10mL sonicate fluid [8] or ge3positive cultures All 197 isolates underwentmolecular testingfor a P acnes hyaluronidase gene and phenotypic testingfor hyaluronidase production three isolates underwent time-course studies for assessment of kinetics of hyaluronidaseproduction

22 Genotypic Detection of Hyaluronidase Gene P acnesisolates were grown under anaerobic conditions in 2mLbrain heart infusion broth supplemented with 1 glucosewith agitation DNA was extracted using the QIAamp UCPPathogen Mini Kit (QIAGEN Valencia CA) with a finalelution into 50 120583L elution buffer A 451-base-pair regionof the hyaluronidase gene sequence [9] was targeted bypolymerase chain reaction (PCR) primers forward primer51015840-TTTCGGGATCCTTGTGGTA-31015840 and reverse primer51015840-TCTGGACAACAAACCTGT-31015840 Primer sequenceswere selected from the published P acnes hyaluronidasegene sequence [9] Basic Local Assignment Search Tool(BLAST) analysis showed that the primer sequences hadcomplete matches only with the two published P acneshyaluronidase gene sequences 5120583L of isolated DNA wasamplified using 42 cycles as follows melting at 95∘C for 30seconds annealing at 54∘C for 60 seconds and amplifyingat 72∘C for 120 seconds The final product was run on a 1agarose gel and the product identified based on amplifiedproduct size Amplified DNA from a representative isolate

was sequenced and the following sequence was obtainedlsquoCGCCCGCCGTGTGCTGTAGGTCTTCGGGGCTGG-GGTCAGGGTTGGTGCTGGTTGTTATGAAGGGCG-AGTGCGCCTTGCGGGGGTCTGCCGAGATCAGAA-CGGTCTGACGGCTCGAGCCGGATACGAGGTCGG-TGAGGATCTCGGCGACCTTCCGGTCGGTCTTGG-CGATGATCGCTCGGGCACGTGGGTCAGAGGTCC-GGGCGGCTCTGCGTCCGGTGATGATGTCGATCC-ATCGCTCGCACAGCGCCGACCAGCTGTCGGGCC-CGGGGGCGGCAATGGCGTCCGTCACTGTGGGAG-AAGCGGCGAGCGCCATGGCTGACAGCGCAGATG-CGGTGAGGAAGGTTCGTCGAGATGGGGTGCCGA-ACAAGGGTCACGCTCCTCGGGAGAGGGTGGCAG-CACTGCCACACGTACAGGTTTGTTGTCCAGA-31015840consistent with the P acnes hyaluronidase gene

23 Phenotypic Detection of Hyaluronidase ProductionHyaluronidase production was evaluated as described bySmith and Willett [5] and used as a binary determinant ofthe ability of each isolate to produce hyaluronic acid Briefly100mL brain heart infusion media was prepared with 1 gof agar autoclaved at 121∘C for 15min and cooled to 46∘Cthen a 2mgmL solution of sodium hyaluronidase boiledfor 10 minutes to achieve solubility was added to the cooledmedium to a final concentration of 400120583gmL 5 bovinealbumin fraction 119881 was added under constant stirring toyield a final concentration of 1 Plates were poured to adepth of 3-4mm and allowed to solidify at 4∘C P acnesisolates were grown on sheep blood agar under anaerobicconditions until growth was observed A single colony wasstreaked onto the hyaluronic acid medium incubated underanaerobic conditions and observed daily until growth wasobserved (24 to 72 hours) On the day growth was firstobserved the plate was flooded with 2N glacial acetic acidwhich binds hyaluronic acid and albumin forming a whiteprecipitate Hyaluronidase production was considered tohave been present if a zone of clearing was observed

