Potential pathogenic bacteria in metalworking fluids and aerosols from a machining facility

12 Pages·2010·0.25 MB·English

RESEARCH ARTICLE
Potentialpathogenicbacteriainmetalworking£uidsandaerosols
fromamachiningfacility
SarahD.Perkins1&LargusT.Angenent2
1DepartmentofEnergy,EnvironmentalandChemicalEngineering,WashingtonUniversityinSt.Louis,St.Louis,MO,USA;and2Departmentof
BiologicalandEnvironmentalEngineering,CornellUniversity,Ithaca,NY,USA
Correspondence:LargusT.Angenent, Abstract
DepartmentofBiologicalandEnvironmental
The metalworking and machining industry utilizes recirculating metalworking
Engineering,CornellUniversity,Ithaca,NY
fluids for integral aspects of the fabrication process. Despite the use of biocides,
14853,USA.Tel.:[phone];fax:11
6072554449;e-mail:[email protected] these fluids sustain substantial biological growth. Subsequently, the high-shear
forcesincurredduringmetalworkingprocessingaerosolizebacterialcellsandmay
Presentaddress:SarahD.Perkins, causedermatologicandrespiratoryeffectsinexposedworkers.Wequantifiedand
EnvironmentalProtectionAgency,National identified the bacterial load for metalworking fluid and aerosol samples of a
HomelandSecurityResearchCenter,Student machining facility in the US Midwest during two seasons. To investigate the
ServicesContract,Cincinnati,OH45268,USA.
presence of potentially pathogenic bacteria in fluid and air, we performed 16S
rRNAgenesurveys.Theconcentrationoftotalbacterialcells(includingculturable
Received28April2010;revised20August
and nonculturable cells) was relatively constant throughout the study, averaging
2010;accepted6September2010.
5.1(cid:2)108cellsmL(cid:3)1 in the fluids and 4.8(cid:2)105cellsm(cid:3)3 in the aerosols. We
Finalversionpublishedonline18October2010.
observed bacteria of potential epidemiologic significance from several different
DOI:10.1111/j.1574-6941.2010.00976.x bacterial phyla in both fluids and aerosols. Most notably, Alcaligenes faecalis was
identifiedthroughbothdirectsequencingandculturingineverysamplecollected.
Y Editor:MaxHa¨ggblom Elucidatingthebacterialcommunitywithgenesurveysshowedthatmetalworking
fluidswerethesourceoftheaerosolizedbacteriainthisfacility.
G
Keywords
O bioaerosols;metalworkingfluids;bacterialload;
16SrRNAgenesurvey;Mycobacterium
L
immunogenum;Alcaligenesfaecalis.
O
C metalworkingapprenticesafter2yearsofexposure,whilea
Introduction
E studybyParketal.(2007)showedadecreaseinpulmonary
Themetalworkingindustryutilizesrecirculatingmetalwork- function with daily shift progression among machining
Y
ingfluidstocool,removemetalfines,lubricate,andprevent workers.Therefore,theNationalInstituteforOccupational
G
corrosion during metal grinding and cutting procedures. Safety and Health has recognized the adverse effects of
O High-shear forces and excess heat create metalworking metalworkingfluidaerosolsonmachinistsandhasissueda
aerosolsduringstandardgrinding,drilling,andcuttingtasks recommended exposurelimitof0.4mgm(cid:3)3thoracicparti-
L
(Simpsonetal.,2003).Metalworkingfluidsaremixturesof culatemass,whichareinhalableparticlesthatarecapableof
O
oils, biocides, dissolved metals (e.g. chromium, nickel, penetrating into the bronchial region of the lung (US
I
B cobalt), microorganisms, antifoaming agents, and other DepartmentofHealthandHumanServices,1998).
O organic and inorganic constituents, which can be toxic to The interest in bacterial exposures to metalworking
operators(Woskieetal.,1996;Kennedyetal.,1999;Gordon, personnelhasintensifiedbecauseanoutbreakofhypersen-
R
2004).Epidemiologicalassessmentswithmachineoperators sitivity pneumonitis was linked to high concentrations of
C
in the metalworking industry have identified dermatologic bacteria in metalworking fluid (Wallace et al., 2002).
MI effects(Godderisetal.,2008)andrespiratoryeffects(Barhad Hypersensitivity pneumonitis is a pulmonary disorder in-
etal.,1975;Woskieetal.,1996;Greavesetal.,1997;Kennedy volving an immunologic reaction of the lung to inhaled
etal.,1999;Woskieetal.,2003;Parketal.,2007;Parketal., sensitizing agents of organic origin, such as bacterial or
2008). Kennedy et al. (1999) reported an increase in fungalantigenswithinaerosols(Bernsteinetal.,1995;Khalil
bronchial responsiveness with symptoms of asthma in etal.,2007).Tocausesuchanimmunologicalresponse,the
FEMSMicrobiolEcol74(2010)643–654 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
644 S.D.Perkins&L.T.Angenent
bacterial cell does not have to be viable. Pseudomonas spp. controlareaofthefacilityhousesatotalof13machines:five
hasbeendiscussedastheprimarygram-negativeculturein Makino A51 horizontal machining center units and eight
some metalworking fluids (Simpson et al., 2003). Gram- other computer numerical control type units with metal-
positive bacteria are also abundant in metalworking fluids workingfluids[theworkshopfloorplan(Fig.S1)withother
and the cell wall of, for example, mycobacteria have been details about this facility can be found in the online
found to induce an inflammatory response in humans SupportingInformation].
(Falkinham, 2003). In fact, the gram-positive Mycobacter-
ium immunogenum has been isolated in multiple metal-
Sampling plan
working facilities across North America and have been
associatedwithatleast98casesofhypersensitivitypneumo- Metalworkingfluid,indoormetalworkingaerosol,andout-
nitis (Chang et al., 2004). The relative abundance of M. door aerosol samples were collected during this study for
immunogenum in metalworking fluid suggests that this twoseasons–winterandsummerof2008.Forbothseasons,
organism is resistant to biocides commonly used in the wesampledforatotalperiodof6daysintwo3-dayintervals
metalworkingindustry(Wallaceetal.,2002;Selvarajuetal., (days 1–3 and days 4–6), whichwere separated by a 2-day
2008). The thick hydrophobic cell wall of mycobacteria weekend.WesampledtwoMakinoA51machines(machines
increasestheirresistancetoconventionaldisinfectants,such AandB)inthecomputernumericalcontrolarea(Fig.S1),
as chlorine, monochloramine, chlorine dioxide, and ozone andbecausebothofthemhadindependentfluidrecircula-
treatments,givingthemacompetitiveedgeinpotablewater tion tanks, the fluids were sampled and tested separately
(Carson et al., 1978; Taylor et al., 2000; Falkinham, 2003; throughout the study. The indoor aerosol samples were
Vaerewijcketal.,2005). collected adjacent to the work area of these two machines.
