ORIGINAL PAPER Need for database extension for

Chemical Papers 68 (11) 1435–1442 (2014) DOI: 10.2478/s11696-014-0612-0

ORIGINAL PAPER

Need for database extension for reliable identification of bacteria from extreme environments using MALDI TOF mass spectrometry‡ a

Anna Kopcakova,

a,b f

a Institute b Department

d Institute

f Faculty

d,e

Andrej Godany,

of Animal Physiology, Slovak Academy of Sciences, Soltesovej 4–6, 04001 Kosice, Slovakia

of Biochemistry, Faculty of Science, Pavol Josef Safarik University, Srobarova 2, 04154 Kosice, Slovakia

c Faculty

e Faculty

Zuzana Stramova, c Simona Kvasnova, Zuzana Perhacova, a,c Peter Pristas*

of Natural Science, Matej Bel University, Tajovskeho 40, 97401 Banska Bystrica, Slovakia

of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 84551 Bratislava, Slovakia

of Natural Sciences, University of SS Cyril and Methodius in Trnava, J. Herdu Square 2, 91701 Trnava, Slovakia

of Ecology and Environmental Sciences, Technical University in Zvolen, T. G. Masaryka 24, 96001 Zvolen, Slovakia Received 6 December 2013; Revised 21 June 2014; Accepted 26 June 2014

The ability of MALDI TOF MS (matrix-assisted laser desorption ionisation time-of-flight mass spectrometry) to identify cultivable microflora from two waste disposal sites from non-ferrous metal industry was analysed. Despite the harsh conditions (extreme pH values and heavy metal content in red mud disposal site from aluminium production or high heavy metal content in nickel sludge), relatively high numbers of bacteria were recovered. In both environments, the bacterial community was dominated by Gram-positive bacteria, especially by actinobacteria. High-quality MALDI TOF mass spectra were obtained but most of the bacteria isolates could be not identified using MALDI Biotyper software. The overall identification rate was lower than 20 %; in two of the environments tested identification rates were lower than 10 %. As a dominant bacterial species, Microbacterium spp. in drainage water from an aluminium red mud disposal site near Žiar nad Hronom, Bacillus spp. in red mud samples from the same site, and Arthrobacter spp. from nickel smelter sludge near Sereď were identified by a combination of the Biolog system and 16S rRNA sequence analysis. As the primary focus of the MALDI TOF MS-based methodology is directed towards medically important bacteria, reference database spectra expansion and refinement are needed to improve the ability of MALDI TOF MS to identify environmental bacteria, especially those from extreme environments. c 2014 Institute of Chemistry, Slovak Academy of Sciences
Keywords: waste, environment, bacteria, identification, MALDI TOF, mass spectrometry

Introduction The rapid and reliable identification of microorganisms is a crucial requirement in many fields of microbiology. In most microbiology laboratories, identification is currently based on Gram-staining, culture and growth characteristics, and biochemical patterns. These procedures are cumbersome and time-

consuming. Molecular methods have recently been introduced, but these are expensive, time-consuming and not currently suitable for routine identification (Ferreira et al., 2011). The possibility of using MS for the identification of microorganisms was proposed in 1975 (Anhalt & Fenselau, 1975). In the mid-1990s, MALDI TOF MS was used in the identification of bacteria for research

*Corresponding author, e-mail: [email protected] ‡ Presented at the International Conference on Applied Natural Sciences 2013, Nový Smokovec, Slovakia, 2–4 October 2013.