24 Time Course Studies of Hyaluronidase Production Themethod was modified from that described by Tolksdorf andMcCready [10] The assay measures turbidity resulting fromhyaluronic acid mixed with albumin in an acidic solutionhyaluronidase activity is measured as lack of turbidity Stan-dard curves of hyaluronic acid turbidity and hyaluronidaseactivity were constructed Hyaluronic acid sodium salt fromStreptococcus equi (Sigma St Louis MO) was used for con-structing the standard curve and for experimental readingsand hyaluronidase from bovine testes (Sigma) was used todefine a standard curve of enzyme concentrationactivity(Figures 1(a) and 1(b)) Based on these standardized curvesthe limit of detection was 00125120583g120583L hyaluronidase andthe assay was fully saturated at 005 120583g120583L hyaluronidase(Figure 1(b))

To quantify production of hyaluronidase over time threeclinical isolates of P acneswere grown anaerobically on sheepblood agar for two days One was selected based on its lackof production of hyaluronidase using the plate method Theother two produced hyaluronidase as determined using the

International Journal of Bacteriology 3

Table 1 Study isolates

Source of isolation number of isolates of all cases Number with clinically significant growth ()Total number 197 100 71 (36)Upper extremity including hardware and native joints 36 18 24 (67)Lower extremity 51 26 7 (14)Spinevertebral hardware 45 23 25 (49)Breast implant 49 25 9 (18)Penile implant 7 4 1 (14)Cardiac valve 3 2 2 (67)Blood 1 1 1 (100)Pacemaker 5 3 2 (40)The table shows the site of isolation of the study isolates and whether they were considered to represent clinically significant growth (ie not contaminants)

0

005

01

015

02

025

03

035

0 01 02 03 04

OD540

Hyaluronic acid concentration (120583g120583L)

(a)

0

005

01

015

02

025

03

035

04

0 20 40 60 80

OD540

Hyaluronidase concentration (120583g)

(b)

Figure 1 (a) Standard curve used tomeasure hyaluronic acid concentration Optical density of hyaluronic acid at 540 nanometers wavelength(OD540

) (b) Standard curve used to measure hyaluronidase concentration Hyaluronic acid will bind with albumin and form a whiteprecipitate when acidified When hyaluronidase is present it digests hyaluronic acid and the hyaluronic acid cannot bind with albuminand precipitate Decreased precipitation is measured as increased light transmission and therefore a lower OD

540reading

plate method one of which was observed to grow quickly(IDRL-7844) and the other of which took longer than 48hours to exhibit visible growth (IDRL-7751) The isolateswere suspended in brain heart infusion broth supplementedwith 1 glucose to a turbidity of 10 McFarland and diluted1 50 with glucose-supplemented brain heart infusion broth200120583L was inoculated in triplicate in a 96-well tissue cultureplate with 7 identical plates prepared in duplicate One halfof the plates was incubated anaerobically using AnaeroPackSystem (Mitsubishi Gas Chemical America Inc New YorkNY) and one half was incubated aerobically At 6 12 24 4872 96 120 and 144 hours a single plate was removed fromeach incubator the OD

600documented to assess planktonic

growth and the supernatant broth collected andfiltered usinga 02 120583mmembrane filter to abort further enzyme productionby removing viable bacteria and prevent turbidity of bacterialgrowth from influencing the OD

540reading done to detect

enzyme activity based on precipitation 85120583L of a 04mgmLsolution of hyaluronic acid was combined with 85 120583L ofsample broth supernatant and allowed to react at 37∘C for10 minutes The samples were then treated with 135120583L of anacidic albumin reagent described byTolksdorf andMcCreadymade of 25 g bovine serum albumin fraction 119881 in 1000mLof 05M sodium acetate buffer at pH 42 for 10 minutesfollowing which the OD

540was measured (Figure 2) [10]

3 Results

31 Genotypic Detection of Hyaluronidase Gene Of the197 clinical isolates evaluated all (100) were positive forhyaluronidase gene (Figure 3)

