Antifungal susceptibility of 175 Aspergillus isolates from various clinical and environmental sources

17 Pages·2016·0.42 MB·English

MedicalMycology,2016,54,740–756
doi:10.1093/mmy/myw024
AdvanceAccessPublicationDate:3May2016
OriginalArticle
Original Article
Antifungal susceptibility of 175 Aspergillus
isolates from various clinical and environmental
sources
D
o
w
Raquel Sabino1,∗, Elisabete Carolino2, Cristina Ver´ıssimo1, n
lo
a
Marife Martinez3, Karl V. Clemons3,4 and David A. Stevens3,4 d
e
d
1NationalInstituteofHealthDr.RicardoJorge–URSZ-InfectiousDiseasesDepartment,Lisbon,Portugal, from
2ScientificAreaofMathematics,LisbonSchoolofHealthTechnology–PolytechnicInstituteofLisbon, http
Lisbon,Portugal,3CaliforniaInstituteforMedicalResearch,SanJose,CA,UnitedStatesand4Stanford s://a
UniversitySchoolofMedicine,Stanford,CA,UnitedStates c
a
d
e
∗Towhomcorrespondenceshouldbeaddressed.RaquelSabino,NationalInstituteofHealthDr-RicardoJorge,Av-Padre m
ic
Cruz,1649-016Lisboa,Portugal.Tel:[phone];Fax:[phone];E-mail:[email protected]; .o
u
[email protected] p.c
o
m
Received16October2015;Revised26January2016;Accepted21February2016
/m
m
y
Abstract /a
rtic
Some environmental Aspergillus spp. isolates have been described as resistant to anti- le
-a
fungals, potentially causing an emerging medical problem. In the present work, the an- bs
tifungalsusceptibilityprofileof41clinicaland134environmentalisolatesofAspergillus tra
c
wasdeterminedusingtheCLSImicrodilutionmethod.Theaimofthisstudywastocom- t/54
/7
pare environmental and clinical isolates with respect to their susceptibility, and assess /7
4
thepotentialimplicationsfortherapyofisolatesencounteredindifferentenvironments. 0/2
2
To our knowledge, this is the first report comparing antifungal susceptibility profiles of 2
2
5
Aspergillus collected from different environmental sources (poultries, swineries, beach 7
2
sand,andhospitalenvironment).Significantdifferenceswerefoundinthedistributionof b
y
g
thedifferentspeciessectionsforthedifferentsources.Significantdifferenceswerealso u
e
s
foundinthesusceptibilityprofileofthedifferentAspergillussectionsrecoveredfromthe t o
n
various sources. Clear differences were found between the susceptibility of clinical and 0
5
environmental isolates for caspofungin, amphotericin B and posaconazole, with clinical A
p
isolatesshowingoverallgreatersusceptibility,exceptforcaspofungin.Incomparisonto ril 2
clinical isolates, hospital environmental isolates showed significantly less susceptibility 01
9
to amphotericin B and posaconazole. These data indicate that species section identity
andthesitefromwhichtheisolatewasrecoveredinfluencetheantifungalsusceptibility
profile,whichmayaffectinitialantifungalchoices.
Keywords:Aspergillus,antifungalsusceptibility,clinical,environment.
Introduction ity and mortality, especially of solid organ transplant pa-
tients or recipients of allogeneic hematopoietic stem cell
Aspergillus is one of the major fungal threats to im-
transplantation. To exacerbate this problem, intrinsic and
munocompromised patients,1 causing significant morbid-
740 (cid:3)C TheAuthor2016.PublishedbyOxfordUniversityPressonbehalfofTheInternationalSocietyforHumanandAnimal
Mycology.Allrightsreserved.Forpermissions,pleasee-mail:[email protected]
Sabinoetal. 741
acquiredresistancehavebeendocumentedandthenumber The goal of the current study was to determine the an-
of clinical strains resistant to the most common antifun- tifungalsusceptibilityprofileof175isolatesofAspergillus
galsisincreasingrapidlyinsomegeographicareas.2Recent identified as belonging to different sections and collected
observationssuggestthatresistancetotriazolesamongAs- from different clinical and environmental sources. Thus,
pergillus spp. may be more common than previously ac- this study aims to contribute to the knowledge of the epi-
knowledged;3 most resistant isolates harbor azole cross- demiology of antifungal resistance patterns of isolates of
resistance.4 Aspergillus collected from different sources and how the
Variableantifungalsusceptibilityprofilesamongspecies environmentalisolatescouldinfluencetheoverallsituation
have been reported,5 and antifungal resistance in As- ofantifungalresistancebyAspergillusspp.
