Eksponering for mikroorganismer i arbejdsmiljøet

Transkript

1 Eksponering for mikroorganismer i arbejdsmiljøet Anne Mette Madsen, Senior Researcher, The National Research centre of the Working Environment NRCWE/NFA

2 Bacteria, µm Environments: Outdoor Bakterier High pressure cleaning (e.g. Legionella spp.) Indoor Hospitals, Homes, Nursery homes Offices Occupational settings Gram-negative bacteria: endotoxin wwtp, animal breading, composting plants, biofuel plants, MPCA Neighbors to: composting plants, pig farms?

3 Plan Målet er at give en status for, hvor vi er i forskningen, mht. : eksponering for mikroorganismer i arbejdsmiljøet, relateret heldbredseffekter, ved vi hvilke mikroorganismer der er problematiske interventions-muligheder Deponering i luftvejen gartneri og landbrug Eksponering - sammenhæng til effekter interventionsmuligheder Landbrug, gartneri, biobrændsel, skraldemænd og græsfrø Størst fokus på danske arbejdsmiljøer

4 Potentiel deponering i luftvejene baseret på størrelse

5 Potentiel deponering i luftvejene baseret på størrelse MRSA i svinestalde cfu of MRSA/m Farm C Weaner, among pigs, 876 Weaner, window, 386 Weaner, fence, 212 Hallway 1, 177 Hallway 2, 502 Hallway 3, 360 Farrowing, fence, Particle size in µm Madsen, Kurdi, Feld, & Tendal (2018). Airborne Staphylococcus aureus and MRSA as associated with particles of different sizes on pig farms

6 Geometric mean diameter (D g ) af luftbåren MRSA og S. aureus (SA) i seks områder Geometric mean diameter (d g ) d g = exp (Σn i log d i /N)

7 Potentiel deponering af MRSA i luftvejene ved arbejde i svinestalde Gang i stald D g =7.0µm Iblandt svin, D g =5.6µm Entry/feed-storage /hallway, D g =5.5µm Bakterier på luftbåren halmstøv

8 Målt nasal deponering

9 MRSA og nasal deponering og clearance

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11 Symptomer i næsen - landmænd

12 Eksponering og inhalation Association between log-transformed concentrations of fungi, β-glucan, and bacteria in NAL and log-transformed exposure to the same components. Studied for Fungi(25 C) β-glucan Bacteria(25 C) Estimate* [confidence limits] p-value Estimate [confidence limits] p-value Estimate [confidence limits] p-value Men and women 0.43 [ ] Men 0.49 [ ] Women [ ] [ ] [ ] [ ] [ ] [ ] [ ] 0.41 *If estimate is larger than 0 and p<0.05 there is a significant association between exposure and concentration in NAL. Control is made for runny nose and person, and in the non-gender stratified analyses control is made also for gender.

13 Association between fungi and β-glucan in NAL and gender, runny nose and time. Estimates indicate ratios of concentration in NAL between the two levels of each factor. Studied for Gender Runny nose Time* Estimate (Men vs women) [confidence limits] p-value Estimate (Yes vs no) [confidence limits] p-value Estimate (Mon. mor. vs Thur. noon) [confidence limits] p-value Fungi(25 C) in NAL Fungi(25 C) in NAL Fungi(25 C) in NAL Fungi(37 C) in NAL Fungi(37 C) in NAL Fungi(37 C) in NAL β-glucan in NAL Men and women 11.6 [ ] [ ] Men [ ] Women [ ] 0.62 Men and women 1.40 [ ] [ ] 0.73 Men [ ] 0.82 Women [ ] 0.81 Men and women 4.40 [ ] < [ ] [ ] < [ ] < [ ] [ ] [ ] [ ] [ ] 0.014

14 Arbejdsrelateret næse symptomer? Landmænd: The prevalence of nasal symptoms significantly after exposure to to fungal spores/m 3 (OR 6.0, 95% CI ) (Eduard et al., 2007). Børnepasning: Nasal congestion and discharge were associated with total fungal levels, and nasal congestion - also with Aspergillus levels (geometric means 1200 and 32 cfu/m 3, respectively). Cladosporium and Penicillium were the dominating species. Li et al. (1997).