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purposes (Holland et al., 1996) but it took a long time for this technology to arrive in routine microbiology. In 2004, the first complete database for the identification of bacteria became available (Keys et al., 2004). The general principle of MALDI TOF MS revolves around the rapid photo-volatilisation of a sample embedded in a UV-absorbing matrix followed by time-of-flight mass spectrometry (Van Belkum et al., 2012). Samples are prepared by mounting microbial analytes (cell protein extracts or whole cells) with a matrix on the target plate then being desorbed and ionised through energy pulses from an ultraviolet laser. Through random collision in the gas phase, the charge is transferred from the matrix to the microbial molecules and the analytes within the massto-charge range specified are measured. The mass spectrum thus generated is microorganism-specific. The spectrum is automatically compared with the appropriate database for identification of the microorganism (Van Belkum et al., 2012). Based on the similarity to database entries, a score is generated, indicating the level of confidence of identification. Depending on this value, the organism is identified at the genus- or species-level. As with other identification systems (e.g. Biolog MicroPlate System), the reliability of identification is based on the availability of comprehensive reference spectra databases. The MALDI TOF MS was originally applied to rapid identification of clinically important bacteria and is still mainly used for this purpose. There are several commercially available platforms, e.g. Microflex LT (Bruker Daltonics, Bremen, Germany), Vitek MS RUO (bioMérieux, Marcy l’Etoile, France), AXIMAiD (Shimadzu, Kyoto, Japan). All these systems are capable of identifying the majority of clinical samples within five hours and similar identification ratios (92.7 % and 93.2 % correct species identifications) were reported for the Biotyper and Vitek MS systems (Martiny et al., 2012). However, for most of these platforms, the reference databases are biased toward clinical rather than environmental organisms. Environmental pollution, caused by the improper treatment of industrial wastes, has caused serious environmental and social problems. In particular, the non-ferrous metals industry produces large quantities of waste. Most of the non-ferrous minerals and ores available in the earth’s crust are in the form of oxides, sulphides or silicates. The metals are extracted from these through various pyro- and hydro-metallurgical processes, such as smelting, leaching, electrolysing, etc. In each stage of these operations, the undesirable constituents are discarded as liquid or solid wastes, frequently in comparatively large quantities. In most cases the wastes are found to contain considerable amounts of toxic and heavy metal compounds. When such hazardous wastes are dumped in landfills, the weathering process leads to them contaminating the

soil and ground, as well as the surface water and the air. Bioremediation is proposed as an inexpensive, effective and environmentally safe technology that offers new ways of cleaning up hazardous wastes. However, the use of bioremediation is limited by an incomplete understanding of bioremediation processes and the participating microflora. The present study sought to test the ability of the MALDI TOF MS-based methodology to identify bacteria isolated from highly alkaline aluminium brown mud and nickel smelter sludge disposal sites. Endogenous bacteria from heavily contaminated industrial waste disposal sites should potentially be used in bioremediation processes. The available data on the bacterial populations of aluminium red mud or nickel sludge are very limited. Hamdy and Williams (2001) reported the presence of multiple bacterial genera including species of Bacillus, Lactobacillus, Leuconostoc, Micrococcus, Staphylococcus, Pseudomonas, Flavobacterium, and Enterobacter in bauxite residue treated by using various added nutrients and/or hay. To the best of our knowledge, there is no report on the indigenous microflora isolated from nickel sludge.

Experimental Sample collection and analysis Brown mud samples (50 g) and samples of drainage water (10 mL) were collected from three different sites at the Slovalco co. disposal site near Žiar nad Hronom (Slovakia). According to the Slovalco data, the average composition of the mud is as follows: SiO2 (11–14 %), Fe2 O3 (30–35 %), TiO2 (3–4 %), Al2 O3 (10–12 %), CaO (24–26 %), Na2 O (3–6 %). The heavy metal content is (mg kg−1 ): 10, 220, 400, 700, 150, and 800 for Hg, Cu, Cr, V, Pb, and As, respectively (data from Slovalco). Nickel sludge samples were collected at the disposal site of the Niklová huta smelter near Sereď (Slovakia). The average composition of nickel sludge is: Fe (50–80 %), Cr2 O3 (2.5–3.5 %), SiO2 (6–8 %), Al2 O3 (6–8 %), CaO (2.5–3.5 %), Ni (0.17 %), P2 O3 (0.6 %) (Michaeli et al., 2012). The samples were transferred to the laboratory immediately and stored in the cold (4 ◦C) prior to microbiological analysis. The pH of the samples was determined according to the ISO standard. Isolation and growth of bacteria A sterile PBS solution (10 mL) was added to the solid sample (0.5 g) or drainage water (1 mL) and, after 20 min of intensive mixing, the aliquots were spread on a non-selective agar medium (TSA – Tryptone Soya Agar, Oxoid, USA). Cultivation proceeded under aerobic conditions at 22 ◦C for 24–72 h. The individual isolates were selected on the basis of cell and