Multiple control measures have been applied in metal- Background air samples were collected outdoors and
workingfacilities toreduceoccupational exposuretoaero- upwindfromthefacility.Quantitativeculturingandmicro-
sols. Machine enclosures, exhaust filters, and electrostatic scopy methodswereperformeddaily forallsamples,while
precipitatorshavebeenadded,whilebiocidesandformalde- samples for DNA extraction were filtered and stored at
hyde in fluids have been used to combat microbial growth (cid:3)801Caftereachsamplingevent.
(Linnainmaa et al., 2003; Cohen & White, 2006).Here, we
quantified and identified bacteria from air samples of one
Aerosol sampling procedure
metalworkingfacilitywithmachineenclosuresandbiocides
within a sampling period of 6 days for two seasons. We Aerosol samples were collected with liquid impingers
investigated the presence of potential pathogenic bacteria (Biosamplers, AGI-30, SKC, Eighty-four, PA) at a rate of
with 16S rRNA gene sequence surveys. We also sampled 12.5LPM for 60min, allowing a total sample volume of
metalworkingfluidstoascertainwhetherthebacterialcom- 0.75m3 to pass through each of three samplers (three
munities of the fluid and aerosol were similar. To our samplers were run simultaneously at each location). Three
knowledge, this would be the second study to analyze flowmeters(RMA-22Dwyer,MichiganCity,IN)wereused
communities for both metalworking fluid and aerosol sam- to control the airflow rate (one flow meter for each
ples. A recent study by Gilbert et al. (2010a,b) utilized Biosampler). The Biosamplers were positioned on a sam-
gradientgelelectrophoresisfingerprintstoassessmetalwork- plingstand1.5mfromthefloorandwerefilledwith20mL
ing fluid communities; and herein, we used comprehensive of sterile phosphate buffer solution (PBS). After a 30-min
gene surveys. We, therefore, did not rely on just culturing sampling period, the volume of PBS for each sampler was
techniquesthathavebeenshowntoonlyidentifyo1%ofthe topped off when necessary to ensure that the sampling
microorganisms in environmental samples (Amann et al., solution was not lost to evaporation. At the end of the
1995; Pace, 1997; Edwards, 2000) and 0.02–0.2% of the samplingperiod,thissolutionwascombinedfromthethree
bacterial cells in aerosol samples (Angenent et al., 2005). samplersandthefinalvolumewasrecorded.Acondensation
Furthermore, with a comprehensive gene survey, we also particlecounter(3760A,TSI,Shoreview,MN)wasusedfor
included nonviable bacteria other than specifically targeted real-time analysis of the total concentration of parti-
generaofPseudomonasandMycobacterium. cles40.01mm in diameter at the same location as the
aerosol sampler. The inlet air tube of the condensation
particle counter was positioned at the center of the same
Materialsand methods
sampling stand. Two high-volume vacuum pumps
(2688VE44, 2669CE44, Thomas, Lake Zurich, IL) were
Facilitydetails
connected to the condensation particle counter and the
TheUSMidwestmetalworkingfacilitythatwassampledin Biosampler flow meters with norprene tubing (Masterflex,
thisstudyemploys(cid:5)100workers.Thecomputernumerical VernonHills,IL).
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol74(2010)643–654
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Potentialpathogensinmetalworkingfluidsandaerosols 645
Bacterial quantification:colonycounts,total contained1.25UofGoTaq(PromegaCorp.,Madison,WI),
bacterial counts,and quantitativePCR(qPCR) 0.4pmolmL(cid:3)1 forwardand reverse primers (8F – 50-AGAG
TTTGATCCTGGCTCAG-30; 1391R – 50-GACGGGCGG
To quantify heterotrophic bacterial growth, we inoculated
TGWGTRCA-30)targetingbacterial16SrRNAgenes(Lane,
duplicate, serial-diluted, aliquots of metalworking fluid,
1991),0.5mMMgCl ,0.2mMdNTPs,0.8mgmL(cid:3)1bovine
aerosols, and sterile PBS onto tryptic soy agar plates 2
serum albumin (BSA), and 2mL of template. Control
amended with 0.1gL(cid:3)1 cyclohexamide (C7698, Sigma,
positive and negative reactions were included with each
St. Louis, MO) to repress fungal growth. We used an
reactionset, whileBSAwasaddedto impedeamplification
Autoplate 4000 (Spiral Biotech, Norwood, MA) to plate
inhibition.DNAfromrepresentativecoloniesgrownonthe
thesesamples.Allinoculatedplateswereincubatedat351C
tryptic soy agar plates were extracted and PCR amplified
for 24h before quantification by colony counts, which we
with universal bacterial primers (8F and 1391R) according
expressedasCFUs.Totalcellcountswereconductedthrough
tothesamePCRprotocol.