32 Phenotypic Detection of Hyaluronidase Production Ofthe 197 isolates tested 191 (97) produced hyaluronidase


0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540

Negative controlIDRL-7676

IDRL-7751IDRL-7844

Figure 2 Kinetics of hyaluronidase production in Propionibac-terium acnes Three isolates were studied including a hyaluronidasenonproducing (as determined by plate assay) isolate (P acnesIDRL-7676) and two hyaluronidase-producing (as determined byplate assay) isolates (P acnes IDRL-7751 and P acnes IDRL-7844)Hyaluronidase production was measured by increased light trans-mission at OD

540 Sterile medium was used as a negative control

characterized by a zone of clearing around colonies (Fig-ure 4) The clinical isolates of P acnes showed some variationin time to achieve robust bacterial growth Most achieveddetectable plate growth by 24 hours however some took aslong as 72 hours of incubation before detectable plate growthwas observed

33 Time Course Studies of Hyaluronidase Production Whenthe twohyaluronidase-producingP acnes isolateswere grownanaerobically hyaluronidase was detectable in solution by96 hours and continued to accumulate over time (Figure 2)suggesting that hyaluronidase production begins when theorganism reaches logarithmic phase growth Hyaluronidaseproduction did not occur under aerobic conditions (Fig-ure 5)

4 Discussion

Hyaluronic acid is a component of skin and extracellularmatrix found throughout the body primarily in soft tissueHyaluronidase is the term used to describe any enzymeable to cleave hyaluronic acid There are several distincthyaluronidases including enzymes found in mammalianspermatozoa in the saliva of parasites such as leechesand in bacteria Bacterial hyaluronidases are similar to oneanother in that the end product of bacterial hyaluronidases

Neg

ativ

e con

trol1 2 3 4 5 6 7 8 9 10

minus500bp

minus100 bp

100

bpla

dder

minus1000 bp

Figure 3 Genotypic detection of P acnes hyaluronidase genePolymerase chain reaction assay was used to detect a 451-base-pairsegment of the P acnes hyaluronidase gene in 197 P acnes clinicalisolates This is a representative gel demonstrating the appearanceof a typical selection of isolates The identity of each organism bylane is as follows 1 IDRL-7421 2 IDRL-7811 3 IDRL-7812 4 IDRL-8403 5 IDRL-9037 6 IDRL-6536 7 IDRL-7599 8 IDRL-7714 9IDRL-9038 10 IDRL-7737

Bacterial growth Zone of clearing

Figure 4 Phenotypic detection of hyaluronidase production Plateswere prepared with brain heart infusion agar supplemented withhyaluronic acid and albumin P acnes isolates were allowed to growuntil there was visible growth thereafter the plate was acidified with2N glacial acetic acid causing undigested hyaluronic acid to bindto albumin forming a white precipitate Hyaluronidase productionwas considered to be present if a zone of clearing was observed

is unsaturated disaccharides This is distinct from mam-malian and parasitic hyaluronidases [7] A variety of Gram-positive human pathogens excrete hyaluronidase an enzymethat may enable bacterial spreading and tissue penetration[7] These include species of Streptococcus Staphylococ-cus Peptostreptococcus and Clostridium listed previouslyin addition to P acnes Gram-negative bacteria may alsoproduce hyaluronidase however rather than excreting theenzyme extracellularly the hyaluronidase they produce is aperiplasmic enzyme and as such is less likely to aid in tissuepenetration than is the enzyme produced by Gram-positive


0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540

IDRL-7751 aerobicIDRL-7844 aerobic

IDRL-7751 anaerobicIDRL-7844 anaerobic

Figure 5 Hyaluronidase production by Propionibacterium acnesunder aerobic and anaerobic conditions Two hyaluronidase-pro-ducing isolates of P acnes were incubated under aerobic and anaer-obic conditions Hyaluronidase production was measured by lighttransmission at OD

540 Hyaluronidase was detected with incubation

under anaerobic but not aerobic conditions

bacteria Approximately 50 of the hyaluronic acid in thehuman body is found in the skin though it is rich in articularspaces as well As such organisms able to cleave hyaluronicacid may be favored as agents of infection in these locationsaided by their enhanced ability to penetrate these spaces