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colony morphology and used for further analyses. Basic microbiology techniques After repeated sub-culturing and checking for purity, the isolates were identified by Gram-staining and phenotypic patterns. Gram-stained slides of the isolate were examined microscopically to study their cellular morphology. The selected isolates were identified on the basis of their phenotypic pattern using the GEN III MicroPlate system (Biolog, Hayward, USA) or on the basis of 16S rRNA sequence analysis. The total DNA of isolates was extracted by BactozolTM (a kit for the isolation of bacterial DNA) (Molecular Research Centre, Inc., Cincinnati, OH). The DNA thus isolated was used as a template for PCR amplification of 16S rRNA gene fragments. The DNA extracted was amplified using PCR technology and the universal 16S rDNA primers fD1 and rp2 (Weisburg et al., 1991). Amplifications and sequencing of amplified products were performed as previously described (Weisburg et al., 1991). 16S rDNA sequences were compared with those in the Ez-Taxon-e database (Kim et al., 2012) available at http://eztaxon-e.ezbiocloud.net/. MALDI TOF MS analysis, samples preparation MALDI target plates were inoculated by applying a thin film of a small amount of a single colony newly grown overnight directly onto a polished steel MALDI target plate. Alternatively, the biological material (one bacterial colony) was re-suspended in distilled water (300 L). Then, absolute ethanol (900 L) was added, the mixture was centrifuged at 13000g for 2 min, and the supernatant was discarded. Formic acid (70 vol. %, 30 L) was added to the pellet and thoroughly stirred by pipetting prior to the addition of acetonitrile (30 L) to the mixture. The mixture was centrifuged once more at 13000g for 2 min. The supernatant (1 L) was placed on a spot of the steel target plate and air-dried at ambient temperature. Both the microbial film and the supernatant of the extracted proteins were overlaid with 1 L of the matrix solution (a saturated solution of α-cyano-4-hydroxycinnamic acid in 50 % acetonitrile and 2.5 % trifluoroacetic acid) and air-dried (Ferreira et al., 2011). MALDI TOF MS was performed using a Microflex LT instrument (Bruker Daltonics GmbH, Leipzig, Germany) with FlexControl software (version 3.0). The spectra were recorded in the positive linear mode (laser frequency: 20 Hz; ion source 1 voltage: 20 kV; ion source 2 voltage: 18.4 kV; lens voltage: 9.1 kV; mass range: 2000–20000 Da). For each spectrum, 240 shots in 40-shot steps from different positions of the target spot (automatic mode) were collected and analysed. The raw mass spectra obtained for each iso-

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late were imported into Biotyper software-version 3.0 (Bruker Daltonics GmbH, Leipzig, Germany; database version 3.3.1.0) and analysed by standard patternmatching with default settings without any user intervention. Each spectrum was matched against all spectra of the analysed set. The list of score values was used to calculate normalised distance values between the analysed species, resulting in a matrix of matching scores; dendrograms were created using the MALDI Biotyper 3.0 software.

Results and discussion Industrial waste is a waste produced by industrial activity such as that of factories, mills or mines. Much industrial waste is neither hazardous nor toxic; however, industrial waste from the non-ferrous metal industry in particular is known for its toxicity and the waste disposal sites form environments especially hostile to life. In the present experiments, microflora from two waste disposal sites in Slovakia – the red mud disposal site from aluminium production near Žiar nad Hronom and the nickel sludge disposal site near to Sereď, were analysed for cultivable bacteria. Despite the extreme conditions pertaining in all two industrial waste disposal sites examined, relatively abundant cultivable bacteria population were detected. In the samples from the aluminium red mud disposal site, bacteria were detected with a frequency of approximately 80 cfu per mL of drainage water and of approximately 3500 cfu per g of mud (Table 1). On the basis of the cells and colony morphologies, 12 or 19 isolates were selected for further analysis from the drainage water and red mud samples, respectively. Using MALDI TOF MS, high quality spectra were obtained for all the isolates from drainage water. The MALDI TOF MS is an analytical method for microbial identification and characterisation based on the rapid and precise assessment of the mass of molecules in a variable range of 0.1–100 kDa (Cobo, 2013). For two isolates from red mud sample, no peaks could be detected using the sample preparation method recommended by the manufacturer. These two isolates consisted of filamentous Gram-positive bacteria and, on the basis of cell and colony morphologies as well as 16S rDNA analysis, could be identified as members of Streptomyces genus (Table 1). Streptomycetes and related genera (e.g. Nocardia, Actinomyces) are usually either not identified or only poorly identified by the MALDI TOF approach (Bizzini et al., 2011). In most cases, from 10 to 30 well-defined peaks were detected using a m/z ratio of 3000–15000 (Fig. 1). A comparison of the spectra obtained against the database of reference spectra in the Biotyper software made it possible to reliably identify a single isolate from the drainage water sample and seven isolates from the red mud sample (Table 1) at species-level. Cluster analysis split the bacterial population from drainage wa-