the epifluorescence microscopy procedure: 0.5mL of a 1/
10th dilutionofmetalworkingfluid or20mLofthecollec-
Cloning,sequencing, andanalyses
tionsolutionwasfilteredthrougha0.22-mmblackpolycar-
bonatefilter(GEWater,Trevose,PA),stainedwith1mMof 16SrRNAgenesurveyswereperformedforapooled(n=6)
DAPIsolution(Sigma),andrinsed.Cellswerethencounted sample of metalworking fluid from each machine during
from 20 fields of view using an epifluorescence microscope each of two seasons (winter fluid machine A; winter fluid
(Olympus BX41, Center Valley, PA). qPCR was used to machine B; summer fluid machine A; and summer fluid
determinethebacterialDNAloadwithineachsample.Wells machine B). The aerosol samples that were collected adja-
contained 2mL of extracted DNA template and 23mL of centtobothmachinesAandB(eachmachinehadaseparate
SYBRgreenmastermix(ABgene,Rockford,IL)supplemen- sampling setup) were pooled (from three sampling days)
tedwith0.25UUDP-N-glycosidaseand10mMofuniversal into four samples based on season and sampling days 1–3
bacterial primers (forward primer – 50-TCCTACGGGAGG anddays4–6.Anadditionalaerosolsample(days4–6)from
CAGCAGT-30; reverse primer – 50-GGACTACCAGGGTATC machine B collected during the winter sampling event was
TAATCCTGTT-30) (Nadkarni et al., 2002). Samples were also surveyed (winter aerosol machine A and B days 1–3;
analyzed with a Stratagene Mx3000P qPCR system (Cedar winter aerosol machine A and B days 4–6; winter aerosol
Creek, TX), using the program outlined, and verified by An machineBdays4–6;summeraerosolmachineAandBdays
etal.(2006)(40cyclesat951Cfor15s,1minat601C,30sat 1–3; and summer aerosol machine Aand B days 4–6). For
721C with data collection temperatures of 85–881C, and eachofthesesamples,PCRproductswerecleaned,concen-
analysisofafinalmeltingcurve).Togenerateastandardcurve trated (Montage PCR Cleaning Kit, UFC7PCR50, Milli-
for qPCR, DNA was extracted from Escherichia coli and pore), and sent to the Genome Sequencing Center at the
quantifiedusingaPicoGreendsDNAQuantitationKit(Mole- WashingtonUniversitySchoolofMedicineinSt.Louis,for
cularProbes,Eugene,OR).ThestandardcurveofE.coliDNA cloning, purification, and Sanger sequencing on an ABI
rangedfrom0.034pgL(cid:3)1to34.7ngL(cid:3)1withanR2of0.94. 3730 capillary sequencer (Applied Biosystems, Foster City,
CA). The 16S rRNA gene PCR products from the single
DNA extractionandPCRamplification culturesgrownonTSAweregelpurified(MontageDNAgel
extraction kit, LSKGEL050, Millipore). Purified amplicons
For each day of sampling, we concentrated 40mL of PBS
weresenttoRetrogenInc.(SanDiego,CA)forDNAsequen-
collection solution (pooled from three samplers), before
cingwith8Fastheprimer.Weobtainedasinglesequencewith
DNAextractionusinganisoporemembranefilterof0.2-mm
a length of (cid:5)700nt for these environmental isolates and,
pore-size (GTTP02500, Millipore, Billerica, MA) mounted
therefore,didnotbuildaconsensussequenceorusethemfor
on polysulfone filter funnel stands (28144-700, Pall, Ann
our phylogenetic analyses. These sequences were identified
Arbor, MI) that were sterilized by a sequential treatment
through RDP (Cole et al., 2007). The 16S rRNA gene
withbleach,DIwaterrinse,UV,andtheautoclave.Samples
sequencesobtaineddirectlyfromenvironmentalsampleswere
were immediately stored at (cid:3)801C until the filter was
analyzed as described by Perkins et al. (2010). A circular
subjectedtoabead-beatingandphenol:chloroformextrac-
neighbor-joiningphylogenetictreewascreatedwiththeonline
tionprotocol(Angenentetal.,2005).The16SrRNAgenes
INTERACTIVETREEOFLIFE(ITOL)program(Letunic&Bork,2007).
were amplified using a 30-cycle touchdown PCR [initial
2-min denaturing step at 941C, followed by 20 cycles at
Statisticalanalysis
921Cfor30s,gradient65–451C((cid:3)11Cpercycle)for90s,
721Cfor90s;thesetouchdowncycles werefollowedby10 Foreachmethodofquantification,astatisticalanalysiswas
cycles at 921C for 30s, 451C for30s, 721Cfor 30s, and a compiledusingtheRProjectforstatisticalcomputingsoft-
final 721C extension for 15min]. The 50-mL solution ware(http://www.r-project.org).
FEMSMicrobiolEcol74(2010)643–654 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
646 S.D.Perkins&L.T.Angenent
Nucleotide sequenceaccession numbers fluidfrommachineB(Table1).Thesummerfluids,which
werehigherintemperature(Table1),containedarelatively
Near-full-length 16S rRNA gene sequences were deposited
higherDNAconcentrationthanthewintersamples.Athree-
in GenBank under accession numbers FJ657671–FJ658996.
Sequences from the cultured plates were deposited in wayANOVAbetweentheseason,machine,andquantification
method determined all three to be significant factors
GenBankunderaccessionnumbersGQ463228–GQ463237.
(Po0.001).
Results
Metalworking aerosols
Bacterial quantification
To investigate whether the bacterial load in the air of the
metalworkingfacilityremainedsimilarduringthesampling
Metalworking fluids
periodof6days,weaveragedthequantitativedatafromthe
We had anticipated that the total bacterial load of the indooraerosolsamplesforeachconsecutive3-daysampling
metalworkingfluidsfor thisstudywouldnotchangemuch intervalperiodduringeachseason(days1–3anddays4–6).
between the first (days 1–3) and the second sampling Wehadnotpreviouslyinvestigatedtheairmixingcharacter-
interval period (days 4–6) within one season, which was a istics of the sampling area, and therefore, we also investi-
correct assumption (P=0.88). Therefore, we averaged our gated the quantitative aerosol data separately for the
quantitative data acquired for each machine during the 6 samplers that were placed in close vicinity to machines A
daysofsamplingforeachseason(n=6;Table1).Thetotal and B. The data from the outdoor aerosol samples were
volumetric cell counts quantified through epifluorescence averaged for the entire 6-day sampling period during each
rangedfrom3.91(cid:2)107to1.55(cid:2)109cellsmL(cid:3)1inthewinter season (n=6). Next, a four-way statistical analysis with all
fluid from machine A and machine B, respectively. These quantitative aerosol data determined that there was a
counts for the summer samples were more similar to each significant difference for the bacterial quantities between
other, ranging from 1.08(cid:2)108 to 3.62(cid:2)108cellsmL(cid:3)1 for seasons or between quantification methods (Po0.001).