Results of our investigation show that all tested clinicalisolates ofP acnes have the described hyaluronidase gene andmost have the ability to cleave hyaluronic acid Organismsthat tested positive by PCR but which were unable to cleavehyaluronic acid may have mutations in the hyaluronidasegene or its regulators The ubiquity of the amplified geneticsequence is noteworthy as it suggests that the hyaluronidaseis important to the core genome of P acnes The widespreadpresence of this gene inP acnesmakes it appealing to considerit as a potential genetic target for detection of P acnes inclinical specimens [11 12]

The nearly universal phenotypic ability to cleavehyaluronic acid is notable not only as a potential functionalvirulence mechanism but also as a potential tool fordetermining whether P acnes isolated from a clinicalspecimen is a contaminant or a pathogen given the results ofthe time-course studies in liquid medium the enzyme mayonly be present in clinical tissue specimens in true infectionsIn our experiments using liquid media hyaluronidaseproduction was detected by approximately 96 hours ofanaerobic growth This contrasted with the observed 24 to72 hours it took for P acnes to produce detectable quantities

of hyaluronidase using the plate method The reason for thedifference in time to detectable hyaluronidase productionmay be attributable to the lower inoculum used in the studiesin liquid compared to solid media Alternatively it may bea consequence of the density of growth which would behigher sooner on plates than in liquid medium In clinicalspecimens collected from discrete physiologic spaces fromwhich P acnes is isolated the presence of hyaluronidasemay hypothetically support the sustained presence andgrowth of P acnes in that site which would not be expectedto be the case when the P acnes isolated is a contaminantsince a newly introduced contaminant would not havehad sufficient time to accumulate detectable quantities ofhyaluronidase This may provide a tool for differentiatingbetween contamination and sustained growth even if noliving organism is isolated from a specimen Of note otherorganisms are known to produce hyaluronidase though theirrequisite growth conditions for hyaluronidase productionand kinetics thereof are outside the scope of this paper

Demonstrating that an enzyme such as hyaluronidase canbe detected as a byproduct of P acnes growth is a novelpotential approach to detecting and characterizing infectionsand could set precedent for using byproducts of growthas a means of differentiating between contamination andsustained growth of this organism and others in clinicalspecimens Describing the secreted and excreted productsassociated with bacterial growth has potential diagnosticimplications not only for P acnes but also for many organ-isms The P acnes secretome has been described and 20proteins have been identified though not all have beencharacterized Exactly how unique each organismrsquos secretomeis is unknown however it is likely that the secretome variesfrom organism to organism and could potentially be usedas a ldquofingerprintrdquo of growth to signify specific organism-types in clinical specimens even in the case of nonculturableorganisms Given a lack of information on the secretomeof common pathogens a discussion of the broader appli-cation of using secreted or excreted proteins for detectingand characterizing bacterial growth from clinical specimensremains academic aside from the established use of ureaseproduction for diagnosing Helicobacter pylori infection Thismay represent an approach to more broadly detect andcharacterize infection particularly in cases where there is aquestion of potential contamination

The environment of the articular space has been a subjectof debate and the question has been raised over time asto what oxygen tension is present in the joint space It hasbeen suggested that articular spaces may have lower oxygencontent than other locations in the body [13]P acnes typicallygrows better under anaerobic than aerobic conditions Ourobservation that P acnes hyaluronidase is produced underanaerobic but not aerobic conditions (Figure 4) may haveclinical relevance in the context of the environment in jointspaces and possibly in biofilms

There are several limitations to our study In cases wherethe gene segment of interest was present but there wasphenotypic negativity we did not sequence the gene Alsoquantification of hyaluronidase production was performed


on just three isolates Further characterization of moreisolates under a variety of conditions might be informative