pergillus strains tends to emerge with varying frequencies
invariouscentersandcountries,especiallyinEurope.6 Re-
Materials and methods
ducedsusceptibilityofstrainstoantifungalshasbeenshown D
o
toberelatedtohighermortalityratesinmurinetreatment Aspergillusisolates w
n
modelsand,presumably,inclinicaltreatment.7,8 Atotalof175isolatesofvariousspeciesofAspergilluswere loa
d
Exposure of Aspergillus spp. to antifungal agents via testedfortheirsusceptibilitytofiveantifungaldrugsinthis ed
medicaloragriculturaluseofthesecompoundsappearsto study.Ofthese,41werefromclinicalsources,and134were fro
m
haveamajorimpactonacquisitionofresistancetotriazoles. collectedfromenvironmentalsourcesinPortugal.Clinical h
Azolefungicidesareusedcommonlyforplantprotectionby isolates(oneisolateperpatient)included20frombronchial ttps
the European Union member states. Apart from being se- secretions or sputum, seven from bronchoalveolar lavage ://ac
a
lected due to local use of azole fungicides, azole-resistant (BAL), seven from auricular exudates, two from biopsies de
m
Aspergillus spp. might be introduced in the environment (skullandpalate),onefromascitesfluid,onefromstool,one ic
viatheuseofimportedazole-treatedcommercialcompost, fromnasalsinusaspirate,andtwofromunknownsources. .ou
p
an ecological niche that has been suggested as a key com- Environmental isolates included 36 from sand, 39 from .co
m
ponent in resistance development.9 Moreover, Aspergillus poultries(air,surfacesandlitter)and29fromswineries(air, /m
isolates are frequently recovered from poultry farms; in surfaces,andlitter),23fromhospitalenvironments(airand my
these settings, fungi can be exposed to antifungal drugs surfaces),andsevenfromothersources(food,stones). /artic
regularly used for the prevention of avian mycosis. The Identificationofthespeciessectionswasbasedonmor- le
-a
consequence of development of resistance is that patients phological observation, achieved through macro- and mi- bs
at risk can be exposed to and infected by azole-resistant croscopic characteristics. Identification to species section trac
strains from the environment, which is one of the biggest level of the 175 isolates was confirmed by sequencing the t/54
recent concerns of the mycological community. Regard- ITS1-5.8S-ITS2 region of the ribosomal DNA after PCR /7/7
ing other classes of antifungals, only sporadic resistance amplificationusingtheuniversalfungalprimers(ITS1and 40
/2
has been detected, although innate amphotericin B resis- ITS4).18 For the Fumigati section (48 isolates), identifica- 22
2
tanceofAspergillusterreus10,11andAspergillusnidulans12 tiontospecieswasdonebysequencingthecalmodulingene, 57
2
iscommon. as described previously.17 In both cases, the obtained se- b
y
The majority of studies on Aspergillus spp. azole- quences were accepted only in case of ≥98% homology gu
e
resistance have been focused on isolates from the As- with sequences deposited in the databases (National Cen- st o
pergillusfumigatussection,Fumigati,whereasfewdataare terforBiotechnologyInformationandCBS-KNAWFungal n 0
available on antifungal susceptibility patterns of the other BiodiversityCentre)thatwereBLASTsearched. 5 A
sinecetniovnirso.1n3m–1e5nFtualrtihsoelramteosreh,atshbeedeentepcetriofonromfeadzobluet-rpersiimstaarniclye pril 20
Antifungalagents 1
focusedontheFumigatisection.Nativeresistancetocertain 9
antifungalsreinforcestheimportanceofknowingthedistri- The antifungal agents studied were caspofungin (CAS;
butionofantifungalsusceptibilitiesofdifferentspeciesfrom Merck & Co. Inc, Whitehouse Station, NJ, USA),
differentenvironments.16Thisgivesinsightintowhetheran amphotericin B (AMB; Bristol-Myers Squibb Co., Prince-
isolateofAspergillusthatonemayencounterinvariousen- ton, NJ, USA), itraconazole (ITC; Janssen Pharmaceutica
vironments is more or less likely to be drug-susceptible or N.V., Beerse, Belgium), voriconazole (VRC; Roerig Divi-
resistant, and may also suggest where environmental ma- sion of Pfizer Inc., NY, USA), and posaconazole (PCZ;
nipulations(e.g.,useofchemicals)haveledtoalterationsin Schering Corporation, Kenilworth, NJ, USA). They were
the susceptibility of the local flora. Moreover, a profile of obtainedaspowders,andstocksolutionswerepreparedin
resistanceinvariousenvironmentsmaybeusefulinguiding 100%dimethylsulfoxideforazolesandAMBorinsteril-
immunocompromised patients with respect to places they izedwaterforCAS.Stocksolutionsofeachdrugwerekept
mightparticularlyavoid. at−70◦C.
742 MedicalMycology,2016,Vol.54,No.7
Antifungalsusceptibilitytesting ysis.Geometricmeans(basedonlog transformations),me-
2
dian,andrangesweredeterminedusingExcelforWindows
Isolates were subcultured on potato dextrose agar slants
XP.
(Becton,DickinsonandCompany,SparksMD,USA)for5
to7daysatroomtemperature.Theantifungalsusceptibil-
ityprofilesweredeterminedusingabrothdilutionreference
Results
methodforsusceptibilitytestingofmoulds.TheM38-A218
The distribution of clinical and environmental isolates is
protocol from the Clinical and Laboratory Standards In-
detailedinTable1.