machinesBandA,respectively,andweresignificantlyhigher However, the analysis did not find a significant difference
thanthePBSbackground(Table1).Thebacterialcellsinthe between the sampling interval days1–3 and 4–6 (P=0.79)
metalworking fluid were culturable at high concentrations or between machines (P=0.77). Therefore, we report the
exceeding 1(cid:2)108CFUsmL(cid:3)1 for the winter fluid for average data for the entire 6-day sampling period for both
machine A and the summer fluids for both machines. indoor samplers (machines A and B combined). The aver-
The concentration of culturable cells for the winter aged epifluorescence cell count for the indoor aerosols
fluid sample from machine B was six orders of magnitude collected during the winter sampling event was 6.04(cid:2)
lower (9.50(cid:2)102CFUsmL(cid:3)1) with a CFUs/cells ratio of 105cellsm(cid:3)3 (Table 2). Compared with the indoor air,
2.43(cid:2)10(cid:3)5 (Table 1). Based on operator information we the background air sample taken outside the facility
obtainedatthefactory,thebiocidesinbothmachineswere had a considerably lower average bacterial load of
the same. qPCR analysis further validated the considerable 1.21(cid:2)105cellsm(cid:3)3(Table2).Thisshowsthatmetalworking
bacterial load for the metalworking fluids, with bacterial activities raised the bacterial load in the indoor air. Com-
DNA concentrations ranging from 194mgmL(cid:3)1 in the pared with the winter indoor air samples, the summer
winterfluidfrommachineAto984mgmL(cid:3)1inthesummer epifluorescencecellcountsfromtheindoorairwereslightly
Table1. Six-dayaverageconcentrationsoftotalcells,CFUs,andDNAfrom16SrRNAgenesforthemetalworkingfluidscollectedfromtherecirculation
tankofmachinesAandBandPBSbackgroundfluidduringthewinterandsummersamplingevent
Temperature
Seasons Machine (1C) pH cellsmL(cid:3)1(cid:2) CFUsmL(cid:3)1w CFUs/cells mgDNAmL(cid:3)1z
Winter A 30.0 8.6 1.55(cid:2)109(cid:6)0.42(cid:2)109 1.16(cid:2)108(cid:6)1.79(cid:2)108 7.53(cid:2)10(cid:3)2 1.94(cid:2)102(cid:6)10.7(cid:2)102
B 25.3 9.3 3.91(cid:2)107(cid:6)1.31(cid:2)107 9.50(cid:2)102(cid:6)0.13(cid:2)102 2.43(cid:2)10(cid:3)5 5.89(cid:2)102(cid:6)8.32(cid:2)102
PBS – – 1.06(cid:2)102(cid:6)3.98(cid:2)102 BD 0 0.92(cid:6)1.70
Summer A 35.3 8.2 3.62(cid:2)108(cid:6)2.11(cid:2)108 2.87(cid:2)108(cid:6)2.00(cid:2)108 7.93(cid:2)10(cid:3)1 8.25(cid:2)102(cid:6)9.68(cid:2)102
B 31.7 8.1 1.08(cid:2)108(cid:6)0.83(cid:2)108 1.38(cid:2)108(cid:6)1.06(cid:2)108 1.29 9.84(cid:2)102(cid:6)0.03(cid:2)102
PBS – – 4.13(cid:2)102(cid:6)18.7(cid:2)102 0.43(cid:6)1.12 1.05(cid:2)10(cid:3)3 BD
(cid:2)
Totalbacterialcellcountswithdirectmicroscopy.
wCFUswithheterotrophicplates.
zQuantitativelevelsof16SrRNAgeneDNAwithqPCR; (cid:6)SDwithn=6.
BD,dataisbelowthelimitofdetection.
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol74(2010)643–654
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Potentialpathogensinmetalworkingfluidsandaerosols 647
Table2. Six-dayaverageconcentrationsoftotalcells,CFUs,particles,andDNAfrom16SrRNAgenesfortheaerosolscollectedfromtwosampling
locations(n=12),andbackgroundoutsideair(upwindfromthefacility;n=6)duringthewinterandsummersamplingevent
Seasons Locations cellsm(cid:3)3(cid:2) CFUsm(cid:3)3w CFUs/cells Particlesm(cid:3)3z ngDNAm(cid:3)3‰
Winter Inside 6.04(cid:2)105(cid:6)1.69(cid:2)105 5.58(cid:2)103(cid:6)8.06(cid:2)103 9.24(cid:2)10(cid:3)3 1.36(cid:2)1010(cid:6)0.28(cid:2)1010 8.58(cid:6)3.97
Outside 1.21(cid:2)105(cid:6)0.33(cid:2)105 3.86(cid:2)101(cid:6)16.3(cid:2)101 3.20(cid:2)10(cid:3)4 4.76(cid:2)109(cid:6)2.79(cid:2)109 1.11(cid:6)0.72
Summer Inside 5.90(cid:2)105(cid:6)1.06(cid:2)105 2.59(cid:2)103(cid:6)3.94(cid:2)103 7.35(cid:2)10(cid:3)3 1.19(cid:2)1010(cid:6)0.36(cid:2)1010 5.06(cid:6)2.46
Outside 9.79(cid:2)104(cid:6)1.73(cid:2)104 3.01(cid:2)102(cid:6)4.99(cid:2)102 3.08(cid:2)10(cid:3)3 9.42(cid:2)108(cid:6)7.18(cid:2)108 0.63(cid:6)0.13
(cid:2)
Totalbacterialcellcountswithdirectmicroscopy.
wCFUswithheterotrophicplates.
zQuantitativelevelsof16SrRNAgeneDNAwithqPCR.
‰Particleconcentrationwithacondensationparticlecounter; (cid:6)SDwithn=12forinsideandn=6foroutside.
lower (5.90(cid:2)105cellsm(cid:3)3, Table 2). The background out- countsbetweenmachineswasshownpossiblyindicatesthat
doorairsamplescollectedinthesummerwerealsoslightly theindoorareaairwaswellmixedandthatthelocationof
lower than the winter outdoor air sample (9.79(cid:2)104 the sampler within the area did not affect the quantitative
cellsm(cid:3)3,Table2).Inaccordancewiththewintersampling aerosoldata.Basedonthisinformation,theairsamplesfor
event, the bacterial load in the summer indoor air was machinesAandBwerepooledintofouraerosolsamplesto
elevated considerably compared with the outdoor air. The differentiate just between season and interval period. We
concentrationofculturablecellsfortheindooraerosolswas checked whether this assumption was valid with the 16S
twoordersofmagnitudelowercomparedwiththetotalcells rRNAgene survey bycomparing thecommunitycomposi-
present for the winter and the summer (5.58(cid:2)103 and tion for a fifth aerosol sample from machine B during the
2.59(cid:2)103CFUsm(cid:3)3,respectively)withaCFUs/cellsratioof winter sampling eventfor the second interval period (days
9.24(cid:2)10(cid:3)3 and 7.35(cid:2)10(cid:3)3, respectively (Table 2). This 4–6), with a pooled samplefrommachine Aand B for the
ratio was slightly lower for the outside aerosols with sameperiod.