5 Conclusions

Most clinical isolates of P acnes are capable of cleavinghyaluronic acid and all tested clinical isolates of P acneshad a hyaluronidase gene The ubiquity of this characteristicin P acnes isolates and the finding that the studied subsetof isolates of P acnes produced detectable quantities ofhyaluronidase by 96 hours of sustained growth could hypo-thetically have diagnostic utility in differentiating betweencontamination with P acnes and its presence as a pathogen inclinical specimens In vivo and clinical studieswould be usefulto characterize the translation of our findings and to explorethe use of excreted proteins in characterizing the significanceof bacterial growth

Disclaimer

Thecontent is solely the responsibility of the authors and doesnot necessarily represent the official views of the NationalInstitute of Health

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Research reported in this publication was supported by theNational Institute of Arthritis and Musculoskeletal and SkinDiseases of the National Institute of Health under Awardno R01 AR056647 and the National Institute of Allergy andInfectious Diseases under Award no R01 A1091594

References

[1] S M Butler-Wu E M Burns P S Pottinger et al ldquoOptimiza-tion of periprosthetic culture for diagnosis ofPropionibacteriumacnes prosthetic joint infectionrdquo Journal of Clinical Microbiol-ogy vol 49 no 7 pp 2490ndash2495 2011

[2] S K Shannon J Mandrekar D R Gustafson et al ldquoAnaerobicthioglycolate broth culture for recovery of Propionibacteriumacnes from shoulder tissue and fluid specimensrdquo Journal ofClinical Microbiology vol 51 no 2 pp 731ndash732 2013

[3] D R Osmon E F Berbari A R Berendt et al ldquoDiagnosisand management of prosthetic joint infection clinical practiceguidelines by the infectious diseases Society of AmericardquoClinical Infectious Diseases vol 56 no 1 p -e25 2013

[4] C Holland T N Mak U Zimny-Arndt et al ldquoProteomicidentification of secreted proteins of Propionibacterium acnesrdquoBMCMicrobiology vol 10 article 230 2010

[5] R F Smith and N P Willett ldquoRapid plate method for screeninghyaluronidase and chondroitin sulfatase-producing microor-ganismsrdquo Applied Microbiology vol 16 no 9 pp 1434ndash14361968

[6] U Hoeffler ldquoEnzymatic and hemolytic properties of Propi-onibacterium acnes and related bacteriardquo Journal of ClinicalMicrobiology vol 6 no 6 pp 555ndash558 1977

[7] W L Hynes and S L Walton ldquoHyaluronidases of Gram-positive bacteriardquo FEMSMicrobiology Letters vol 183 no 2 pp201ndash207 2000

[8] C Cazanave K E Greenwood-Quaintance A D Hanssen etal ldquoRapidmolecularmicrobiologic diagnosis of prosthetic jointinfectionrdquo Journal of Clinical Microbiology vol 51 no 7 pp2280ndash2287 2013

[9] B Steiner S Romero-Steiner D Cruce and R GeorgeldquoCloning and sequencing of the hyaluronate lyase gene fromPropionibacterium acnesrdquo Canadian Journal of Microbiologyvol 43 no 4 pp 315ndash321 1997

[10] S Tolksdorf and M H McCready ldquoThe turbidimetric assay ofhyaluronidaserdquoThe Journal of Laboratory andClinicalMedicinevol 34 no 1 pp 74ndash89 1949

[11] P Schafer B Fink D Sandow A Margull I Berger andL Frommelt ldquoProlonged bacterial culture to identify lateperiprosthetic joint infection a promising strategyrdquo ClinicalInfectious Diseases vol 47 no 11 pp 1403ndash1409 2008

[12] Y Achermann M Vogt M Leimig J Wust and A TrampuzldquoImproved diagnosis of periprosthetic joint infection by multi-plex PCR of sonication fluid from removed implantsrdquo Journalof Clinical Microbiology vol 48 no 4 pp 1208ndash1214 2010