stitutewasappliedfordeterminingtheMinimalInhibitory
Concentrations(MIC)forAMB,VRC,ITC,PCZ,andthe
Minimal Effective Concentration (MEC) for CAS. The fi-
Comparisonofantifungalsusceptibilityofclinical
nalconcentrationsofdrugsinthewellsrangedfrom0.5to
andenvironmentalisolates
8μg/ml(ITC),0.25to8μg/ml(PCZandVRC),0.125to D
o
8 μg/ml (AMB), and 0.39 to 50 μg/ml (CAS). An internal The probability of encountering resistance as a dependent wn
controlstrain(CandidakefyrstrainSA)withknownsuscep- variable of the source of the isolate was studied. The me- load
tpiobsiliittiyveacnodnMtroElCo/fMthIeCarnatnifguensgwalass’pinoctleundceyd.Sinabeoauchraruudndaesxa- deaiacnhaanntdifugenogmaleatrgiecnmtaegaanin(sGtMth)eMcliInCicaolroMreEnCvirvoanlumeesnftoarl ed from
tMtrhoeDsne,uUamgSaAbre)prwloaeftreecsoin(lBooneccyut-lofaontre,mdDwiincigkthiuntnshoitensfiiannnadtlhiCenooincmoupcluuamnluymt,oS.1cp8haerckks isshoolAawtnneasli,ynwsTisitahboolfeut2thr.eegasurdscteopttihbeiliirtyspepchieensootfypAesspebrgyillMusa,nanr–e https://ac
a
Plateswereincubatedat35◦Candexaminedafter48h WhitneyUtestdemonstratedcleardifferencesbetweenclin- de
m
incubation.Absenceofvisualgrowth(forAMB,VRC,ITC, icalandenvironmentalisolates.ForCAS,asignificantdif- ic
andPCZ)oraberrantgrowth(forCAS)definedtheMICor ference (P = .047) was found between the MECs of clini- .oup
calandenvironmentalisolates,withclinicalisolateshaving .c
MEC,respectively.Incaseofupperendofrangeofvalues o
m
ofMECorMIC,thetestswereperformedasecondtimeto highervalues.AlthoughtheMECrangeswereidentical,the /m
geometric mean of clinical isolates was three times higher m
confirm the previous results. A selected sample of isolates y
wbyasotneestdeidluttoioanssoesrslreesps.roducibility,andalltheresultsvaried t2lah.t6aensμght/hamdalt,MoreEfsCptehcvetailveuenelvysi)rf.ooTnrmwCeeAnnSttya-l>seisv6oe.3lnatμ(e6sg6/m%(6l.),3wcμlihgne/imrcealalsais5no4d- /article-ab
s
(40%) environmental isolates had MEC values above this tra
c
value. When different environmental sources were com- t/5
Statisticalanalysis pared,theMECrangeswerethesameforCAS. 4/7
/7
4
Comparisons of MIC were done among the various clini- Significant differences were found between MICs of 0
calandenvironmentalgroupsofisolates,aswellasspecies AMB for clinical and environmental sources (P < .001); /22
2
2
section,usingnonparametricstatisticalmethods.AMann– environmental strains were less susceptible. Their MICs 5
7
Whitney U test was used to compare the antifungal drug rangeduptoMIC>8μg/mlandthegeometricmeanwas 2 b
y
MICs,19 regardless of Aspergillus species, between two higherthatoftheclinicalstrains(2.2μg/mlvs.1.5μg/ml). g
u
groups: clinical and environmental isolates (all environ- No clinical isolates had MICs > 2 μg/ml to AMB, as es
t o
mental sources were considered as only one group). A opposed to 31 (23%) environmental isolates with MICs n
0
Kruskal–Wallis test19 was used to compare the MICs for >2μg/ml. 5
A
espacehcieasn-tsiefcutniognasl.aImfosniggndifiifcfaenretndtisffoeurrecnecseosrwaemreonfogudnidffewreitnht comNpoarsiinggnitfihceacnltindicifafleraenndceesnv(Piro>nm.0e5n)tawlegrreoufopusnfdorwthheeinr pril 20
1
the initial Kruskal–Wallis comparison, all pairs were then susceptibilitytoITCorVRC.Despitetheslightdifferences 9
compared using a Kruskal–Wallis multiple comparison intheMICranges,thegeometricmeanvaluesweresimilar.
test.19Agoodnessoffitχ2forhomogeneitywasusedtode- For ITC, 21 (51%) clinical isolates and 72 (54%) of the
terminewhetherthedistributionofthedifferentspeciessec- environmentalisolateshadanMIC>2μg/ml.ForVRC,no
tions (e.g., Fumigati, Flavi, Circumdati, Versicolores, and clinicalisolateshowedaMICabove1μg/ml,andonlythree
Nigri)ineachofthesourceswasskewed.Whentheassump- environmentalisolatesshowedaMICvalueof4μg/ml.
tionsofχ2 testwereviolated,theContingencyChi-square ForPCZ,asignificantdifference(P<.001)wasfound
test by Monte Carlo simulation was used.19 A .05 signifi- betweentheMICsfromclinicalorenvironmentalsources.
cancelevelwassettoattainsignificance.ThesoftwareSPSS The median and the geometric mean of environmental