3.20(cid:2)10(cid:3)4 and 3.08(cid:2)10(cid:3)3 for the winter and summer, We generated 1326 near-full-length, nonchimeric 16S
respectively (Table 2). The total particle counts (including rRNA gene sequences, which were distributed between a
both cells and inorganic particles) in the metalworking totalofsevendifferentphyla(Table3).Themostabundant
facilityweresimilarduringthewinterandsummersampling phylum for the combined nine samples was Proteobacteria
events, and were four to five orders of magnitude higher with 490% of the total number of sequences (Table 3
than the epifluorescence cell counts (Table 2). Finally, and Fig. S2). Eight out of our nine surveyed samples
quantitativeDNAconcentrationsintheaerosolsamplewere consistedprimarilyofproteobacterialsequenceswitheither
slightlyhigherduringthewintersamplingeventcompared the class Alphaproteobacteria or Betaproteobacteria as the
withthesummersamplingevent(Table2). most abundant group. There was one exception – the
sequences for the winter metalworking fluid sample from
Sequenceanalysis machineBcontained89%ofthephylumActinobacteriaand
only10%ofthephylum Proteobacteria(Table3).Thiswas
Thesampleswereobtainedduringthewinterandsummer for the same fluid sample that was characterized with a
seasons and consisted of four metalworking fluid samples considerably lower CFUs/cells ratio than the other three
and five aerosol samples within the computer numerical fluid samples (Table 1). Besides being abundant in the
controlareaofthefacility(Fig.S1).Ourstatisticalanalysis winterfluidsampleformachineB,Actinobacteriawasonly
hadshownthatthequantitativedata for themetalworking foundtobeabovedetectioninthewinteraerosolmachines
fluids was significantly different between seasons and ma- AandBduringdays4–6andthesummeraerosolmachines
chines, but not between sampling intervals, which resulted A and B during days 1–3 (both at levels (cid:5)1%, Table 3).
insurveyingthepooledintervalsamplesforeachseasonand Regardless of sample type, Alphaproteobacteria, Betaproteo-
machine separately (four samples). For the metalworking bacteria, and Gammaproteobacteria were present in all
aerosols,ouranalysisdidshowadifferencebetweenseasons, samples. The phyla Bacteroidetes and Firmicutes were each
but not between interval periods and sampling locations found in seven samples between levels of 0.5–4%. Finally,
(machines). Even thoughwe showed that the variabilityof thephylaFusobacteria,Chloroflexi,andAminanaerobiawere
quantitativeaerosoldatawithinthesamplingeventwasnot each only detected in one sample at low abundance
significant, we still surveyed the aerosol samples from the (0.5–1%)(Table3).
two interval periods separately to gauge the variability in We used a color-coded neighbor-joining phylogenetic
communitycompositionwithintheseasonalsamplingper- tree (with all 1326 sequences) in which each of the nine
iod. The fact that no quantitative difference in aerosol colors represents a different sample type to show the
FEMSMicrobiolEcol74(2010)643–654 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
648 S.D.Perkins&L.T.Angenent
Table3. Phylumandclass-levelcomparisonofsequencesfromeachmetalworkingsample
Winterfluid(%) Summerfluid(%) Winteraerosol(%) Summeraerosol(%)
MachineA MachineA Machine MachineA MachineA
Machine Machine Machine Machine andB andB B andB andB
Phylum A B A B Days1–3 Days4–6 Days4–6 Days1–3 Days1–3
Alphaproteobacteria 14.2 1.4 13.0 55.2 78.0 34.0 20.6 32.6 36.4
Betaproteobacteria 82.7 1.4 78.3 31.0 16.7 63.5 73.5 52.4 38.8
Gammaproteobacteria 1.6 7.2 0.0 10.3 0.7 0.3 2.0 12.3 19.8
Firmicutes 1.6 0.0 0.0 3.4 0.7 0.7 2.9 0.9 4.1
Bacteroidetes 0.0 1.4 4.3 0.0 3.3 0.7 1.0 0.4 0.8
Actinobacteria 0.0 88.4 0.0 0.0 0.0 0.7 0.0 0.9 0.0
Fusobacteria 0.0 0.0 4.3 0.0 0.0 0.0 0.0 0.0 0.0
Chloroflexi 0.0 0.0 0.0 0.0 0.7 0.0 0.0 0.0 0.0
Aminanaerobia 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.0
Totalsequences 127 138 23 29 150 288 102 227 242
Thepercentagesofphylumorclassdistributionforsamplesfromthemetalworkingfluidsandaerosols.ThephylumProteobacteriawassubdividedinto
Alphaproteobacteria,Betaproteobacteria,andGammaproteobacteria.
Fig.1. Weighted normalized UniFrac analysis
for differentiating each community in the fluid
andaerosolsamples.
considerableoverlapofindividualsequencesforthemetal- was not observed for the winter aerosol samples (Fig. 1).
workingfluidandaerosolsamplesbetweenthetwoseasons Therefore, the abundance of different phyla was different
(Fig. S2). This is also shown by a weighted environmental betweentheintervalperiodsforthewinteraerosolsamples,
clustering test that further demonstrates the considerable which is also apparently based on the phyla distribution
overlap between the fluid and aerosol communities for results(Table3).
