Systematic and Applied Microbiology 45 (2022) 126339

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Systematic and Applied Microbiology

journal homepage: www.elsevier.com/locate/syapm

Nocardioides carbamazepini sp. nov., an ibuprofen degrader isolated from

a biofilm bacterial community enriched on carbamazepine
Tibor Benedek a,⇑,1, Márton Pápai a,1, Kholood Gharieb a, Anna Bedics a, András Táncsics a, Erika Tóth b,
Hussein Daood c, Gergely Maróti d, Roland Wirth d, Ofir Menashe e,f, Károly Bóka g, Balázs Kriszt h
a
Department of Molecular Ecology, Hungarian University of Agriculture and Life Sciences, Gödöllo }, Hungary
b
Department of Microbiology, Eötvös Loránd University, Budapest, Hungary
c
}, Hungary
Institute of Horticultural Sciences, Laboratories of Food Analysis, Hungarian University of Agriculture and Life Sciences, Gödöllo
d
Institute of Plant Biology, Biological Research Center, ELKH, Szeged, Hungary
e
Water Industry Engineering Department, Engineering Faculty, Kinneret Academic College on the Sea of Galilee, D.N. Emek Ha’Yarden, Israel
f
BioCastle Water Technologies Ltd., Edison Park, Israel
g
Department of Plant Anatomy, Eötvös Loránd University, Budapest, Hungary
h
Department of Environmental Safety, Hungarian University of Agriculture and Life Sciences, Gödöllo }, Hungary

a r t i c l e i n f o a b s t r a c t

Article history: From the metagenome of a carbamazepine amended selective enrichment culture the genome of a new to
Received 12 February 2022 science bacterial species affiliating with the genus Nocardioides was reconstructed. From the same enrich-
Revised 11 May 2022 ment an aerobic actinobacterium, strain CBZ_1T, sharing 99.4% whole-genome sequence similarity with
Accepted 31 May 2022
the reconstructed Nocardioides sp. bin genome was isolated. On the basis of 16S rRNA gene sequence sim-
ilarity the novel isolate affiliated to the genus Nocardioides, with the closest relatives Nocardioides
kongjuensis DSM19082T (98.4%), Nocardioides daeguensis JCM17460T (98.4%) and Nocardioides nitropheno-
Keywords:
licus DSM15529T (98.2%). Using a polyphasic approach it was confirmed that the isolate CBZ_1T repre-
Nocardioides carbamazepini
Ibuprofen
sents a new phyletic lineage within the genus Nocardioides.
Carbamazepine According to metagenomic, metatranscriptomic studies and metabolic analyses strain CZB_1T was
Biodegradation abundant in both carbamazepine and ibuprofen enrichments, and harbors biodegradative genes involved
Metagenomics in the biodegradation of pharmaceutical compounds. Biodegradation studies supported that the new spe-
Metatranscriptomics cies was capable of ibuprofen biodegradation. After 7 weeks of incubation, in mineral salts solution sup-
Binning plemented with glucose (3 g l
1) as co-substrate, 70% of ibuprofen was eliminated by strain CBZ_1T at an
initial conc. of 1.5 mg l
1.
The phylogenetic, phenotypic and chemotaxonomic data supported the classification of strain CBZ_1T
to the genus Nocardioides, for which the name Nocardioides carbamazepini sp. nov. (CBZ_1T = NCAIM
B.0.2663 = LMG 32395) is proposed.
To the best of our knowledge, this is the first study that reports simultaneous genome reconstruction of
a new to science bacterial species using metagenome binning and at the same time the isolation of the
same novel bacterial species.
Ó 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction tems, and it makes obvious that pharmaceutical residues are

present in surface water, groundwater and even in drinking water.
An unprecedented increase in the consumption and simultane- The most commonly detected pharmaceuticals in the freshwa-
ous release and accumulation of pharmaceuticals in the environ- ter ecosystems are diclofenac (DIC), ibuprofen (IBU) and carba-
ment can be witnessed worldwide due to the growing mazepine (CBZ), they can be detected in surface- and
population. The review of Patel et al. [1] summarizes the occur- groundwater, as well as finished drinking water [2–4]. These com-
rence of pharmaceutical residues in aquatic and terrestrial ecosys- pounds enter the environment mainly through municipal and
industrial wastewater effluents [2].
Pharmaceutical residues are gaining more and more attention
⇑ Corresponding author.
since they can produce adverse effects on aquatic organisms.
E-mail address: [email protected] (T. Benedek).
1
Both authors contributed equally to this work.
It has been demonstrated that at environmentally relevant

