Filters collection
The analyses were performed using filters removed from the air conditioning system of passenger cars during two measurement periods: In: “winter season”, namely a 3-month period from January to March, when the average ambient air temperature was below 10ºC for at least 7 consecutive days, and in “summer season”, slightly longer than the calendar summer, namely a 5-month period from May to September, when the average ambient air temperature continued to be above 10ºC for at least 7 consecutive days. During each measurement period, the new/unused filters were also collected and subjected to further comparative analysis. In each season, the collection of filters was performed from air conditioning systems of 15 randomly selected passenger cars manufactured between 2019 and 2020.
The tested vehicles were equipped with two types of the air conditioning systems:
(a) manual – single-zone air conditioning system, which is controlled directly by the driver. Indoor temperature is regulated by mixing warm air flow (from the heater) with cold air and the driver is not able to set the exact temperature inside the vehicle;
(b) automatic air conditioning system, which is electronically controlled using readings from sensors placed all over the car (e.g. external temperature, internal temperature, and sun sensors). Depending on the specific system used in the cabin, the supplied air is directed either to the front window, the side windows or to the leg space. In order to avoid excessive load on the conditioning system, approx. 20% of the air is taken from the outside, the rest deriving from recirculation of the indoor air. The automatic air conditioning systems may be equipped with a multi-segment heater, with a separate control for the driver and the passenger zones (either dual - or four-zone air conditioning systems). Detailed characteristics of the studied vehicles are presented in Table 1.
Table 1
Characteristics of the examined passenger cars
Car no. | Year of manufacture | Car mileage [km] | Car mileage since the replacement of cabin air filter [km] | Type of air conditioning system |
“winter season” measurements |
1/W | 2008 | 165 000 | 15 000 | Automatic dual-zone |
2/W | 2011 | 37 000 | 10 000 | Automatic four-zone |
3/W | 2016 | 18 295 | 6 595 | Manual |
4/W | 2009 | 207 300 | 20 000 | Automatic dual-zone |
5/W | 2011 | 148 741 | 120 892 | Automatic dual-zone |
6/W | 2009 | 141 000 | 40 000 | Manual |
7/W | 2014 | 37 000 | 15 000 | Manual |
8/W | 2018 | 115 000 | 30 000 | Automatic dual-zone |
9/W | 2007 | 155 000 | 30 000 | Automatic four-zone |
10/W | 2012 | 158 900 | No data available | Manual |
11/W | 2016 | 150 204 | 11 000 | Manual |
12/W | 2015 | 135 737 | 119 000 | Automatic dual-zone |
13/W | 2007 | 173 718 | 162 094 | Automatic four-zone |
14/W | 2000 | 281 021 | 80 000 | Automatic four-zone |
15/W | 2009 | 148 000 | 135 000 | Automatic dual-zone |
“summer season” measurements |
1/S | 2019 | 41 750 | 11 750 | Manual |
2/S | 2000 | 250 000 | No data available | Manual |
3/S | 2013 | 136 200 | 15 000 | Automatic four-zone |
4/S | 2007 | 260 000 | 30 000 | Automatic dual-zone |
5/S | 2010 | 242 500 | 25 000 | Manual |
6/S | 2011 | 88 500 | 15 000 | Manual |
7/S | 2010 | 289 600 | 263 500 | Automatic dual-zone |
8/S | 2013 | 133 000 | 10 000 | Automatic dual-zone |
9/S | 2014 | 201 237 | 24 737 | Manual |
10/S | 2007 | 171 000 | 15 000 | Automatic dual-zone |
11/S | 2006 | 255 600 | 28 000 | Automatic dual-zone |
12/S | 2000 | 282 688 | about 180 000 | Manual |
13/S | 2005 | 155 000 | 50 000 | Automatic dual-zone |
14/S | 2011 | 103 600 | No data available | Automatic dual-zone |
15/S | 2009 | 196 400 | No data available | Automatic dual-zone |
Filter samples processing and analyses
A total of 32 samples of AC filters were separately collected into separate sterile polyethylene bags and subsequently stored in a dry dark place at room temperature (250C ± 2) until further analysis.