both seasons because samples did not clearly cluster by Furtherphylogeneticanalyseswereperformedtoclassify
sampletype(fluidvs.aerosols–Fig.1).Fromthisinforma- individual16SrRNAgenesequencesbasedonsequencesin
tion, it is clear that the fluids are the source of aerosols publicdatabases. We found 200 different operational taxo-
inthemetalworkingfacility.Ourweightedandnormalized nomic units (OTUs) for the 1326 nonchimeric full-length
UniFrac clustering results also suggest that the air in the sequences (Table 4). To investigate whether OTUs from
machining room was well mixed because the bacterial differentsampletypeswerecloselyrelated,weperformeda
communities for the winter aerosol samples for days 4–6 maximum likelihood analysis on the sequences that
that were taken close to machines A and B clustered wereidentified withinthree bacterialphyla(Fig. 2).Again,
closely with thesample that was takenclose to machine B, sequencesfromwinterfluid,summerfluid,winter aerosol,
even though the winter fluid sample from machine B was andsummeraerosolwereoftencloselyrelated.Forexample,
very different (the most separated sample with UniFrac) Alcaligenes faecalis, which is one of the most abundant
(Table 3 and Fig. 1). The UniFrac results were less clear in species in our gene survey, was represented in all nine
answering the question whether the communities for samples.Thus,ingeneral,oursurveysupportsthefactthat
the aerosol samples varied between interval periods the metalworking fluid is the likely source of the bacterial
days 1–3 and days 4–6. The summer aerosol samples aerosolsandthatnoobviousdifferencebetweenseasonsand
clustered together well for the two periods, but this machines was observed.However, as we had shown above,
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol74(2010)643–654
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Potentialpathogensinmetalworkingfluidsandaerosols 649
Table4. Sequencedetailsandpotentialpathogensfoundineachsample
Nonchimeric Detection Matching RDP%matchto
Sample sequences OTUs method sequences knownspecies Potentialpathogen
Winterfluid 127 22 Direct 81 97.5 Alcaligenesfaecalis;DQ857898
MachineA Cultured – 97.8 Alcaligenesfaecalis;DQ110882
Cultured – 499.9 Morganellamorganii;AJ301681
Winterfluid 138 21 Direct 2 99.2 Alcaligenesfaecalis;AY959943
MachineB Cultured – 97.8 Alcaligenesfaecalis;DQ110882
Direct 1 97.6 Brevundimonasdiminuta;AY730717
Cultured – 499.9 Morganellamorganii;AJ301681
Direct 1 96.5 Mycobacteriumchelonae;AJ419969
Direct 84 99.5 Mycobacteriumimmunogenum;AJ812215
Cultured – 499.9 Ochrobactrumanthropi;AJ276036
Direct 1 97.6 Pseudochrobactrumasaccharolyticum;AM180485
Winteraerosol 150 31 Direct 3 97.3 Alcaligenesfaecalis;DQ857898
MachineAandB Cultured – 97.8 Alcaligenesfaecalis;DQ110882
Days1–3 Direct 34 499.9 Brevundimonasdiminuta;X87274
Cultured – 499.9 Morganellamorganii;AJ301681
Direct 2 97.5 Pseudochrobactrumasaccharolyticum;AM180485
Direct 1 97.9 Wautersiellafalsenii;AM238673
Winteraerosol 288 40 Direct 132 98.5 Alcaligenesfaecalis;DQ857898
MachineAandB Cultured – 97.8 Alcaligenesfaecalis;DQ110882
Days4–6 Direct 2 98.3 Bordetellatrematum;AJ277798
Direct 37 98.3 Brevundimonasdiminuta;EF191247
Direct 4 98.8 Ochrobactrumanthropi;AB120120
Direct 3 98.5 Pseudochrobactrumasaccharolyticum;AM180485
Winteraerosol 102 19 Direct 58 99.7 Alcaligenesfaecalis;DQ857898
MachineB Cultured – 97.8 Alcaligenesfaecalis;DQ110882
Days4–6 Direct 2 99.4 Ochrobactrumanthropi;AJ294349
Direct 1 98.3 Brevundimonasdiminuta;EF1911247
Direct 2 98.0 Pseudochrobactrumasaccharolyticum
Summerfluid 23 9 Direct 14 98.5 Alcaligenesfaecalis;DQ857898
MachineA Cultured – 97.7 Alcaligenesfaecalis;DQ857898
Direct 1 98.8 Ochrobactrumanthropi;AB120120
Summerfluid 29 11 Direct 3 99.0 Alcaligenesfaecalis;DQ857898
MachineB Cultured – 97.7 Alcaligenesfaecalis;DQ857898
Summeraerosol 227 24 Direct 77 96.8 Alcaligenesfaecalis;DQ857898
MachineAandB Cultured – 98.8 Alcaligenesfaecalis;DQ110882
Days1–3 Direct 19 98.2 Brevundimonasdiminuta;DQ857897
Direct 1 98.0 Corynebacteriumxerosis;X81914
Direct 3 99.4 Ochrobactrumanthropi;AY730720
Direct 2 98.7 Pseudochrobactrumasaccharolyticum;AM180485
Direct 1 96.8 Wautersiellafalsenii;AM238670
Summeraerosol 242 23 Direct 62 98.0 Alcaligenesfaecalis;DQ857898
MachineAandB Cultured – 97.7 Alcaligenesfaecalis;DQ857898
Days4–6 Direct 6 99.0 Brevundimonasdiminuta;EF191247
Direct 1 97.0 Citrobacteramalonaticus;AF025370
Cultured – 499.9 Ochrobactrumanthropi;AB120120
Direct 2 98.3 Ochrobactrumanthropi;AM114409
Direct 2 98.5 Staphylococcusepidermidis;CP000029
Direct 1 97.2 Wautersiellafalsenii;AM238670
(cid:2)
Total 1326 200 646
(cid:2)
TotalnumberofdistinctOTUsfoundamongall1326sequences.
Thetotalnumberofnonchimericsequencescollected,numberofOTUs,methodofdetection,andnumberofsequencesthatmatchedwithanidentity
497%toaknownpotentialpathogenintheRDPdatabasearegiven.
FEMSMicrobiolEcol74(2010)643–654 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
650 S.D.Perkins&L.T.Angenent
Fig.2. Maximum likelihood phylogenetic
distributionofpotentiallypathogenic16S
rRNAgenesequencescollectedinthisstudy.
ReferencesequencesfromtheNCBIdatabase
areincludedwiththeiraccessionnumbers.