15 Gartneriarbejde og endotoksin-eksponering 10EU ca. 1 ng endotoxin OEL about 5 ng/m 3 corresponds to about 50EU/m 3

16 Inflammation - gartnere Inflammation markers in acute phase response Much higher during infections Cardiovascular diseases SAA: Serum amyloid A CRP: C-reaktiv protein in response to inflammatory stimuli Up 13 blood samples per worker through 3 years, and exposure measurement

17 Interventionsmulighed Eksempel, rydning af udtjente planter straks:

18 Chips 1 Chips 2 Straw 1 Straw 2 Straw 3 Straw 4 Briquettes Pellets Bark chips Industry chips Chips 1 Chips 2 Straw 1 Straw 2 Straw 3 Straw 4 Briquettes Pellets Endotoxin (EU) /kg biofuel Endotoxin (EU) relaesed per kg biomass Total number of fungi/kg biofuel Chips 1 Chips 2 Straw 1 Straw 2 Straw 3 Straw 4 Briquettes Pellets Total number of bacteria/kg biofuel Mikrobiel støvning fra biobrændsler Wood chips and straw were agitated in a rotating drum with a volume of 3.3 m ,0E+06 S1 S2 S3 S4 S5 S6 C1 C2 C3 C ,0E ,0E ,0E+03 Straw 1,0E+02

19 Koncentrationer i områder

20 Partikelkoncentration i områder

21 Eksponering målt på personer

22 Biobrændsel og luftveje Does the use of biofuels affect respiratory health among male Danish energy plant workers? Vivi Schlunssen, Anne Mette Madsen, Simon Skov, Torben Sigsgaard

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24 mg/m Eksempel på interventioner til at reducere eksponeringen på biobrændselsanlæg 2,4 Opriver 0,27 Kaj 1,0 Støv 0,86 0,6 Modtagelse Før Efter 0,44 Før Efter Cfu/m Total antal mikroorganismer Opriver Actinomyceter-mesofile Kaj 2900 Modtagelse Før Efter Antal/m Efter Opriver Kaj Før Modtagelse

25 Eksponering ved arbejde som skraldemand Measurements on 1 or 2 drivers per truck Spend 37% of their working hours in the truck cab GSP-inhalable samplers MALDI-TOF MS for identification at species level Temp. during sampling between 0 and 8 C, RH between %

26 Eksponerings-niveauer cfu/m Fungi Maks=1.2x Maks=4.6x10 4 Maks= Maks= cfu/m Bacteria Maks=4.8x Maks=2.6x10 3 Maks= Maks=160 1 Personal Truck cab Outdoor Reference 1 Personal Truck cab Outdoor Reference Maks=146 Endotoxin 3 EU/m 3 1 Maks= Maks =1.8 Maks =1.4 Personal Truck cab Outdoor Reference

27 Person A.flavus 0.008%(1/13) A.glaucus 0.65% (1/13) A.nidulans 0.86% (3/13) A.niger 3.9% (2/13) A. ochraceus 0.01% (1/13) A.versicolor 3.0% (2/13) Ch. funicola 0.06% (1/13) Cl.herbarum 1.0% (3/13) M. circinelloides 0.007% (1/13) P. brevicompactum 16.8% (8/13) P. camemberti 12.1% (5/13) P. chrysogenum 2.7% (2/13) P. commune 15.7% (10/13) P. digitatum 18.8% (9/13) P. expansum 4.1% (5/13) P. glabrum 3.1% (3/13) P. italicum 9.2% (7/13) P. lanosum 0.22% (1/13) P. olsonii 1.3% (1/13) P. roqueforti 0.22% (1/13) Unidentified 6.2% (6/13) Svampearter Identificeret ved MALDI-TOF-MS Førerhus A. glaucus 0.56% (1/10) A.nidulans 0.56% (1/10) A.niger 0.97% (2/10) A.versicolor 0.81% (2/10) Cr. magnus 0.15% (1/10) P. brevicompactum 10.0% (5/10) 6.7 P. camemberti 4.4% (2/10) P. chrysogenum 6.7% (2/10) P. commune% 10.2 (7/10) P. digitatum% 23.4 (7/10) 10.2 P. expansum 7.6% (4/10) P. glabrum 5.7% (2/10) P. italicum 8.6% (4/10) P. olsonii 15.3% (4/10) 23.4 Pa. variotii 0.85% (1/10) Unidentified 4.0% (6/10) 28 different fungal species Penicillium constituted 84% and 92% of all fungi in personal and truck cab 15 Penicillium species were found 6 Aspergillus species