[13] K Lund-Olesen ldquoOxygen tension in synovial fluidsrdquo Arthritisand Rheumatism vol 13 no 6 pp 769ndash776 1970

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International Journal of

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Zoology


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BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


a component of the extracellular matrix Hyaluronic acid ispresent in tissues throughout the body including bones andjoints contributing to the viscosity of synovial fluid and thelubrication of joints It is also a part of the extracellular struc-ture of chondrocytes involved in tissue healing The ability ofbacteria to degrade hyaluronic acidmay be a virulence factorenabling penetration of hyaluronidase-producing organismsinto tissues rich in hyaluronic acid including joints creatingan advantage for establishing growth in articular spaces Inaddition to P acnes other Gram-positive pathogens such asStreptococcus pneumoniae Streptococcus intermedius Strepto-coccus constellatus Streptococcus dysgalactiae Staphylococcusaureus Peptostreptococcus species Clostridium perfringensandClostridium septicum as well as other species of Propioni-bacterium including P granulosum produce hyaluronidase[7]

Herein we defined the prevalence of a hyaluronidase geneand of hyaluronidase production in a collection of clinicalisolates of P acnes We also defined the time course of in vitrohyaluronidase production by P acnes using a subset of threeclinical isolates

2 Methods

21 P acnes Isolate Collection 197 clinical isolates of P acneswere studied including isolates associated with clinicallysignificant monomicrobial infections such as PJI spineinfections and endocarditis as well as isolates from clinicalspecimens with polymicrobial growth and isolates represent-ing contamination (Table 1) Of the 197 isolates 71 were asso-ciated with clinically significant growth defined as growthof ge20 colony forming units10mL sonicate fluid [8] or ge3positive cultures All 197 isolates underwentmolecular testingfor a P acnes hyaluronidase gene and phenotypic testingfor hyaluronidase production three isolates underwent time-course studies for assessment of kinetics of hyaluronidaseproduction

22 Genotypic Detection of Hyaluronidase Gene P acnesisolates were grown under anaerobic conditions in 2mLbrain heart infusion broth supplemented with 1 glucosewith agitation DNA was extracted using the QIAamp UCPPathogen Mini Kit (QIAGEN Valencia CA) with a finalelution into 50 120583L elution buffer A 451-base-pair regionof the hyaluronidase gene sequence [9] was targeted bypolymerase chain reaction (PCR) primers forward primer51015840-TTTCGGGATCCTTGTGGTA-31015840 and reverse primer51015840-TCTGGACAACAAACCTGT-31015840 Primer sequenceswere selected from the published P acnes hyaluronidasegene sequence [9] Basic Local Assignment Search Tool(BLAST) analysis showed that the primer sequences hadcomplete matches only with the two published P acneshyaluronidase gene sequences 5120583L of isolated DNA wasamplified using 42 cycles as follows melting at 95∘C for 30seconds annealing at 54∘C for 60 seconds and amplifyingat 72∘C for 120 seconds The final product was run on a 1agarose gel and the product identified based on amplifiedproduct size Amplified DNA from a representative isolate

was sequenced and the following sequence was obtainedlsquoCGCCCGCCGTGTGCTGTAGGTCTTCGGGGCTGG-GGTCAGGGTTGGTGCTGGTTGTTATGAAGGGCG-AGTGCGCCTTGCGGGGGTCTGCCGAGATCAGAA-CGGTCTGACGGCTCGAGCCGGATACGAGGTCGG-TGAGGATCTCGGCGACCTTCCGGTCGGTCTTGG-CGATGATCGCTCGGGCACGTGGGTCAGAGGTCC-GGGCGGCTCTGCGTCCGGTGATGATGTCGATCC-ATCGCTCGCACAGCGCCGACCAGCTGTCGGGCC-CGGGGGCGGCAATGGCGTCCGTCACTGTGGGAG-AAGCGGCGAGCGCCATGGCTGACAGCGCAGATG-CGGTGAGGAAGGTTCGTCGAGATGGGGTGCCGA-ACAAGGGTCACGCTCCTCGGGAGAGGGTGGCAG-CACTGCCACACGTACAGGTTTGTTGTCCAGA-31015840consistent with the P acnes hyaluronidase gene