(Statistic Package for Social Sciences) V21.0 (IBM Corp., strains had higher values. No isolates, other than four en-
Armonk,NY,USA)wasusedtoperformthestatisticalanal- vironmental ones, had MICs > 2 μg/ml to PCZ. Those
Sabinoetal. 743
Table1.MolecularidentificationandantifungalsusceptibilityoftheAspergillusisolatescollectedfromdifferentclinicaland
environmentalsources.
Source Sections MECCAS MICAMB MICICZ MICVCZ MICPCZ
(ITSsequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)
Sputum Fumigati 25 2 4 ≤0.25 ≤0.25
Sputum Fumigati 25 2 2 ≤0.25 0.5
Bronchialsecretions Fumigati 25 1 2 ≤0.25 ≤0.25
Bronchialsecretions Fumigati 25 1 2 ≤0.25 ≤0.25
Sputum Fumigati ≤0.39 1 4 ≤0.25 ≤0.25
Bronchoalveolarlavage Fumigati 25 1 4 ≤0.25 ≤0.25
Bronchoalveolarlavage Flavi ≥50 2 2 0.5 0.5
Bronchoalveolarlavage Flavi ≤0.39 1 2 ≤0.25 1 D
Sputum Flavi ≥50 2 2 ≤0.25 0.5 ow
n
Sputum Nigri ≤0.39 1 4 ≤0.25 1 lo
a
Notreferred Flavi 25 2 2 ≤0.25 0.5 d
e
BStrooonlchialsecretions NFliagvrii ≤≥05.309 12 42 00..55 01.5 d from
BAruorniccuhloaarlevxeouldaartelavage FFuummiiggaattii 2255 11 44 ≤00..525 ≤00..525 https
Sputum Nigri ≤0.39 1 4 ≤0.25 1 ://a
c
Auricularpus Fumigati 1.6 2 4 ≤0.25 ≤0.25 ad
e
Bronchialsecretions Fumigati 25 2 2 ≤0.25 ≤0.25 m
ic
Sputum Fumigati 12.5 2 4 ≤0.25 ≤0.25 .o
u
Sputum Fumigati 12.5 2 4 0.5 0.5 p
.c
Biopsypalate Fumigati 12.5 2 4 ≤0.25 ≤0.25 om
Bronchoalveolarlavage Versicolores 25 1 2 ≤0.25 0.5 /m
m
Sputum Fumigati ≤0.39 2 4 ≤0.25 0.5 y
BSprountucmhialsecretions FFuummiiggaattii 2255 22 14 ≤≤00..2255 ≤≤00..2255 /article
Bronchoalveolarlavage Fumigati 12.5 1 4 0.5 0.5 -ab
s
Sputum Fumigati 25 2 2 0.5 1 tra
Asciticfluid Fumigati 25 2 4 ≤0.25 ≤0.25 ct/5
Earexudate Flavi 25 2 2 ≤0.25 0.5 4/7
Bonebiopsy Flavi ≥50 2 2 0.5 0.5 /7
4
Bronchialsecretions Flavi ≥50 2 2 0.5 0.5 0/2
Notreferred Flavi ≤0.39 2 2 0.5 0.5 22
2
Earexudate Flavi ≤0.39 1 1 ≤0.25 ≤0.25 5
7
Bronchialsecretions Nigri ≤0.39 1 8 1 2 2 b
Nasalsinusaspirate Flavi ≤0.39 2 2 ≤0.25 0.5 y g
u
Bronchoalveolarlavage Fumigati 12.5 2 4 1 0.5 e
s
Earexudate Nigri ≤0.39 1 4 ≤0.25 1 t o
n
Earexudate Nigri ≤0.39 1 4 ≤0.25 1 0
5
Earexudate Nigri ≤0.39 2 8 1 2 A
p
BSprountucmhialsecretions FFuummiiggaattii 1≥25.05 22 12 ≤00..525 ≤≤00..2255 ril 20
1
Beachsand Fumigati 25 2 2 1 1 9
Beachsand Nigri ≤0.39 1 >8 0.5 2
Beachsand Nigri ≤0.39 1 4 ≤0.25 1
Beachsand Versicolores 3.1 4 4 1 1
Beachsand Circumdati ≤0.39 8 2 ≤0.25 2
Beachsand Versicolores ≤0.39 2 4 ≤0.25 1
Beachsand Circumdati 25 2 4 0.5 1
Beachsand Circumdati ≤0.39 >8 2 ≤0.25 ≤0.25
Beachsand Circumdati 0.78 >8 4 ≤0.25 1
Beachsand Nigri ≤0.39 1 4 0.5 1
Beachsand Nigri ≤0.39 1 4 ≤0.25 1
Beachsand Versicolores 25 2 2 ≤0.25 0.5
744 MedicalMycology,2016,Vol.54,No.7
Table1– continued
Source Sections MECCAS MICAMB MICICZ MICVCZ MICPCZ
(ITSsequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)
Beachsand Circumdati ≤0.39 >8 4 0.5 1
Beachsand Fumigati 25 2 2 0.5 0.5
Beachsand Fumigati 25 2 4 0.5 1
Beachsand Nigri ≤0.39 1 4 ≤0.25 1
Beachsand Fumigati 12.5 2 2 0.5 0.5
Beachsand Nigri ≤0.39 1 4 ≤0.25 1
Beachsand Versicolores ≤0.39 2 4 ≤0.25 1
Beachsand Notidentified ≤0.39 2 >8 1 8
Beachsand Circumdati 0.78 >8 4 ≤0.25 1
D
Beachsand Fumigati 12.5 2 2 ≤0.25 ≤0.25 ow
n
Beachsand Fumigati 25 4 1 0.5 0.5 lo
Beachsand Fumigati 12.5 2 1 ≤0.25 0.5 ad
e
BBeeaacchhssaanndd CFuirmcuigmadtiati 12.56 >28 42 ≤00..525 01.5 d from
Beachsand Nigri ≤0.39 1 4 ≤0.25 1 h
Beachsand Circumdati 1.6 >8 4 ≤0.25 1 ttps
Beachsand Flavi ≥50 2 2 ≤0.25 0.5 ://a
c
Beachsand Notidentified 3.1 4 4 0.5 0.5 ad
e
Beachsand Fumigati ≤0.39 4 4 ≤0.25 0.5 m
ic
Beachsand Fumigati 25 2 2 0.5 0.5 .o
Beachsand Nigri ≤0.39 1 4 0.5 1 up
Beachsand Aspergilli ≤0.39 1 1 ≤0.25 ≤0.25 .co
m
Beachsand Circumdati 1.6 >8 4 ≤0.25 1 /m
Beachsand Nigri 12.5 1 4 ≤0.25 1 m
y
OOtthheerreennvviirroonnmmeennttaall FNuigmriigati ≤02.539 12 82 ≤01.25 ≤01.25 /article
Otherenvironmental Nigri ≤0.39 1 8 1 0.5 -a
b
Otherenvironmental Nigri ≤0.39 1 4 0.5 1 stra
OOtthheerreennvviirroonnmmeennttaall FFuummiiggaattii 1225.5 22 22 ≤00..525 ≤≤00..2255 ct/54
/7
Otherenvironmental Fumigati 12.5 2 2 ≤0.25 ≤0.25 /7
4
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5 0
/2
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5 2