28 Person Acinetobacter 0.18% (1/13) Arthrobacter 3.9% (2/13) Bacillus 5.7% (4/13) 2.4 Brevibacterium 6.8% (2/13) 6.8 Kocuria 3.5% (2/13) 3.5 Microbacterium 5.1% (3/13) Førerhus Micrococcus 36.3% (12/13) Moraxella 3.3% (2/13) Pseudomonas 1.1% (2/13) Solibacillus 0.78% (1/13) Staphylococcus 21.4% (8/13) Streptococcus 2.4% (1/13) Streptomyces 7.1% (6/13) Unidentified 2.6% (4/13) Acinetobacter 0.81% (1/10) Bacillus 6.1% (2/10) Cellulosimicrobium 2.0% (1/10) Microbacterium 0.67% (1/10) Bakteriearter 81 different bacterial species 11 different Staphylococcus species : S. capitis, S. epidermis, S. equorum, S. haemolyticus S. hominis, S. lentus S. saprophyticus, S. sciuri S. succinus S. warneri S. xylosus Micrococcus 50.3% (8/10) Staphylococcus 27.1% (4/10) Streptomyces 5.9% (3/10) Unidentified 7.0% (4/10) Only few Gram-negative bacteria

29 Affaldssorteringsanlæg, NGS Sorting plant Penicillium Outdoor

30 ODTS og græsfrø

31 Organisk Dust Toxic Syndrome (ODTS) - symptomer: 4-12 hours after exposure Dry Cough Mucosal irritation Shortness of breath Chest Tightness Fatigue Flu-like symptoms Fever Duration of symptoms (2-days- a week)

32 Hvad forårsager ODTS The aetiology of ODTS is unknown Mouldy grain, wood chips, animal farming Organic dust, endotoxin and fungi seem to have been implicated Are certain microorganisms involved? What level of exposure causes ODTS? Organic dust Endotoxin Fungi

33 Formodet udbrud af ODTS på et græsfrøanlæg Few hours after unloading and cleaning of a specific lot of grass seeds 6 out of 12 workers began to experience airway symptoms and flu-like symptoms along with general unease. After coming home the symptoms were worsen. Cleaning was stopped the following morning as two workers were brought to the hospital due to respiratory symptoms.

34 Formål Get knowledge on amounts of microorganisms, endotoxin, and microbial enzymes in dust released from grass seeds causing ODTS versus reference dust Get knowledge on which microbial species are present in dust released from grass seeds causing ODTS versus reference dust Problematic Reference Problematic - cleaned Reference - cleaned

35 Characterisation of aerosols using different approaches Fungi Total number of fungi microscopy Cfu of fungi Identification by MALDI-tof and Sanger Sequencing β-glucan Fungal diversity - sequencing of total fungal DNA Bacteria Total number of bacteria microscopy Cfu of bacteria Identification by MALDI-tof and Sanger Sequencing Bacterial diversity - sequencing of total bacterial DNA Endotoxin Dust Gravimetric Microbial enzymes Total protein Serine protease NAGase β-glucosidase β-xylosidase Xylanase The cultivation and MALDI approach Metabolically active microorganisms produce extracellular enzymes Germinating conidia release allergens Some microorganisms are infectious Measured as cfu Identification by MALDI-tof The sequencing microbial diversity approach Not all species are cultivable on agar media The diversity in problematic versus reference seeds may shift

36 Step 1: kilde til eksponering 40g grass seeds per sample Triplicate Problematic Reference Problematic - cleaned Reference - cleaned

37 Endotoxin units/g seed Endotoxin units/g seed Step 1 Kilde til eksponering, Endotoxin Problematic uc Problematic cunproblematic ucunproblematic c Problematic uc Problematic c Unproblematic uc Unproblematic c

38 Step 1: Kilde til eksponering Table 1. Concentrations (amount kg -1 seed) of endotoxin and microorganisms in grass seed extracts (step 1) Source Endotoxin Mesophilic Aspergillus Thermophilic Mesophilic fungi fumigatus actinomycetes actinomycetes [EU] [cfu] [cfu] [cfu] [cfu] Problematic seeds, uncleaned Problematic seeds, cleaned Reference seeds, uncleaned Reference seeds, cleaned 3x10 9 a 2x10 10 a 5x10 9 a 1x10 11 a 3x10 11 a 2x10 9 b 1x10 10 a 3x10 9 a 1x10 11 a 4x10 10 b 8x10 7 c 3x10 7 c 9x10 3 b 4x10 5 b 2x10 6 d 2x10 7 d 4x10 8 b 9x10 3 b 9x10 3 c 5x10 7 c

39 Step 2: Emission fra græsfrø Four kinds of seeds: cleaned and uncleaned problematic and reference seeds European standard EN using a rotating drum (HSE Rotating Drum Dustiness Tester model EDT38L, Hertfordshire, UK). Bulk samples weighing 200 g ( 270 ml) (as suggested in MDHS 81). The drum: 0.3 m in diameter, equipped with eight internal longitudinal vanes, which lifted the sample and subsequently let it fall at a rate of 4 rpm for 1 minute.