A square piece of 10 x 10 cm of the filter fabric was cut out using sterile scalpel, weighed and placed in 100 ml of 1 g/l of peptone + 5 g/l of NaCl solution. The sample was then shaken on an orbital laboratory shaker for 15 minutes at 250 rpm, diluted and then 100 µl of the resulted suspension was inoculated on malt extract agar (MEA, Oxoid Ltd., Basingstoke, UK) supplemented with 0.05g chloramphenicol. After inoculation, agar plates were incubated for 4 days at 30°C, followed by 4 days at 22°C. The concentrations of total culturable fungi were expressed as the number of colony forming units (cfu) on the culture medium per m2 of the examined filter (cfu/m2). A few yeast colonies were identified by Gram stain and then by the API test (API 20 C AUX, BioMérieux, Marcy l'Etoile, Francja). Moulds were stained with lactophenol and identified according to their morphology, using several identification keys (Fisher and Cook 1998; Samson et al. 2004; St-Germain and Summerbell 2011).
During each measurement session, the new/unused filters were also included and analysed in the same way as the filters from studied cars. Species affiliation of the moulds (identified using microscopic methods) was also confirmed with molecular technics.
DNA was isolated from pure fungal cultures grown on MEA plates, using Fungi DNA Mini-Kits (Syngen Biotech, Wroclaw, Poland), according to manufacturer protocol. DNA samples were stored at -800C until further analysis. The isolated fungal DNA was used as a template in PCR with ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primer sets which allow amplification of the fungal genome fragment located between 18S and 28S rRNA genes, covering the ITS1, 5.8S rRNA and ITS2 fragments. Amplified PCR products were purified, sequenced using DNA analyzer (model 3730, Applied Biosystems, Waltham, USA), and compared with the genotypes from the GenBank database (National Center for Biotechnology Information, US National Library of Medicine, Bethesda, USA) using the BLAST (Basic Local Alignment Search Tool) algorithm (White et al. 1990).
Molecular detection of selected Aspergillus species
In order to confirm the presence of at least one of the four species of the Aspergillus genus: Aspergillus fumigatus, A. niger, A. terreus and/or A. flavus in the tested isolates, real-time PCR testing was performed using Aspergillus - Selective screening kit (Genesig, Chandler's Ford, UK), with modifications to increase the sensitivity of the method. An amount of 20 µl of reaction mixture contained 0.5 µl Aspergillus_SCRN primer/probe mix (included in the kit), 2 µl of nuclease-free water (included in the kit), 2.5 µl of isolated DNA, and 5 µl of iTaq Universal Probes Supermix (Bio-Rad, Hercules, USA). Apart from the control strains, the positive control attached to the Aspergillus_SCRN positive control template (FAM) was used. The amplification was carried out using StepOne RT-PCR System (Thermo Scientific, Waltham, USA), under the following conditions: Pre-PCR Read (holding stage) − 30 seconds at 60°C, then holding stage − 2 minutes at 50°C and 10 minutes at 95°C followed by 40 cycles: 15 seconds at 95 ° C and 1 minute at 60°C, and finally Post PCR-Read − 30 seconds at 60°C.
The TaqMan probes used in the experiment were labelled with FAM dye (6-carboxylfluorescein), and fluorescence reading was performed on the blue channel according to manufacturer protocol. The applied tests are characterized by a high sensitivity, of ≥ 90, which allows detection from 1x102 to 1x108 copies of the sought genes in the sample. The test result was considered positive when the amplification curve crossed the threshold line, showing the value of the threshold cycle (Cq). The samples with Cq = 28 ± 3, in the absence of amplification in the negative control and with the values of 16 ≤ Cq ≤ 23 for the positive control (as recommended in the protocol), were considered positive. Only the reactions, for which the amplification efficiency was ≥ 90%, were analysed.
Detection of aflatoxin-producing fungi
The presence of gene fragments regulating the production of aflatoxins in the isolates obtained from fungal cultures was assessed using the PCR method as described by Sherm et al. (2005). The pairs of primers were used that were complementary to the fragments of structural genes aflD and aflO or regulatory aflS and aflR genes, involved in the biosynthesis of aflatoxin B1. The primer sequences are listed in Table 2.