2
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5 25
7
Poultry-Surface Flavi ≤0.39 2 2 ≤0.25 0.5 2 b
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5 y g
Poultry-Air Flavi ≥50 2 2 0.5 1 ue
s
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5 t o
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5 n 0
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5 5 A
PPoouullttrryy--ALiitrter FFllaavvii ≥≥5500 22 22 ≤≤00..2255 00..55 pril 20
1
Poultry-Litter Fumigati 12.5 2 2 ≤0.25 ≤0.25 9
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Litter Clavati ≤0.39 1 4 0.5 1
Poultry-Surface Versicolores 12.5 4 >8 4 2
Poultry-Surface Versicolores ≤0.39 4 4 0.5 1
Poultry-Litter Fumigati 12.5 2 2 ≤0.25 0.5
Poultry-Air Aspergilli ≤0.39 1 4 1 1
Poultry-Air Candidi ≤0.39 4 2 ≤0.25 1
Poultry-Air Versicolores ≤0.39 4 4 1 2
Poultry-Air Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Versicolores ≤0.39 4 >8 0.5 1
Sabinoetal. 745
Table1– continued
Source Sections MECCAS MICAMB MICICZ MICVCZ MICPCZ
(ITSsequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)
Poultry-Air Fumigati 12.5 2 2 ≤0.25 0.5
Poultry-Litter Fumigati ≤0.39 2 2 ≤0.25 0.5
Poultry-Surface Fumigati 25 2 2 ≤0.25 0.5
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5
Poultry-Air Aspergilli 3.1 2 >8 2 >8
Poultry-Air Versicolores ≤0.39 1 4 ≤0.25 1
Poultry-Air Usti ≤0.39 8 8 1 2
Poultry-Surface Versicolores ≤0.39 1 4 ≤0.25 1
Poultry-Litter Fumigati ≤0.39 2 2 ≤0.25 1
D
Poultry-Air Versicolores 0.78 2 4 ≤0.25 1 ow
Poultry-Litter Candidi ≤0.39 1 0.5 ≤0.25 ≤0.25 nlo
Poultry-Litter Fumigati ≤0.39 2 2 ≤0.25 ≤0.25 ad
e
PPoouullttrryy--SSuurrffaaccee NFuigmriigati ≤≤00..3399 21 28 ≤00..525 11 d from
Poultry-Litter Versicolores 25 2 4 ≤0.25 1 h
Poultry-Litter Candidi ≤0.39 1 0.5 ≤0.25 ≤0.25 ttps
Poultry-Surface Flavi ≥50 2 2 ≤0.25 0.5 ://a
c
Swinery-Air Flavi ≥50 2 2 ≤0.25 0.5 ad
e
Swinery-Surface Nigri ≤0.39 1 4 ≤0.25 1 m
Swinery-Air Fumigati 12.5 2 2 ≤0.25 0.5 ic.o
Swinery-Surface Candidi ≤0.39 2 2 ≤0.25 1 up
Swinery-Surface Circumdati ≤0.39 >8 4 ≤0.25 1 .co
m
Swinery-Surface Circumdati ≤0.39 8 4 ≤0.25 1 /m
Swinery-Air Candidi ≤0.39 2 2 ≤0.25 1 m
y
SSwwiinneerryy--ASuirrface FVuemrsiicgoaltoires ≤102..359 22 44 ≤00..525 11 /article
Swinery-Air Fumigati 25 2 2 ≤0.25 0.5 -a
b
Swinery-Air Fumigati 25 2 2 ≤0.25 1 stra
SSwwiinneerryy--SSuurrffaaccee NFliagvrii ≤≥05.309 21 28 ≤00..525 01.5 ct/54
/7
Swinery-Air Versicolores ≤0.39 2 4 ≤0.25 1 /7
4
Swinery-Surface Versicolores 1.6 4 4 ≤0.25 1 0
/2
Swinery-Air Flavi ≥50 2 2 ≤0.25 0.5 2
2
Swinery-Air Flavi 25 2 2 ≤0.25 0.5 25
7
Swinery-Air Usti 0.78 4 >8 2 >8 2 b
Swinery-Air Versicolores ≤0.39 4 4 ≤0.25 1 y g
Swinery-Air Clavati 12.5 ≤0.5 4 1 2 ue
s
Swinery-Air Flavi ≥50 2 2 ≤0.25 0.5 t o
Swinery-Air Candidi 1.6 1 1 ≤0.25 ≤0.25 n 0
Swinery-Surface Versicolores ≤0.39 2 4 ≤0.25 1 5 A
SSwwiinneerryy--SAuirrface VFlearvsiicolores ≤02.539 22 42 ≤≤00..2255 01.5 pril 20
1
Swinery-Air Nigri ≤0.39 1 8 ≤0.25 1 9
Swinery-Surface Usti ≤0.39 2 8 4 >8
Swinery-Surface Flavi ≥50 2 2 ≤0.25 0.5
Swinery-Surface Versicolores 12.5 4 4 ≤0.25 1
Hospitalenvironment-Air Nigri ≤0.39 1 4 ≤0.25 1
Hospitalenvironment-Surface Nigri ≤0.39 1 4 ≤0.25 1
Hospitalenvironment-Air Nigri ≤0.39 1 4 ≤0.25 1
Hospitalenvironment-Air Nigri ≤0.39 1 4 0.5 1
Hospitalenvironment-Air Flavi ≤0.39 2 2 0.5 0.5
746 MedicalMycology,2016,Vol.54,No.7
Table1– continued
Source Sections MECCAS MICAMB MICICZ MICVCZ MICPCZ
(ITSsequencing) (μg/ml) (μg/ml) (μg/ml) (μg/ml) (μg/ml)
Hospitalenvironment-Surface Flavi ≥50 2 2 0.5 1
Hospitalenvironment-Surface Flavi ≥50 2 2 0.5 1
Hospitalenvironment-Surface Flavi ≤0.39 2 2 0.5 1
Hospitalenvironment-Air Flavi ≥50 2 2 ≤0.25 0.5
Hospitalenvironment-Air Nidulantes ≤0.39 2 2 ≤0.25 0.5
Hospitalenvironment-Air Notidentified ≤0.39 4 4 0.5 0.5
Hospitalenvironment-Air Circumdati ≤0.39 2 4 0.5 1
Hospitalenvironment-Air Circumdati ≤0.39 2 4 ≤0.25 1
Hospitalenvironment-Air Versicolores ≤0.39 2 4 1 2
D
Hospitalenvironment-Air Aspergilli 1.6 4 8 0.5 1 ow
Hospitalenvironment-Air Circumdati 6.3 >8 4 ≤0.25 1 nlo
Hospitalenvironment-Air Circumdati ≤0.39 >8 4 ≤0.25 1 ad
e