https://doi.org/10.1016/j.syapm.2022.126339
0723-2020/Ó 2022 The Author(s). Published by Elsevier GmbH.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

concentrations DIC, IBU and CBZ may exert ecotoxic effect on rain- (0.5 g) and the enrichment cultures of each month was extracted
bow trout (Oncorhynchus mykiss), mussels (Mytilus galloprovincialis using the DNeasyÒ PowerBiofilm Kit (Qiagen, Germany) following
and Dreissena polymorpha), crustacean (Gammarus pulex), adult the instructions of the manufacturer. From enrichment cultures,
zebrafish (Danio rerio), Japanese medaka (Oryzias latipes) among 40 ml were centrifuged at 2360 g for 15 min using a Rotanta 460
others [5–11]. Therefore, it must be a priority to understand the R centrifuge (Hettich, Germany) and the community DNA was
ultimate environmental fate of pharmaceutical residues and to extracted from the pellet.
investigate the microbiology behind their biodegradation. The quantity of DNA samples was estimated using a NanoDrop
We recently committed to the selective enrichment, identifica- ND-1000 spectrophotometer (NanoDrop Technologies, Wilming-
tion and isolation of pharmaceuticals-, especially DIC, IBU and CBZ, ton, USA) and a Qubit 2.0 Fluorimeter (Life Technologies, Carlsbad,
degrading bacteria. In the present paper we report on a novel USA). Paired-end fragment libraries were prepared using the NEB-
Nocardioides species capable of ibuprofen biodegradation obtained NextÒ UltraTM II DNA Library Prep Kit for Illumina. Paired-end frag-
from a biofilm bacterial community selectively enriched on carba- ment reads were generated on an Illumina NextSeq sequencer
mazepine as sole source of carbon and energy. using TG NextSeqÒ 500/550 High Output Kit v2 (300 cycles).
Based on the List of Prokaryotic Names with Standing Nomen- For the metatranscriptomic study, total RNA from the pellet
clature (www.bacterio.net; [12]), the genus Nocardioides currently (40 ml of enrichment culture centrifuged at 2360 g) of the three
comprises 136 validly described species. The genus was proposed months old enrichment cultures was extracted using PureLinkTM
by Prauser [13]. Nocardioides species are Gram-positive, coccus- RNA Mini Kit (Invitrogen, Thermo Fisher Scientific). Metatranscrip-
shaped actinobacteria often isolated from diverse habitats such tome sequencing was performed as follows: paired-end libraries
as seawater, freshwater spring, soil, sediments or even from xeno- were prepared using the Zymo-Seq RiboFreeTM Total RNA Library
biotics contaminated environments [14–23,14,24]. Some members Kit (the protocol includes the universal rRNA depletion step as
of the genus such as N. aromaticivorans H-1T, N. nitrophenolicus NSP well). Sequencing was done on an Illumina NextSeq sequencer
41T, N. oleivorans BAS3T, N. daeguensis JCM 17460T, N. pyridinolyti- using the TG NextSeqÒ 500/550 High Output Kit v2 (300 cycles).
cus OS4T and N. soli mbc-2T were described as dibenzofuran, p- Primary data analysis (base-calling) was carried out with ‘‘bcl2-
nitrophenol, crude-oil, chlorophenols, pyridine and carbendazim fastq” software (v2.17.1.14, Illumina).
degrading organism, respectively [19–21,25,26]. Other Nocardiodes
isolates have been reported as atrazine, phenanthrene, s-triazine Raw sequence filtering and co-assembly
herbicide, deoxynivalenol and 2–4-dinitroanisole degrading bacte- Galaxy Europe server was employed to pre-process the raw
ria [27–31]. To the best of our knowledge no Nocardioides pure iso- sequences (i.e., sequence filtering, mapping, quality checking).
lates have been reported so far capable of ibuprofen Low-quality reads were filtered by Prinseq (min. length: 100;
biodegradation. min. score: 15; quality score threshold to trim positions: 20; slid-