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Table 1. Characteristics of bacterial populations in environments studied, as identified by MALDI TOF MS analysis RMDS-ZHa NSSDS-SDb

Characteristics

pH Cultivable bacteria counts Total number of isolates among them Gram-negative Gram-positive Actinomycetes Producing high quality MALDI spectra Identifiedc at species-level (score ≥ 2.0) Identifiedc at genus-level (score range: 1.7–2.0) Not reliable identification (score ≤ 1.7) Total number of species Dominant genus Shannon diversity index

Drainage water

Red mud

13.1 80 cfu per mL 12 2 10 0 12 1 (8.5 %) 2 (17 %) 9 (74.5 %) 6 Microbacterium 2.35

11.6 3500 cfu per g 19 3 14 2 17 7 (41 %) 7 (41 %) 3 (18 %) 14 Bacillus 3.60

8.1 32000 cfu per g 23 0 22 1 22 2 (9.5 %) 10 (45 %) 10 (45 %) 7 Arthrobacter 2.23

a) Red mud disposal site near Žiar nad Hronom (RMDS-ZH); b) nickel smelter sludge disposal site near Sereď (NSSDS-SD); c) identified by MALDI Biotyper. Table 2. Comparison of identification of selected isolates obtained by MALDI TOF MS analysis and 16S rDNA analysis MALDI TOF MS identification

16S rDNA-based identification

Isolate

V6 V9 V10 V11 V12 V13 K1 K6 K7 K20

Organism (best match)

Score

Blastn best hit

not reliable identification not reliable identification Micrococcus luteus Bacillus megaterium not reliable identification not reliable identification Acinetobacter lwofii Acinetobacter calcoaceticus no peak found not reliable identification

1.33 1.52 2.24 2.00 1.40 1.21 1.96 1.98 – 1.36

Brevundimonas bullata Microbacterium hydrogenocarboxydans Micrococcus luteus Bacillus megaterium Microbacterium testaceum Micrococcus lactis Acinetobacter lwoffii Acinetobacter oleivorans Streptomyces malaysiensis Isoptericola halotolerans

ter into six biotypes (species), whereas the population from red mud was separated into 14 biotypes. For the delineation of biotypes (species) a distance level of 500 was used. This distance level is an arbitrary distance limit set for secure reliable species identification (Sauer et al., 2008). For closely related species, however, lower distance limits are observed. Christensen et al. (2012) observed distances as low as 100 between species of Gram-positive, catalase-negative cocci not belonging to the Streptococcus or Enterococcus genera. In the present experiment, isolates K2 and K4 exhibited a distance level of 400 and were identified as Arthrobacter polychromogenes and A. tumbae with identification scores of 2.375 and 1.887, respectively. Selected isolates were identified by 16S rDNA analysis. The 16S rDNA gene-sequence comparison has emerged as a favoured genetic technique for the identification of bacteria. This analysis can readily identify poorly described, rarely isolated or phenotypically aberrant strains (Clarridge, 2004). For most isolates, a good correlation was observed between identifica-

Similarity/% 99.6 99.7 99.6 99.7 99.8 100.0 99.7 99.1 99.2 99.7

tion by MALDI TOF MS and 16S rDNA analyses. Isolates V10 and V11 were identified as Micrococcus luteus and Bacillus megatherium, respectively, using both methods. Similarly, for the isolate K1 which was identified as Acinetobacter lwofii at genus-level only (MALDI TOF MS score: 1.96), identification was confirmed by the 16S rDNA sequence comparison, confirming the identification usefulness of MALDI TOF MS analysis. On other hand, isolate K6, identified as Acinetobacter calcoaceticus using MALDI with a similar score value, was shown to be Acinetobacter oleivorans using 16S rDNA analysis. Other isolates not identifiable using the MALDI TOF MS approach were identified as members of Brevundimonas, Microbacterium, and Isoptericola genera (Table 2.) The V12 isolate was identified as Microbacterium testaceum using 16S rDNA analysis, a species which is included in the MALDI Biotyper database. However, neither the MALDI TOF MS nor the Biolog systems identified this isolate as M. testaceum and it is probably a new species of Microbacterium genus. No other non-