HHoossppiittaalleennvviirroonnmmeenntt--ASuirrface CVeirrcsuicmoldoarteis 02.758 >48 >48 ≤04.25 12 d from
Hospitalenvironment-Air Versicolores ≤0.39 4 4 0.5 2 h
Hospitalenvironment-Surface Versicolores 6.3 2 4 0.5 1 ttps
Hospitalenvironment-Surface Versicolores ≤0.39 2 4 ≤0.25 0.5 ://a
c
Hospitalenvironment-Air Versicolores ≤0.39 2 4 0.5 1 ad
e
m
ic
.o
four isolates showed MICs ≥ 8 μg/ml. In addition, these ingly,12(33%)environmentalisolateswithMIC>2μg/ml up
same isolates had cross-resistance with ITC (MICs ≥ for AMB were collected from beach sand (Table S1, .co
m
8μg/ml);thesefourisolateswerecollectedfrombeachsand Table 2). For collections from both these environments, /m
m
(one isolate), poultry (one isolate), or swineries (two iso- theclinicalisolateshadlowerMICsforAMB. y
/a
lates). One isolate, collected from a swinery surface, also A significant difference was found for PCZ (χK2W(4)= rtic
showedareducedsusceptibilitytoVRC(MIC=4μg/ml) 30.857,P≤.001)whencomparingallthesources.Namely, le-a
b
(Table1). between clinical isolates and those from hospital environ- s
ment(P<.001),swineries(P<.001),orbeachsand(P= trac
t/5
.001).Inthesecomparisons,theenvironmentalisolateshad 4
/7
Comparisonofantifungalsusceptibilityof higherMICs(TableS1). /74
isolatescollectedfromeachsourceanalyzed No significant differences in MIC values were found 0/2
2
among the isolates from different specific sources for the 2
2
Because we had large numbers of isolates from distinct 5
antifungalsITCandVRC(datanotshown). 7
2
sources,wecomparedthesusceptibilityoftheisolatesfrom b
y
the various sources with each other. When comparing the g
u
Comparisonofantifungalsusceptibilityfrom e
MECsofCASforisolatescollectedamongstallthesources, s
significant differences were found (χ2 (4) = 9.510, strainsbelongingtodifferentspecies-sections, t on
P = .050), specifically between hospitalKenWvironmental (P regardlessofthesource 05
A
=isol.a0t0e8s.)Ionrbboetahchinsstaanndce(sP, c=lin.i0ca4l8)isocolamtepsasrheodwteodchliingihcearl DtheetseermstiundaiteisonwaosfsdpoenceiebsyseDctNioAnfsoeqrueeancchinogf.tTheheissoelaretesuslitns pril 20
1
MEC values (Table S1). Among environmental isolates, a showedthatninespeciessectionswererepresentedamong 9
significant difference was also found between hospital en- the175isolates.TheMICorMECvaluesforthemostrep-
vironmental and isolates from poultries (P = .023), with resentativespeciessections(i.e.,Fumigati,Flavi,Nigri,Ver-
the latter isolates having four-times higher MECs to CAS. sicolores,Circumdati)weredetermined.Thegroup“other
Twenty(51%)isolatescollectedfrompoultrieshadaMEC speciessections”includesthespeciessectionsUsti,Clavati,
above6.3μg/ml(TableS1). AspergilliandCandidi(Table3).Multiplecomparisontests
Significant differences were also found when compar- showedsignificantdifferencesamongtheMICorMECof
ing the MICs for AMB of isolates collected among all the thosesections(TableS2).
sources (χ2 (4)= 13.097, P = .011), especially between Regardless of the source, the sections Fumigati and
KW
clinicalisolatesandisolatesfromthehospitalenvironment Flavi presented higher MEC values for CAS than did the
(P=.029)orisolatesfrombeachsand(P=.049).Interest- other species sections (geometric mean of 10.4 μg/ml and
Sabinoetal. 747
Table2.Minimalinhibitoryconcentrations(MIC)orminimaleffectiveconcentrations(MEC)ofvariousantifungaldrugsagainst
clinicalandenvironmentalisolatesofAspergillus.
Clinical Hospital Poultries Swineries Beachsand Otherenvironments
(N=41) (N=23) (N=39) (N=29) (N=35) (N=8)
MEC(cid:2) orMICparameters CASMEC(μg/ml)
Median 25 0.39 12.50 1.60 1.19 18.75
Geometricmean 6.2 1.2 4.4 2.9 2.2 17.7
Range ≤0.39–>50 ≤0.39–>50 ≤0.39–>50 ≤0.39–>50 ≤0.39–>50 12.5–25
No.isolates(%)withMIC>6.3μg/ml 27(65.8) 4(17.4) 20(51.3) 13(44.8) 13(36.1) 4(100.0)
P-valueclinicalvs.environmentalisolates 0.047∗
AMBMIC(μg/ml)
D
Median 2 2 2 2 2 2 ow
n
Geometricmean 1.5 2.4 2.0 2.1 2.4 2.0 lo
Range 1–2 1–>8 1–8 ≤0.5–>8 1–>8 2 ad
e
NP-ov.ailsuoelactleisn(i%ca)lwvsi.thenMviIrCon>me2nμtagl/misollates 0(0.0) 7(30.4) 6(15.4) <0.600(12∗0.4) 12(33.3) 0(0.0) d from
ITCMIC(μg/ml) http
s
Median 4 4 2 4 4 2 ://a