40 Endotoxin EU/g Fungi cfu/g Emission fra græsfrø Problematic uc Problematic c Unproblematic uc Unproblematic c Problematic uc Problematic c Unproblematic ucunproblematic c

41 Step 2: NGS - 16S sequencing Bacterial genera in higher reads GENUS Reference seeds - clean Reference seeds -unclean Problematic seeds - clean Problematic seeds -unclean Achromobacter Acinetobacter Bacillus Brachybacterium Cellulosimicrobium Cupriavidus Enterococcus Erwinia Klebsiella Laceyella Lactococcus Leucobacter Methylobacillus Ochrobactrum Olivibacter Promicromonospora Sphingobacterium Staphylococcus Stenotrophomonas Streptomyces Tetrathiobacter Thermoactinomyces Xylanimicrobium

42 Step 2: NGS - 16S sequencing Bacterial genera in lower reads GENUS Reference Reference Problematic seeds Problematic seeds seeds - clean seeds -unclean - clean - unclean Agrococcus Clavibacter Devosia Dyadobacter Exiguobacterium Flavobacterium Frigoribacterium Hymenobacter Janthinobacterium Kineococcus Luteimonas Methylobacterium Mycoplana Pedobacter Planomicrobium Propionibacterium Rathayibacter Rhodobacter Salinibacterium Sanguibacter Sphingomonas Streptococcus Variovorax

43 Step 2: svampe-slægter i højere reads (NGS ITS) i frø der gav ODTS GENUS Reference seeds - clean Reference seeds - unclean Problematic seeds - clean Problematic seeds - unclean Alternaria Aspergillus Lichtheimia Mucor Penicillium Rhizomucor Rhizopus Sarocladium Trichothecium

44 EndotoxinEU/m 3 Step 3: Eksponering igennem gentagelse af arbejdet der gav ODTS TWA of 8-timers eksponeringsmåling Actinomycetes cfu/m Dust mg/m person person stat. stat. stat. stat. Outdoor

45 Step 3: NGS - 16S sequencing Bakterie-slægter i højere reads - arter med MALDI MALDI-TOF B.cereus B. licheniformis B. subtilis C. cellulans E. casseliflavus K. pneumonia S. epidermidis S. capitis S. pasteuri etc

46 Total 6.5% 48% 73% 97% 11% 9.5% *See Table 3. Step 3: svampe forskelle NGS - ITS sequencing samt MALDI Workers Reference dust Problematic dust Fugal genera 1 2 seeds - clean seeds - unclean seeds - clean seeds - unclean Alternaria MALDI-TOF 1.6%* Al.alternata 0.36% 0.17% 0.067% 1.1% 0.44% Aspergillus A. fumigatus 13% 32% 5.8% 0.39% 31% 28% Lichtheimia A. glaucus 0.39% A. nidulans 0.066% bd bd 0.84% 0.50% Mucor A. niger 0.11% A. penicilloides 0.026% bd bd 0.58% 0.37% Penicillium A. Versicolr 0.71% 0.90% 0.37% 0.053% 2.0% 0.50% Rhizomucor Rhizopus microspores 0.22% 0.053% bd bd 0.54% 0.39% Rhizopus 64% 14% 1.5% 0.48% 39% 53% Sarocladium 0.11% 0.36% 0.10% 0.040% 0.63% 0.98% Trichothecium 7.6% 0.74% 0.22% 0.040% 6.9% 3.5% Total 87% 49% 8.1% 1.1% 83% 87% *See Table 3. Workers Reference dust Problematic dust Fungal genera 1 2 seeds - clean seeds - unclean seeds - clean seeds - unclean Cladosporium MALDI-TOF 5.3%* Cryptococcus neoformans 45% 48% 91% 9.0% 7.9% Cryptococcus 0.85% 1.9% 16% 2.4% 1.1% 0.67% Sporobolomyces 0.32% 1.4% 6.2% 3.1% 0.38% 0.87% Wallemia 0.095% 0.066% 2.2% 0.17% 0.012% bd

47 Afrunding Mange miljøer er forhøjede eksponeringer for endotoksin, svampe og bakterier den forøgede eksponering i arbejdsmiljøer er ofte Penicillium-arter Klare sammenhænge mellem endotoksin eksponering og symptomer på luftveje, inflammationsmarkører Ikke så klare sammenhænge mellem symptomer og svampe og bakterie eksponering nogen gange sammenhæng til svampeeksponering Bør laves interventioner til at reducere eksponering Tak til alle arbejdspladser der har deltaget Tak til kollegaer på projektet Og tak for opmærksomheden