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Fig. 1. Normalised MALDI TOF mass spectra of selected isolates from aluminium red mud disposal site near Žiar nad Hronom: isolate K9 identified as Bacillus cereus with high probability (identification score: 2.369) (A); isolate V11 not reliably identified (identification score: 1.601) (B). The m/z ratio of dominant peaks is shown.

identifiable species is included in the MALDI Biotyper database version 3.3.1.0, used for the identification of isolates, which would explain why no reliable identification was obtained by the MALDI TOF MS approach. In the drainage water sample, only Micrococcus luteus was identified reliably at species-level but the standard microbiological approaches combined with 16S rDNA analysis identified the actinobacteria of Microbacterium spp. as dominant members of this community. In the red mud sample, bacteria of Arthrobacter polychromogenes, Kocuria rosea, M. luteus, B. megaterium and B. cereus species were identified at reliable species-identification level. The bacterial population was dominated by Bacillus spp. The cluster analysis implemented in the MALDI Biotyper soft-

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ware grouped isolates according to the similarity of their mass spectra. The analysis showed that isolates V3, V8, V9, and V17 are practically identical with a distance-level below 50. Another group was formed by V4, V11, and V16 showing higher distance-levels (Fig. 2). The isolates were identified as Bacillus megaterium albeit with low confidence (identification score in the range of 1.9–2.1). On the basis of phenotypic and 16S rDNA identifications, all three isolates were confirmed as B. megaterium. Surprisingly, analysis indicated the bacterial populations of the drainage water and the red mud from the aluminium red mud disposal site to be substantially different (Fig. 1). Analysis showed that different bacterial populations reside in red mud and drainage water environments when only a single common species, M. luteus, was detected. This bacterium is capable of long-term survival under stress conditions (Kaprelyants & Kell, 1993). Data on the bacterial populations of aluminium red mud are very limited. A single report by Hamdy and Williams (2001) described the presence of multiple bacterial species in treated bauxite residues. In a difference from the present results, a much higher occurrence of Gram-negative bacteria was detected; however, the red mud samples were treated by using various added nutrients and/or hay, so not all species could be considered autochthonous for the red mud environment. As in the present data, Hamdy and Williams (2001) reported the presence of Bacillus spp. and Micrococcus spp. in the red mud environment. In alkaline and heavy metal-contaminated environments, actinobacteria are frequently detected as a dominant cultivable bacteria. Borsodi et al. (2005) reported that actinobacteria-related species dominated within alkali-philic and alkali-tolerant bacteria cultivated from decomposing reed rhizomes in a Hungarian soda lake. Similarly, actinobacteria were found in heavily contaminated soils in Scotland (Ellis et al., 2003) or in heavy metal-contaminated soil in the Upper Silesia region in Poland (Margesina et al., 2011). A less varied bacterial population was detected in the nickel sludge disposal site. Despite the cultivable bacteria counts being higher than in the other environment (more than 32000 cfu per g), the MALDI TOF MS analysis detected the presence of seven biotypes (species) within the 22 isolates studied. As with the red mud sample, one isolate belonging to the actinomycetes group was found not to produce reliable mass spectra upon analysis. The bacterial population of the nickel smelter sludge disposal site was dominated by actinobacteria, namely strains of two species of Arthrobacter genus (A. sulphureus and A. psychrophenolicus); however, only two out of 22 isolates were identified at species-level. No Gram-negative bacteria were detected in this environment (Table 1). To the best of our knowledge, there are no reports on the microflora of nickel smelter sludge but actinobacteria were found to belong to the dominant nickel-resistant

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Fig. 2. Dendrogram of relatedness of selected isolates from aluminium red mud disposal site near Žiar nad Hronom based on MALDI TOF protein mass spectra comparison. Identification results and scores (average from two experiments) are shown. Isolates from drainage water are marked by letter V, isolates from red mud by letter K.