Individualmetalworkingfluidsequencesare
labeledwith‘fluid’followedbytheirunique
sequenceidentity.Metalworkingaerosol
samplesareidentifiedby‘aerosol’followed
bytheiruniquesequenceidentity.Thetotal
numberofsimilarOTUs(basedona3%
cutoff)fromthisstudyisinparenthesis
followingthesequenceidentity.Thescalebar
representsbasechangespersite.The
bootstrapnumbersarebasedonthe
neighbor-joiningmethod.
there is one exception – the winter fluid machine B was dochrobactrum asaccharolyticum, Brevundimonas diminuta,
enrichedwithActinobacteria.Thisenrichmentdidnotresult Wautersiella falsenii, Morganella morganii). The maximum
in considerably higher levels of these Actinobacteria in the likelihoodphylogeny identifiedandverifiedtherelatedness
indooraerosoloftheroom(Table3). of all the potential pathogens in our samples to cultured
Using the RDP algorithm, we identified 646 full-length sequences in public databases (Fig. 2). Besides the six
sequences (out of a total of 1326 full-length sequences) to pathogens that were found in more than one sample, five
be497%identicaltoknownpotentialpathogens(Table4). sequences that were closely related to potential pathogenic
Six of the potentially pathogenic bacteria were found in bacterialspecieswerefoundinonlyonesample(Bordetella
multiplesamples(A.faecalis,Ochrobactrumanthropi,Pseu- trematum; Citrobacter amalonaticus; Staphylococcus
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol74(2010)643–654
PublishedbyBlackwellPublishingLtd.Allrightsreserved
Potentialpathogensinmetalworkingfluidsandaerosols 651
epidermidis; Corynebacterium xerosis; and M. immunogen- should be further verified with more metalworking fluid
um) (Fig. 2). The enrichment of M. immunogenum in the samplesfromadditionallocations.
winter fluid machine B is of importance because it is this
actinobacterial species that caused the winter fluid sample Metalworking bioaerosol concentrationswere
for machine B to be an outlier compared with the other comparableto other studies
samples.Interestingly,M.immunogenumwasnotpresentin
The concentration of total bacterial cells, including cultur-
anyother metalworking fluid or aerosol samples (not even
able and nonculturable cells, within the facility averaged
intheothersampleswithActinobacteria)(Fig.2).
4.8(cid:2)105(cid:6)1.4(cid:2)105cellsm(cid:3)3orapotentialinhalationdose
of1(cid:2)106(cid:6)3(cid:2)105microorganismsh(cid:3)1forstaff,assuming
Discussion an average inhalation rate for moderate activity to be
2.1m3h(cid:3)1(EPA,1997).Thisconcentrationisslightlylower
Consistentlyhigh bacterial concentrationswere than the Abrams et al. (2000) assessment, which ranged
found for the metalworking fluids from8.7(cid:2)105to2.7(cid:2)106totalthoracicbacteriam(cid:3)3with-
in a metalworking facility. An aerosol sample at an indoor
Therearenosuggestedmaximumcontainmentlevelsspeci-
hospitaltherapypoolcontainednearlythesameconcentration
fied for bacterial concentrations in metalworking fluids;
of cells (106cellsm(cid:3)3) compared with our study (Angenent
however, numerous studies have shown the propensity of
et al., 2005), while a lower concentration of
metalworkingfluidstofostermicrobialgrowth.Thefacility
slightlyo2.1(cid:2)105cellsm(cid:3)3wasfoundinsideasingle-family
assessed in this study had expressed concern regarding
home (Fabian et al., 2005). The average outdoor aerosol
biomass accumulation in the fluid of multiple machines
samplecollectedinthisstudy(1.1(cid:2)105(cid:6)0.2(cid:2)105cellsm(cid:3)3)
and had observed a decreased air quality in the computer
wassimilartooutdoorsamplescollectedinSaltLakeCity,UT
numerical control area of the facility. In our analysis, the
(105–107cellsm(cid:3)3) (Radosevich et al., 2002) and East St.
concentrationsofbacteria(culturableandnonculturable)in
Louis,IL(1.5(cid:2)105cellsm(cid:3)3)(Rauer,2005).
the fluids were relatively constant throughout the study
(averaging 5.1(cid:2)108(cid:6)7.0(cid:2)108cellsmL(cid:3)1 with epifluores-
Potentially pathogenic bacteriawerefound in
cencemicroscopyand1.4(cid:2)108(cid:6)1.2(cid:2)108CFUsmL(cid:3)1with
the metalworking fluidandaerosol samples
culture quantifications). These findings are in accordance
with previous metalworking fluid studies. Using fluores- Typicalcultureanalysesofmetalworkingfluidsandaerosols
cence microscopy, Thorne et al. (1996) found the total have shown high concentrations of clinically significant
(culturable and nonculturable) bacterial concentration of Pseudomonas spp. and Mycobacterium spp. (Lewis et al.,
metalworking fluids to range between 1.5(cid:2)107 and 2001).Thefindingspresentedheredidnotyieldsignificant
1.7(cid:2)108organismsmL(cid:3)1, while other studies have shown quantitiesofeithergenus:2%ofthesequencesinthisstudy
culturinganalysesofmetalworkingfluidstoyieldanaverage werefromthegenusPseudomonas(30/1326)andonefluid
of107CFUsmL(cid:3)1(Mattsby-Baltzeretal.,1989;Sandinetal., samplecontainedsequencefromthegenusMycobacterium.
1991;Thorneetal.,1996).TheCFUstocellsratiosforthree However, other organisms previously cultured in metal-
ofthemetalworking fluidsampleswerebetween0.075 and working fluid samples were found during this analysis.
1.29 (Table 1), which shows that 7.5–100% of all counted Acinetobacterspp.,Alcaligenesspp.,Morganellaspp.,Ochro-
cells were culturable in our lab. Thorne et al. (1996) and bactrum spp., and Staphylococcus spp. have each been
Abrams et al. (2000) found very similar percentages of isolated from metalworking fluids (Mattsby-Baltzer et al.,
4–100%, respectively. However, the winter fluid sample 1989;Sandinetal.,1991;Laitinenetal.,1999;Brackeretal.,
collected from machine B showed that only 0.002% 2003), while Brevundimonas spp., Corynebacterium spp.,
of the bacterial cells were cultured in our lab (Table 1). andOchrobactrumspp. havebeenidentifiedinmetalwork-
Veillette et al. (2004) also noted relatively low percentages ing aerosols (Gorny et al., 2004). 3.3% (30/907) of the
for their metalworking fluid characterization. We showed aerosol sequences identified here were from the genus
that460% of the sequences from our winter fluid for Acinetobacter, but none were identical at the species level
machine B sample were identified to be M. immunogenum (497%)toaspeciesofAcinetobacterintheRDPdatabase.
at the species level (497% identical) (Table 4), while Over 40% of the sequences (546/1326) in this study were
Veillette et al. (2004) also detected M. immunogenum in fromthegenusAlcaligenes(Table4).Inparticular,A.faecalis
their metalworking fluid samples with selectively designed was found within both the cultured and the directly
PCR probes. Therefore, the relatively low CFUs/cells ratio extracted sequences of all metalworking fluid and aerosol
may be a marker for M. immunogenum presence partly samples.Alcaligenesfaecalisistypicallynonpathogenic,but
because of the slow growth rate of Mycobacterium spp. under certain circumstances, it has been identified in
(Falkinham, 2003; Veillette et al., 2008). However, this conjunction with bone abscesses (Fenollar et al., 2006).