ing window used to calculated quality score: 1). Filtered sequences
were checked with FastQC. The filtered sequences produced by
Materials and methods
Prinseq were co-assembled with Megahit [36] (min. contig length:
1500; min k-mer size: 21; max k-mer size: 141). Downstream tax-
Selective enrichment cultures
onomical analysis and visualization of the retained metagenomic
sequence reads were performed by the MEGAN6 software [37].
The selective enrichment of potentially pharmaceutical degrad-
Raw metagenomic and metatranscriptomic sequence reads are
ing bacteria from a microbial biofilm was conducted in mineral
available on NCBI under the following BioProject accession num-
salts solution amended with vitamins [32]. The enrichment cul-
bers PRJNA782474.
tures contained as sole source of carbon and energy either diclofe-
nac sodium, ibuprofen or carbamazepine (100 mg l
1). The biofilm
Reconstruction of bacterial genomes from the metagenomes
sample was collected from a Pump & Treat system treating BTEX
(metagenome binning)
(benzene, toluene, ethyl-benzene, xylenes) contaminated ground-
The quality filtered, trimmed and assembled reads obtained
water. Both the groundwater remediation system and the starting
from the previous step were used for assembling genomes from
biofilm bacterial community were thoroughly characterized ear-
the metagenomes. Binning of the contigs was carried out with
lier. The biofilm bacterial community has already been proven to
three different binning algorithms: Metabat2 [38], Maxbin2 [39]
be the source of isolation of simple- and polycyclic aromatic hydro-
and Concoct [40]. The result of each binning procedure was further
carbon degrading bacteria [33–35].
improved with Metawrap [41]. Bin qualities were estimated with
The enrichments were done in 300 ml Erlenmeyer flasks (sealed
CheckM [42] and bin taxonomy was determined using the GTDB
with cotton wool) containing 100 ml of enrichment media inocu-
taxonomic database [43]. The metagenome assembled genome rel-
lated with 1 ml of biofilm bacterial suspension (0.5 g biofilm sus-
evant to this study, Nocardioides sp. bin2, can be accessed through
pended in 50 ml saline solution – 0.9% NaCl). The enrichment
NCBI under the accession number JAJTIU000000000.
cultures were incubated at 28 °C on a rotary shaker (150 rpm)
for 3 months in total. After one and two months of incubation
Calculation of RPKM values based on metagenome and
sub-cultivations took place; 10 ml of the enrichment culture was
metatranscriptome data
transferred to 90 ml of fresh enrichment medium and incubated
Phylogenetic diversity of metagenomes indicated that the rela-
again for one additional month.
tive abundance of Nocardioides spp. was considerable in CBZ and
IBU enrichments. In connection with this, genome-resolved
Metagenomic and metatranscriptomic studies of enrichment cultures metagenomics resulted in a high quality Nocardioides related bin
genome (100% completeness, NCBI accession number
Genomic DNA and mRNA purification JAJTIU000000000).
To assess the phylogenetic diversity of the samples, shotgun To get information about the incidence and activity of the bin
metagenome sequencing was performed on the total community related Nocardioides species in different enrichment cultures dur-
DNA of the initial biofilm sample and of the pharmaceutical ing the whole enrichment period, RPKM (read per kilobase per mil-
amended enrichment cultures using Illumina platform (Illumina lion mapped reads) values were calculated using metagenome data
Inc., USA). Total community DNA from the initial biofilm sample obtained from enrichment cultures and the reconstructed bin
2
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