Geometricmean 2.8 3.5 2.6 3.1 3.0 2.0 c
a
Range 1–8 2–>8 ≤0.5–>8 1–>8 1–>8 2 de
m
No.isolates(%)withMIC>2μg/ml 21(51.2) 17(73.9) 13(33.3) 16(55.2) 23(63.9) 0(0.0) ic
.o
P-valueclinicalvs.environmentalisolates 0.4 u
p
VRCMIC(μg/ml) .co
m
Median 0.3 0.5 0.25 0.25 0.25 0.25 /m
m
Geometricmean 0.3 0.4 0.3 0.3 0.3 0.3 y
RNaon.igseolates(%)withMIC>2μg/ml ≤00(.205.0–)1 ≤10(.245.3–)4 ≤10(.225.6–)4 ≤10(.235.4–)4 ≤00(.205.0–)1 ≤00.2(05.–00).5 /article
P-valueclinicalvs.environmentalisolates 0.8 -ab
s
PCZMIC(μg/ml) tra
c
Median 0.5 1 0.5 1 1 0.25 t/5
4
Geometricmean 0.5 0.9 0.7 0.9 0.8 0.2 /7
/7
Range ≤0.25–2 0.5–2 ≤0.25–>8 ≤0.25–>8 ≤0.25–8 0.25 4
0
No.isolates(%)withMIC>2μg/ml 0(0.0) 0(0.0) 1(2.6) 2(6.9) 1(2.8) 0(0.0) /2
2
P-valueclinicalvs.environmentalisolates <0.001∗ 22
5
7
∗Statisticallysignificantdifferencesat5%significancelevelusingtheMann–WhitneyUtest. 2 b
(cid:2)MECappliesonlytocaspofungin. y g
u
e
s
19.5μg/ml,respectively;Table3).Thesevalueswere10or the other species sections (geometric mean = 3.0 μg/ml). t on
0
20timeshigherthanthoseobtainedwithisolatesbelonging Interestingly,27(100%)isolatesintheNigricomplexand 5
A
tootherspecies-sections.IsolatesfromtheCircumdaticom- 24 (92%) isolates in the Versicolores complex had MIC p
plex showed, on average, three-times higher median MIC values≥4μg/mlforITC(Table4).Theisolatesbelonging ril 2
0
valuestoAMB(geometricmean=6.2μg/ml)thantheoth- to Fumigati and Flavi presented the lowest MIC values to 19
ers species-sections (Table 3). Thirteen isolates (81%) in ITC (geometric mean = 2.4 and 2.0 μg/ml, respectively).
the Circumdati complex had a MIC to AMB ≥ 8 μg/ml For VRC, the Versicolores and Usti sections had isolates
(Table4).AMICvalueof8μg/mlforAMBwasdetected withthehighestMICvaluestoVRC(4μg/ml).Oneofthe
foronlyoneotherisolate,whichbelongedtotheUstisec- four isolates that presented a MIC value of ≥8 μg/ml for
tion.Thatisolatewascollectedfromtheairatonepoultry both ITC and PCZ could not be identified to the species
farm and showed a MIC value of 8 μg/ml also for ITC. levelbuttheotherthreewereidentifiedasbelongingtothe
The susceptibility to ITC differed among species sections, Usti and Aspergilli sections. Two other isolates, identified
with higher values for Nigri complex (geometric mean = as A. versicolor and A. sidowii, presented ITC MICs val-
4.9 μg/ml), followed by Versicolores (geometric mean = ues≥8μg/ml,togetherwithhighMICstoVRC(4μg/ml)
4.1μg/ml),Circumdati(geometricmean=3.7μg/ml)and (Table4).
748 MedicalMycology,2016,Vol.54,No.7
Table3.Distributionofminimalinhibitoryconcentrations(MIC)andminimaleffectiveconcentrations(MEC)(μg/ml)amongthe
differentAspergillussections.
Fumigati Flavi Nigri Versicolores Circumdati Othersections
(N=48) (N=39) (N=27) (N=26) (N=16) (N=19)
CAS(cid:2)
Median 18.75 50.00 ≤0.39 ≤0.39 0.59 0.39
Geometricmean 10.36 19.49 0.44 1.26 0.89 0.70
Range ≤0.39–50 ≤0.39–50 ≤0.39–12.5 ≤0.39–25 ≤0.39–25 ≤0.39–12.5
AMB
Median 2.00 2.00 1.00 2.00 >8.00 2.00
Geometricmean 1.86 1.93 1.03 2.41 6.17 1.93
D
Range 1–4 1–2 1–2 1–4 2–>8 ≤0.5–8 ow
n
ITC lo
a
d
Median 2.00 2.00 4.00 4.00 4.00 4.00 ed
Geometricmean 2.41 1.96 4.91 4.11 3.67 2.99 fro
Range 1–4 1–2 4–>8 2–>8 1–2 ≤0.5–>8 m h
VRC ttp
s
Median ≤0.25 ≤0.25 ≤0.25 ≤0.25 ≤0.25 0.50 ://a
c
a
Geometricmean 0.33 0.30 0.38 0.41 0.28 0.55 d
e
Range ≤0.25–1 ≤0.25–0.5 ≤0.25–1 ≤0.25–4 ≤0.25–0.5 ≤0.25–4 m
ic
PCZ .o
u
p
Median 0.50 0.50 1.00 1.00 1.00 1.00 .c
o
Geometricmean 0.43 0.54 1.05 1.05 0.96 1.12 m
/m
Range ≤0.25–1 ≤0.25–1 0.5–2 0.5–2 ≤0.25–2 ≤0.25–>8 m
y
(cid:2)MECappliesonlytocaspofungin. /artic
le
-a
Distributionofspeciessectionsindifferent houses(30.8%)orswineries(26.9%),andtheisolatesbe- bs
environments longingtothespeciessectionNigriweremorefrequentin trac
Theanalysisofdifferencesinencounteringresistanceindif- thebeachsandisolates(33.3%)orclinical(25.9%)sources t/54/7
ferentenvironmentsjustelucidatedappeartobedependent (Table4). /74
0
ondifferencesinresistanceamongspecies-sections,andthe /2
2
2
differentdistributionofspeciessectionsinvariousenviron- 2
ments. The goodness of fit χ2 for homogeneity test was Differencesinsusceptibilityofspeciessections 572
fromdifferentsources b