bacteria in serpentine soil in central Italy (Mengoni et al., 2001) and Arthrobacter spp. are typical representatives of bacteria even in non-polluted soil (Su et al., 2004). In general, despite the extremely harsh conditions (extreme pH values and heavy metal content in the red mud disposal site from aluminium production or the high heavy metal content in nickel sludge), relatively high numbers of bacteria were recovered. The cell counts observed (up to 104 cultivable bacteria per gram of nickel sludge) are significantly lower than those for pristine soils. For example, Su et al. (2004) reported bacterial counts as high as 107 cultivable bacteria per gram of arid soil in China. In both the red mud and nickel sludge environments, the bacterial community was dominated by Gram-positive bacteria, especially by actinobacteria. Actinobacteria are typical representatives of soil microflora; in the study by Su et al. (2004) referred to above, actinobacteria represented up to 30 % of cultivable microflora. As expected, due to heavy contamination, the diversity of the bacterial population was found to be very low. For pristine soil, the Shannon diversity index is usually over 4.0 and decreases as the contamination level increases (Wang et al., 2007). High-quality MALDI TOF mass spectra were obtained for all the bacteria other than actinomycetes. Identification rates were rather low; in two environments (drainage water from red mud disposal site and nickel smelter sludge disposal site) they were lower than 10 %. A higher identification rate was observed for bacteria from the red mud sample, probably due to the prevalence of Bacillus spp. in this environment.

These bacilli are Gram-positive bacteria of clinical as well as of industrial importance (Logan, 2012), hence are well-represented in the MALDI Biotyper reference database. There are (as in database version 3.2.1.0) 141 entries (species or subspecies) of Bacillus spp. as opposed to 81 or 84 entries for Microbacaterium and Arthrobacter spp., respectively. Overall, 43 % of bacterial isolates from the extreme environments in the present experiments were not reliably identified using MALDI TOF MS and fewer than 20 % were identified at species-level. The MALDI TOF MS was found to be a fast and responsive diagnostic tool for the typing but not for the identification of bacteria from extreme environments. The method was originally developed for the rapid identification of clinically important bacteria and is still mainly used for this purpose. For example, Eigner et al. (2009) reported over 95 % of correctly identified bacterial species of clinical importance using the same approach as in the present study. Accuracy in the identification of Enterobacteriaceae, nonfermenting Gram-negative rods, staphylococci, enterococci, and streptococci with the MALDI Biotyper was found to be 95.5 %, 79.7 %, 99.5 %, 100 % and 93.7 %, respectively. Similarly, high identification rates were reported by Van Veen et al. (2010). Correct species identification by MALDI TOF MS was observed in 97.7 % of Enterobacteriaceae, 92 % of non-fermentative Gram-negative bacteria, 94.3 % of staphylococci, 84.8 % of streptococci, 84 % of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella and Kingella) and 85.2 % of yeasts.

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A. Kopcakova et al./Chemical Papers 68 (11) 1435–1442 (2014)

The application of MALDI TOF MS in environmental microbiology remains limited. MALDI TOF MS has been successfully applied to a number of taxa, e.g. of Arthrobacter species (Vargha et al., 2006), Leuconostoc spp., Fructobacillus spp. and Lactococcus spp. (De Bruyne et al., 2011) or even for archae or extremophiles (Krader & Emerson, 2004). In all these reports, however, MALDI TOF MS was applied to pure cultures identified by the 16S rRNA-sequencing approach. On the other hand, Edouard et al. (2012), who used the MALDI TOF MS approach mainly for environmental isolates of Propionibacterium spp., reported identification rates as low as 18.7 % and proposed database-enrichment for the reliable identification of environmental bacteria. MALDI TOF MS has confirmed its potential as a rapid screening method for determining similarities between bacterial isolates comparable with and sometimes surpassing the discriminatory power of the 16S-based approach. Koubek et al. (2012) compared MALDI TOF MS with two other methods for the taxonomic identification of bacterial isolates obtained from sediment samples contaminated with polychlorinated biphenyls. As with the other method, MALDI TOF MS was capable of discriminating between four groups of isolates but was unable to identify them down to the species level. In general, environmental bacteria have not been widely evaluated using MALDI TOF MS and are largely absent from the identification databases, limiting the practical extent of this new technique.

Conclusions The MALDI TOF MS proves a reliable method for the typing but not for the identification of most bacterial isolates from heavily contaminated non-ferrous metal industry waste disposal sites. The present data indicate that reference database spectra expansion and refinement are needed to improve the ability of MALDI TOF MS to identify environmental bacteria, especially those occurring in extreme environments. Acknowledgements. This publication is the result of the implementation of project no. 26220120001 supported by the Research & Development Operational Programme funded by the ERDF. A part of the data was presented at the 4th International Scientific Conference “Applied Natural Sciences 2013” in Nový Smokovec, High Tatras, Slovakia and appeared in the Conference Proceedings CD-ROM.

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