genome. In the case of the three-months-old enrichment cultures lyzer (Life Technologies, USA). The resulted sequences were edited
the RPKM values were calculated also on metatranscriptome data. and assembled using MEGA X [44], then homology BLAST searches
The RPKM values were calculated as follows: [45] were made in the GenBank database (https://www.ncbi.nlm.
nih.gov/BLAST/). EzTaxon-e server carried out the determination of
RPKM ¼ number of mapped reads= the closest type strains of isolates on the basis of 16S rRNA genes
 
genome length total number of reads (https://eztaxon-e.ezbiocloud.net/, [46]. 16S rRNA gene sequence of

1000 1; 000; 000 strain CBZ_1T was deposited in the GenBank under the accession
number MZ408918.
where the ‘‘number of mapped reads” were measured by Bowtie2. Multiple alignment of sequences, calculation of evolutionary
First, Bowtie2 was used to create library from the Nocardioides sp. distance by Kimura’s two parameter model [47], and construction
bin2 related bin genome, then it was mapped back to the whole of a neighbor-joining [48] phylogenetic tree were performed using
gDNA sequences and mRNA sequences originated from the enrich- MEGA X. The topology of the trees was evaluated by bootstrapping
ment cultures. The ‘‘genome length” is the size of the used bin gen- with 10,000 resamplings [49].
ome, and the ‘‘total number of reads” are the number of filtered
reads produced by Prinseq. Phylogenomic tree reconstructions

Bacterial isolation and studied bacterial strains As a first attempt, for genome-based phylogeny the UBCG (up-
to-date bacterial core gene) phylogenomic pipeline and 92 core
The isolation of bacterial strains from the enrichment cultures genes (UBCGs) were used. The phylogenetic tree was done for each
was performed after each month of incubation; ten-fold serial dilu- gene as well as a concatenated sequence of the 92 UBCGs (UBCG
tions in saline solutions were made and inoculated onto R2A agar tree) using the available genomes on the NCBI of those Nocardioides
plates (proteose peptone 0.5 g, casamino acids 0.5 g, yeast extract species that were used for the generation of the 16S rRNA gene
0.5 g, dextrose 0.5 g, soluble starch 0.5 g, dipotassium phosphate based phylogenetic tree. The tree generated from the concatenated
0.3 g, magnesium sulfate7H2O 0.05 g, sodium pyruvate 0.3 g, agar alignment is representing the true evolutionary history of whole
15 g, pH 7 ± 0.2). The plates were incubated at 28 °C for 48 h. The genomes, however it may be different from those generated using
developed colonies were purified by streak plating and maintained individual gene trees. To estimate the robustness of each branch
on R2A agar slants at 4 °C, and stored long-term at
80 °C in a the Gene Support Index (GSI) was calculated. The higher the GSI
glycerol-R2A solution (30% v/v). the more robustly the branch is supported [50].
A bacterial strain forming whitish, translucent, flat colonies To confirm the obtained UBCG phylogenetic inference, a second
with irregular margins on R2A agar, the representative of the phylogenomic tree was generated using 101 bacterial whole gen-
new bacterial species on which this study is based on, Nocardioides omes and 120 bacterial marker genes belonging to type strains of
sp. nov. CBZ_1T, was isolated from the one-month-old carba- different Nocardioides species. As a first step, the GTDB-Tk clas-
mazepine enrichment. sify_wf workflow was used for the generation of GTDB-Tk MSA file
The reference organisms used for taxonomic studies were type that was used for the generation of the phylogenetic tree using the
strains of Nocardioides species such as N. kongjuensis DSM19082T, IQTree program (number of bootstrap alignments 1000, maximum
N. nitrophenolicus DSM15529T and N. daeguensis JCM17460T. The iteration 1000, minimum correlation coefficient 0.99, perturbation
strains with DSM and JCM numbers were obtained from the strength 0.5, IQTree stopping rule 100). Subsequently, the obtained
DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkul- Newick file was visualized with the iTOL program.
turen GmbH (Braunschweig, Germany) and the Japan Collection
of Microorganisms, RIKEN (Wako, Japan), respectively. Pan-genomic study and the environmental relevance of the novel
At different stages of the study, for the cultivation of all strains Nocardioides sp.
R2A medium was used, unless specified otherwise.
To see the difference in genomic content between the novel
16S rRNA gene sequencing and phylogenetic analyses species and other Nocardioides spp. pan-genome analysis was per-
formed with the closest 23 Nocardioides spp. determined on the
Genomic DNA of isolate CBZ_1T was extracted by using the basis of the whole-genome based phylogenetic tree (Fig. S1, thick-
DNeasyÒ UltraCleanÒ Microbial DNA isolation Kit (Qiagen, Germany). ened branch).
Bacterial species specific 16S rRNA genes were PCR amplified by Using Anvi’o 7 a database was created from the 25 genomes
using the universal bacterial primers 27F (50 -AGAGTTTGATC(A/C)TG (anvi-gen-contigs-database and anvi-gen-genomes-storage). From
GCTCAG-30 ) and 1492R (50 -TACGG(C/T)TACCTTGTTACGAC TT-30 ). the obtained database a pan-genome database was generated
The PCR reaction mixture in a final volume of 50 ll contained, 5 ll using anvi-pan-genome. The pan-genome database was supple-
10  DreamTaqTM buffer (ThermoFisher Scientific, Lithuania) with mented with ANI calculations using the FastANI program within
MgCl2 (2 mM), 0.2 mM of each dNTP, 0.1 lM of each primer, 1 U Anvi’o (anvi-compute-genome-similarity). The obtained results
of DreamTaqTM DNA Polymerase (ThermoFisher Scientific, Lithua- were visualized with anvi-display-pan and summarized using
nia), 30 ng extracted DNA and nuclease free water up to the final anvi-summarize. Unique genomic islands (UGIs) characteristic to
volume. The temperature profile used for the amplification was an the novel species were also determined.
initial annealing for 3 min at 95 °C followed by 32 cycles of denatu- In order to see if the obtained novel Nocardioides sp. is present
ration for 30 sec at 94 °C, annealing for 30 s at 52 °C, elongation for in other metagenomes, e.g. those exposed to pharmaceutical com-
1 min at 72 °C and then a final extension for 10 min and 10 s at 72 °C. pounds, recruitment of metagenomic reads against the genome of
All amplifications were analyzed under UV light after electrophoresis strain CBZ_1T and the reconstructed Nocardioides sp. bin2 genome
in 1% (w/v) agarose gel stained with ethidium-bromide. The PCR was done and the relative abundance values were calculated using
amplicons were purified by using NucleoSpinÒ Gel and PCR Clean- the CoverM pipeline (https://github.com/wwood/CoverM). For this
up set (Macherey-Nagel, Germany). Subsequently, 16S rRNA gene step metagenomic sequence reads from pharmaceutical (trimetho-
nucleotide sequences were determined with Sanger-sequencing by prim, sulfamethoxazole and carbamazepine) amended liquid
using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technolo- enrichment cultures were used from a previous study ([51];
gies, USA) the sequences were analyzed with ABI 3130 Genetic Ana- BioProjectAccession PRJNA286671).
3
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