FEMSMicrobiolEcol74(2010)643–654 (cid:4)c2010FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
652 S.D.Perkins&L.T.Angenent
WautersiellafalseniiandP.asaccharolyticumwereidentified Understandingthemicrobialecologywithinthe
in both our fluid and aerosol samples, but are the first metalworking fluids andaerosolswill help
potentialpathogens(Kampferetal.,2006a,b)inthisstudy, guideinterventionstopreventfluidovergrowth
that had not been previously isolated from metalworking
TheconsiderablepresenceofA.faecalisinthemetalworking
fluids or aerosols. From these analyses, it is evident that
fluid and aerosol samples was a significant finding. The
potentiallypathogenicbacteriainmetalworkingfluidswere
abundance of this bacterium could be one reason why
aerosolizedintothemachiningworkplacetopossiblycause
Pseudomonas spp. and Mycobacterium spp. were not the
health concerns for severely immunocompromised metal-
most important pathogens to inhabit these metalworking
workingpersonnel.
fluids.Now,withtheknowledgethatAlcaligenesspp.isthe
abundant bacterium in the fluids and aerosols, a more
AbundantM.immunogenumin metalworking targetedengineeringcontrol(e.g.biocide)canbedeveloped.
fluid did not result inaerosol enrichment Itneedstoberealized,however,thatadiversecommunityof
bacteriainhabitthemetalworkingfluid,andthatcontrolling
EventhoughM.immunogenumhadbecomeenrichedinthe
onegroupofmicroorganismsmaystimulateanothergroup,
winterfluidmachineB,nomycobacteriawerefoundinthe
andthat,therefore,acomprehensivegenesurvey,whichwe
aerosol samples (both the pooled aerosol sample and the
performed in this study, is necessary to ascertain the real
aerosolsampletakeninthevicinityofonlymachineB).This
effectsofmitigationmeasures.
is in contrast to Shelton et al. (1999), who found high
concentrations of Mycobacterium spp. in air samples col-
lectednearmycobacteria-contaminatedmachines.Aerosoli-
Acknowledgements
zationofMycobacteriumspp.haslongbeenassociatedwith
pool and whirlpool environments where a ‘bubble burst’
ThefinancialsupportforS.D.P.wasthrougha2005gradu-
mechanism at the water surface is the primary droplet
ate fellowship from the National Science Foundation. We
source (Schafer et al., 2003; Angenent et al., 2005). The
would like to thank the personnel of the metalworking
hydrophobic mycobacterial cells, which may become
facility for their assistance in this study. We would like to
enriched around the bubble (and therefore may become
acknowledge Elizabeth Campbell and Matt Sculnick for
enriched in aerosols), are ejected in water droplets after
their assistance in lab analyses and anonymous reviewers
whichtheybecomebioaerosolswhenthewaterbubbledries
forexcellentcomments.
up (Vaerewijck et al., 2005). Here, we did not find this
phenomenon possibly because aerosol formation through
metalgrindinghasbeenexplainedbyadifferentmechanism
References
–acombinationof‘jetmist’and‘shearforce’mechanisms,
which generated a bimodal particle distribution in metal- AbramsL,SeixasN,RobinsT,BurgeH,MuilenbergM&
workingaerosols(Heitbrinketal.,2000;Wangetal.,2007). FranzblauA(2000)Characterizationofmetalworkingfluid
Similarly, no Mycobacterium spp. were found in shower exposureindicesforastudyofacuterespiratoryeffects.Appl
aerosolsgeneratedbyajetmistmechanism,whiletheywere OccupEnvironHyg15:492–502.
found at high concentrations in a shower water sample AmannRI,LudwigW&SchleiferKH(1995)Phylogenetic
(Perkinsetal.,2009).Instead,Proteobacteriawas,byfar,the identificationandinsitudetectionofindividualmicrobialcells
mostabundantphylumintheaerosols,whichsupportedthe withoutcultivation.MicrobiolRev59:143–169.
workofthreeoutofthesevenpublishedoutdoorairsurveys AnHR,MainelisG&WhiteL(2006)Developmentand
calibrationofreal-timePCRforquantificationofairborne
and an indoor air survey that showed Proteobacteria to be
microorganismsinairsamples.AtmosEnviron40:7924–7939.
exceeding 60% of the bacterial composition (Maron et al.,
AngenentLT,KelleyST,StAmandA,PaceNR&HernandezMT
2005;Baertschetal.,2007;Fiereretal.,2008).Furthermore,
(2005)Molecularidentificationofpotentialpathogensin
our work agrees with that of Gilbert et al. (2010a,b), who
waterandairofahospitaltherapypool.PNatlAcadSciUSA
found an overall lack of anticipated Mycobacterium spp.,
102:4860–4865.
withonlytwoofthe44sitesbeingpositiveformycobacteria
BaertschC,Paez-RubioT,ViauE&PecciaJ(2007)Source
inthefluidsandnoneintheaerosols.However,wecannot
trackingaerosolsreleasedfromland-appliedclassBbiosolids
ruleoutthatthediscrepancybetweenourstudyandthatof
duringhigh-windevents.ApplEnvironMicrob73:4522–4531.
Sheltonetal.(1999),whodidfindaerosolizedMycobacter-
BarhadB,PilatL&TeculescuD(1975)Recentprogressinthe
iumspp.inthemetalworkingfacility,canalsobeexplained studyofoccupationallungdiseasesinRomania.BritJIndMed
due to different mycobacterial fluid concentrations, 32:164–168.
machine designs, machining operations, room tempera- BernsteinDI,LummusZL,SantilliG,SiskoskyJ&BernsteinIL
tures,andrelativehumiditylevels(Brackeretal.,2003). (1995)Machineoperator’slung.Ahypersensitivity
(cid:4)c2010FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol74(2010)643–654
PublishedbyBlackwellPublishingLtd.Allrightsreserved