usedtoverifythatthedistributionofthedifferentsections y
g
u
(Fumigati, Flavi, Circumdati, Versicolores, and Nigri) for To evaluate if there were differences in the susceptibil- e
s
thedifferentsourceswasnothomogeneous(χ2 =19.833, ity profile of the species-sections recovered from the var- t o
4 n
P = .001). Regarding these data, the Contingency Chi- ious sources, we applied a Kruskal–Wallis test. Statisti- 0
5
squaretestbyMonteCarlosimulationwasusedtoanalyze cally significant differences were found for Fumigati and Ap
the existence of significant differences in species-sections’ Flavi species-sections). Regarding the Fumigati complex, ril 2
0
distributionamongdifferentsources.Itwasconcludedthat differences were found for AMB (χ2 (3)=10.381, P = 1
KW 9
thereweresignificantdifferencesamongthedifferentenvi- 0.016), with isolates from beach sand having the highest
ronments(P=.000105,C.I. =(0.00009;0.00012)). MIC and the clinical ones the lowest (P = .015); for ITC
95%
SectionFumigatiwasmorecommoninclinical(53.7%) (χ2 (3)=8.853, P =0.031),withclinicalisolateshaving
KW
and beach sand (20.8%) sources, the Flavi species section thehighestMICandthosefromthepoultryandbeachsand
was more frequently found among clinical (28.2%) and (P = .015 and P = .02, respectively) the lowest; for VRC
poultry (38.5%) isolates, whereas Circumdati species sec- (χ2 (3)=10.329, P =0.016), with isolates from beach
KW
tionwasmorefrequentamongtheisolatesrecoveredfrom sand having the highest MIC and the poultry ones the
beachsand(56.3%)andthehospitalenvironment(31.3%). lowest (P = .011); and for PCZ (χ2 (3)=14.581, P =
KW
Isolates belonging to the Versicolores species section were 0.002)withtheisolatescollectedfromswineriesandbeach
more frequently found in isolates recovered from poultry sand having the highest MIC and the clinical isolates the
Sabinoetal. 749
nsandamong Otherenvironments=(N0) –––– –––– –––– –––– –––– Otherenvironments=(N0) ––––
o
ml)amongthedifferentAspergillussecti =Flavi(N39)SandHospitalPoultriesSwineriesbeach====(N5)(N15)(N7)(N1)(cid:2)μCASMEC(g/ml) 505050507.236.241.050≤≤0.39–500.39–5025–50503(60)1(7)7(100)1(100) μAMBMIC(g/ml) 2222222222220(0)0(0)0(0)0(0) μITCMIC(g/ml) 2222222222220(0)0(0)0(0)0(0) μVRCMIC(g/ml) 0.50.250.250.250.430.260.250.25≤≤≤≤0.25–0.50.25–10.250.250(0)0(0)0(0)0(0) μPCZMIC(g/ml) 10.50.50.50.750.520.50.50.5-10.5-10.50.50(0)0(0)0(0)0(0) =Versicolores(N26) HospitalPoultriesSwineriesSandbeach====(N6)(N8)(N7)(N4) (cid:2)μCASMEC(g/ml) 0.390.400.391.71.21.10.781.8≤≤≤≤0.39–250.39–250.39–12.50.39–251(17)2(25)1(14)1(25) Downloaded from http
μMEC)(g/ Clinical=(N11) 257.5≤0.39–507(64) 21.81–20(0) 21.91–20(0) 0.250.34≤0.25–0.50(0) 0.50.5≤0.25-10(0) Clinical=(N1) 2525251(100) s://academ
( ic
veconcentrations Otherenvironments=(N4) 18.717.712.5–254(100) 2220(0) 2220(0) 0.250.30≤0.25–0.50(0) 0.250.25≤0.250(0) Otherenvironments=(N3) 0.390.39≤0.390(0) .oup.com/mmy/artic
centrations(MIC)andminimaleffecti =Fumigati(N48)SandHospitalPoultriesSwineriesbeach====(N0)(N8)(N4)(N10)(cid:2)μCASMEC(g/ml) –6.418.725–2.417.713.4≤≤–0.39–2512.5–250.39–25–4(50)4(100)9(90) μAMBMIC(g/ml) –222–222.3–222–4–0(0)0(0)2(20) μITCMIC(g/ml) –222–22.42–22–41–4–0(0)1(25)2(20) μVRCMIC(g/ml) –0.250.250.5–0.250.300.43≤≤≤–0.250.25–0.50.25–1–0(0)0(0)2(20) μPCZMIC(g/ml) –0.50.750.5–0.50.700.54≤≤–0.25-10.5-10.25-1–0(0)0(0)0(0) =Nigri(N27) HospitalPoultriesSwineriesSandbeach====(N4)(N1)(N3)(N9) (cid:2)μCASMEC(g/ml) 0.390.390.390.390.390.390.390.57≤≤≤≤0.390.390.390.39–12.50(0)0(0)0(0)1(14) le-abstract/54/7/740/2222572 by guest on
inhibitorycon Clinical=(N22) 2512.9≤0.39–5019(86) 21.61–20(0) 42.91-414(64) 0.250.31≤0.25–10(0) 0.250.32≤0.25-10(0) =Clinical(N7) 0.390.39≤0.390(0) 05 April 2019
al
minim μg/ml μg/ml μg/ml μg/ml μg/ml μ3g/ml
of >6.3 >2 >2 >2 >2 >6.
stribution Cparameters an %)withMIC an %)withMIC an %)withMIC an %)withMIC an %)withMIC an %)withMIC
Di MI me ( me ( me ( me ( me ( me (
Table4. sources. (cid:2)MECor MedianGeometricRangeNo.isolates MedianGeometricRangeNo.isolates MedianGeometricRangeNo.isolates MedianGeometricRangeNo.isolates MedianGeometricRangeNo.isolates MedianGeometricRangeNo.isolates