Morphological, physiological, and biochemical tests into fatty acid methyl esters (FAMEs) by saponification, methyla-
tion and extraction using minor modifications of the method of
Cell size, shape and arrangement of the type strain CBZ_1T were Miller [59] and Kuykendall [60]. The fatty acid methyl esters mix-
studied by native preparations and by Gram staining according to tures were separated by gas chromatography and detected by a
Claus [52]. The cell morphology was investigated using transmission flame ionization detector using Sherlock Microbial Identification
electron microscopy (Morgagni 268). For transmission electron System (MIS) (MIDI, Microbial ID, Newark, DE 19711 U.S.A.). Peaks
microscopy cells were negatively stained with 1% (w/v) uranyl acet- were automatically integrated and fatty acid names and percent-
ate [53]. The growth of strain CBZ_1T in different growth media such ages calculated by the MIS Standard Software (Microbial ID).
as R2A, nutrient (proteose peptone 5 g, beef extract 3 g, NaCl 5 g), LB
(tryptone 10 g, yeast extract 5 g, NaCl 5 g) and TSA – trypticase soy Whole genome sequencing (WGS) and analysis
(tryptone 15 g, soy peptone 5 g, NaCl 5 g) was determined. The tem-
perature tolerance of the isolate was assessed in R2A medium at 4, The genome of strain CBZ_1T was sequenced as described ear-
15, 23, 28 and 37 °C. The optimum pH for growth was assessed in lier in Borsodi [61]. Briefly, Nextera Mate Pair Sample Preparation
R2A broth and the medium was adjusted to pH 4.0–12.0 (at intervals Kit (Illumina, U.S.A) was used to generate mate-paired libraries
of 1 pH unit) using citrate/NaH2PO4 buffer (0.1 M, for pH range 4.0– according to the manufacturer’s protocol for gel-plus version with
5.0), phosphate buffer (0.1 M, for pH range 6.0–7.0), Tris buffer slight modifications. A total of 13 ll of Mate-Paired Tagment
(0.2 M, for pH range 8.0–10.0) and NaOH (5 M, for pH range 10.0– Enzyme was used to produce a robust smear within the 7–11
12.0). Tolerance towards salinity was determined by inoculating kbp region. The 7–11 kbp DNA fraction was excised from the gel
the strain into R2A broth supplemented with 0–4% (w/v) NaCl at using the Zymoclean Large Fragment DNA Recovery kit (Zymo
1% intervals. During determination of optimal cultivation conditions Research, U.S.A) and the circularized DNA was sheared using Cov-
and tolerance tests the bacterial growth was followed by measuring aris S2. All quality measurements were performed on a TapeStation
the optical density (OD) of the bacterial suspension at 600 nm. 2200 instrument (Agilent, U.S.A). Final libraries were quantified
Growth under anaerobic conditions was assessed in R2A broth with using Qubit (ThermoFisher, U.S.A) and sequenced on an Illumina
and without the addition of 0.15% (w/v) KNO3 at 28 °C similar to MiSeq instrument using MiSeq Reagent Kit v2 (500 cycles)
Révész et al. [54]. To ensure anaerobic conditions 100 ml serum bot- sequencing chemistry. De novo assembly and scaffolding were per-
tles containing 50 ml of R2A broth were hermetically closed and formed with CLC Genomics Workbench Tool v11 (Qiagen, Ger-
sparged with N2/CO2 (80:20, v/v) under sterile conditions. Dissolved many). The genome accession number of Nocardioides sp. nov.
oxygen concentration in the bottles was measured non-invasively by CBZ_1T is: JAHTLF000000000.
using a Fibox 3 trace version 3 fibre optic oxygen meter with PSt3 To ascertain the precise taxonomic position of strain CBZ_1T,
sensor spots (PreSens). using the available bacterial genomes, the ANI (orthologous aver-
Physiological and biochemical tests such as production of age nucleotide identity) and dDDH (in silico digital DNA-DNA
hydrogen sulphide from cysteine, hydrolysis of Tween 80, gelatin, hybridization) values between strain CBZ_1T and the closest rela-