23 Phenotypic Detection of Hyaluronidase ProductionHyaluronidase production was evaluated as described bySmith and Willett [5] and used as a binary determinant ofthe ability of each isolate to produce hyaluronic acid Briefly100mL brain heart infusion media was prepared with 1 gof agar autoclaved at 121∘C for 15min and cooled to 46∘Cthen a 2mgmL solution of sodium hyaluronidase boiledfor 10 minutes to achieve solubility was added to the cooledmedium to a final concentration of 400120583gmL 5 bovinealbumin fraction 119881 was added under constant stirring toyield a final concentration of 1 Plates were poured to adepth of 3-4mm and allowed to solidify at 4∘C P acnesisolates were grown on sheep blood agar under anaerobicconditions until growth was observed A single colony wasstreaked onto the hyaluronic acid medium incubated underanaerobic conditions and observed daily until growth wasobserved (24 to 72 hours) On the day growth was firstobserved the plate was flooded with 2N glacial acetic acidwhich binds hyaluronic acid and albumin forming a whiteprecipitate Hyaluronidase production was considered tohave been present if a zone of clearing was observed

24 Time Course Studies of Hyaluronidase Production Themethod was modified from that described by Tolksdorf andMcCready [10] The assay measures turbidity resulting fromhyaluronic acid mixed with albumin in an acidic solutionhyaluronidase activity is measured as lack of turbidity Stan-dard curves of hyaluronic acid turbidity and hyaluronidaseactivity were constructed Hyaluronic acid sodium salt fromStreptococcus equi (Sigma St Louis MO) was used for con-structing the standard curve and for experimental readingsand hyaluronidase from bovine testes (Sigma) was used todefine a standard curve of enzyme concentrationactivity(Figures 1(a) and 1(b)) Based on these standardized curvesthe limit of detection was 00125120583g120583L hyaluronidase andthe assay was fully saturated at 005 120583g120583L hyaluronidase(Figure 1(b))

To quantify production of hyaluronidase over time threeclinical isolates of P acneswere grown anaerobically on sheepblood agar for two days One was selected based on its lackof production of hyaluronidase using the plate method Theother two produced hyaluronidase as determined using the




0

005

01

015

02

025

03

035

0 01 02 03 04

OD540


(a)

0

005

01

015

02

025

03

035

04

0 20 40 60 80

OD540


(b)



540reading







3 Results




0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540


IDRL-7751IDRL-7844





4 Discussion


Neg

ativ

e con

trol1 2 3 4 5 6 7 8 9 10

minus500bp

minus100 bp

100

bpla

dder

minus1000 bp






0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540















5 Conclusions


Disclaimer




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




0

005

01

015

02

025

03

035

0 01 02 03 04

OD540


(a)

0

005

01

015

02

025

03

035

04

0 20 40 60 80

OD540


(b)



540reading







3 Results




0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540


IDRL-7751IDRL-7844





4 Discussion


Neg

ativ

e con

trol1 2 3 4 5 6 7 8 9 10

minus500bp

minus100 bp

100

bpla

dder

minus1000 bp






0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540















5 Conclusions


Disclaimer




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540


IDRL-7751IDRL-7844





4 Discussion


Neg

ativ

e con

trol1 2 3 4 5 6 7 8 9 10

minus500bp

minus100 bp

100

bpla

dder

minus1000 bp






0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540















5 Conclusions


Disclaimer




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

005

01

015

02

025

03

6 12 24 48 72 96 120 144Time (hours)

OD540















5 Conclusions


Disclaimer




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions


Disclaimer




Acknowledgments


References





















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article hyaluronidase in clinical isolates of

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