Table 2
Primers, target genes, sequences, and expected PCR/RT-PCR product sizes in detection of aflatoxin-producing fungi
Primer code | Gene | Primer sequence (5’ → 3’) | PCR product size (bp) | RT-PCR product size (bp) |
Nor1-F | aflD | 5'-ACGGATCACTTAGCCAGCAC-3 ' | 990 | 812 |
Nor1-R | 5'-CTACCAGGGGAGTTGAGATCC-3' |
OmtB (F) - F | aflO | 5'-GCCTTGACATGGAAACCATC-3' | 1333 | 1131 |
OmtB (F) - R | 5'-CCAAGATGGCCTGCTCTTTA-3' |
AflJ-F | aflS | 5'-GAGTCCCTGAGTGTCGGCTA-3' | 1450 | 1004 |
AflJ-R | 5'-TCGGTTGTCATGGTTATCCA-3' |
AflR-F | aflR | 5'-TCGGTTGTCATGGTTATCCA-3' | 999 | 999 |
AflJ-R | 5'-TCGGTTGTCATGGTTATCCA-3' |
The reaction mix for aflO, aflR and aflS genes, at a total volume of 25 µl contained 0.2 µl of Taq DNA polymerase (1U, Qiagen, Germantown, USA), 2.5 µl of 10x PCR buffer containing 15 mM MgCl2, 2.5 µl of 2 mM dNTPs (final concentration 0.2 mM), 0.5 µl of 10 µM of each primer pair (final concentration 0.2 µM), 2 µl of isolated DNA and 16.8 µl of nuclease-free water (Qiagen). The 25 µl of reaction mixture for the aflD gene contained: 0.2 µl Taq DNA polymerase (1U; Qiagen), 2.5 µl 10x PCR buffer containing 15 mM MgCl2, 1.5 µl 2 mM dNTPs (final concentration 0.12 mM), 0.25 µl of 10 µM of each primer pair (final concentration 0.1 µM), 2 µl of isolated DNA and 18.3 µl of nuclease-free water (Qiagen).
PCR testing for the aflO gene was performed in the Mastercycler nexus GSX1 thermocycler (Eppendorf, Hamburg, Germany), and for the remaining genes (aflD, aflR and aflS) in the C1000 Thermal Cycler (BioRad), under the following conditions: 5 minutes at 95°C (initial denaturation), 35 cycles, each of which included specific denaturation (30 seconds at 94°C), primer annealing (60 seconds at 55°C), elongation (90 seconds at 72°C), and final extension for 7 minutes at 72° C. Positive control was the reference strain Aspergillus flavus (ATCC-8062) and nuclease-free water (Qiagen) was used as a negative control.
The detection of amplification products was carried out on 1.5% agarose gels (Prona, Basica LE, ABO, Sp. Z o.o., Gdańsk, Poland) and their separation, in a horizontal electrophoresis device (BioRad), with the following parameters: voltage 80V, current 400mA, time: 55 minutes. In order to visualize the results, the gel after electrophoretic separation was soaked in ethidium bromide solution for 20 min, with a final concentration of 2 µg/ml. The electrophoretic separation image was analysed using an Ingenius Syngene Bio Imaging (SYNGENE) gel analysis and documentation system. The product was read in the SYNGENE system in the presence of the following markers: GeneRules 100bp (Thermo Scientific) for aflD and aflR genes (the amplification products were 990 bp and 999 bp, respectively) and DNA Marker Lambda/BstE II (A&A Biotechnology, Gdańsk, Poland) for aflO (1333 bp product) and aflS (1450 bp product) genes.
Scanning electron microscope (SEM) imaging (photos enclosed)
A 5x5 mm fragment of selected air filters (10 samples) was cut out using a sterile scalpel. Filter surface tests were performed with the use of SU8010 cold-field-emission scanning electron microscope (Hitachi, Tokyo, Japan). To improve conductivity, the material was gold-plated using Q150T ES ion sputter gun (Quorum Technologies, Ltd. Lewes, UK). The observations were carried out at the voltage of 10 kV, WD 10 mm, and at magnifications of x100, x500, x1000 and x5000.
Statistical analysis
The data collected during the study were analyzed using Kruskal-Wallis test, Mann-Whitney U test, Spearman correlation analysis and Fisher test. The STATISTICA data analysis software, version 7.1. (StatSoft, Inc., Tulsa, USA, 2006). All the relationships with p < 0.05 were considered statistically significant.