casein and starch, and DNase activity, were performed according tives were calculated using Ez-Biocloud (https://www.ezbiocloud.
to the protocols of Barrow and Feltham [55]. Catalase activity net/tools/ani; [62]) and the server-based genome-to-genome dis-
was determined by bubble production from H2O2 (3%, v/v) and oxi- tance calculator version 2.1 (https://ggdc.dsmz.de/ggdc.php#;
dase activity was tested using 1% (w/v) tetramethyl- [63]), respectively. ANI and dDDH values between strain CBZ_1T
phenylenediamine oxalate. and the reconstructed Nocardioides sp. bin2 were also determined.
APIÒ 50CH, APIÒ 20NE and APIÒ ZYM strips (bioMérieux, France) No publicly accessible genome sequence of N. daeguensis JCM
were used to further determine biochemical characteristics of 17460T was available. Consequently, for genome-based phyloge-
strains CBZ_1T and the selected closest relatives following the netic studies the genomic DNA of strain JCM 17460T was extracted
instructions of the manufacturer. and sequenced during this study similar to WGS of strain CBZ_1T.
The genome accession number of N. daeguensis type strain JCM
Chemotaxonomic analyses 17460T is: JAHUBO000000000.
Annotation of the genome of strain CBZ_1T was performed by
Chemotaxonomic analyses were conducted by the Leibniz Insti- the Microbial Genome Annotation & Analysis Platform MicroScope
tute, DSMZ (Braunschweig, Germany). (MaGe) [64]. Additionally, putative functions of genes associated in
Respiratory quinones were extracted from freeze dried cell the metabolism of xenobiotics were identified and bioinformati-
material using hexane and were further purified by a silica-based cally analyzed by using MaGe in conjunction with the UniProt
solid phase extraction. Purified samples were further analyzed by database (https://www.uniprot.org/; [65]) and BLAST searches.
HPLC using a reverse phase column recording absorption spectra.
270 nm for ubiquinones and 326 nm for menaquinones were used Testing pharmaceutical biodegradation capacity of strain CBZ_1T
for a relative quantification.
Polar lipids were extracted from freeze dried biomass using a The type strain CBZ_1T was tested for its ability to degrade car-
chloroform: methanol:0.3% aqueous NaCl mixture, and were bamazepine and ibuprofen. Initially, the biodegradation tests were
recovered into the chloroform phase (modified after [56]). Polar conducted in triplicates in 50 ml of Bushnell-Haas mineral medium
lipids were separated by two-dimensional silica gel thin layer (CaCl22H2O 0.002 g, MgSO47H2O 0.02 g, NH4NO3 1 g, KH2PO4 1 g,
chromatography. The first direction was developed in chloro- K2HPO4 1 g, FeCl3∙6H2O 0.005 g, H2O 1 l, with pH 7) supplemented
form:methanol:water, and the second in chloroform:methanol:ac with one of the before mentioned pharmaceuticals as sole source
etic-acid:water. Total lipid material was detected using of carbon and energy (1.5 mg l
1). Subsequently, co-metabolic
molybdatophosphoric acid [57]. biodegradation tests were also conducted, easily assimilable car-
The peptidoglycan structure of strain was determined from wet bon sources such as yeast extract (0.05 or 0.3 g l
1), or glucose
biomass (centrifugal pellet suspended in isopropanol/water 1:1, (0.5 or 3 g l
1) were added to the test solutions mentioned above
v/v) as described by Schumann [58]. next to the pharmaceutical compounds.
For cellular fatty acid determinations all strains were cultivated The test solutions were inoculated with 50 ll of bacterial cell
on the same growth medium (R2A). Cellular fatty acids of strain suspensions (OD600 = 1) obtained in physiological saline solution.
CBZ_1T and the closest relatives were analyzed after conversion Abiotic controls, containing all the above mentioned except the
4
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

bacterium were also set up. Test runs were incubated for several
weeks on a rotary shaker at 145 rpm and 27 °C.
The concentration of pharmaceutical compounds in the bulk
solution was determined weekly using high performance liquid
chromatography (HPLC). Prior to injection into the HPLC instru-
ment the aqueous samples were filtered by passing through What-
man cellulose acetate syringe filters (0.45 lm). A Chromaster
Hitachi instrument consisting of a Model 5110 pump, a Model
5210 autosampler, and a Model 5430 Diode-array detector (DAD)
was used. The separation and data processing were operated by a
EZChom Alite software. The separation of the pharmaceutical com-
pounds was performed on Ascentis C18, 150  0.46 mm column
with isocratic elution of 50:50% (v/v) 0.02 M KH2PO4-acetonitrile
at a flow rate of 0.8 ml min
1. The DAD was operated at a wave-
Fig. 1. The relative abundance of Nocardioides spp. in the enrichment cultures as
length range between 190 and 400 nm. For the quantitative deter- assessed by shot-gun metagenome sequencing and analysis. DIC – diclofenac
mination a calibration curve for each compound was done between enrichment; IBU – ibuprofen amended enrichment; CBZ – carbamazepine amended
concentration and absorbance at maximum wavelengths (ibupro- enrichment.
fen 196 nm and carbamazepine 214 nm). The compounds were
identified based on comparison of retention time and spectral
On the other hand, in the DIC amended enrichments the relative
characteristics with those of standard solution.
abundance of the genus Nocardiodies remarkably decreased and
At the end of the biodegradation experiment the amount of
varied between only 0.02 and 0.004%. (Fig. 1).
pharmaceuticals that adsorbed to the biomass was determined.
At the genus level, DIC enrichments were dominated by Pseu-
For this purpose, the bacterial suspensions were centrifuged
domonas, Methyloversatilis and Azospirillum, reaching a maximum
(2360 g 10 min) and the biomass (pellet) was resuspended in
of 34.5% (1st month), 8.3% (3rd month) and 3.1% (3rd month) rel-
5 ml HPLC grade acetonitrile. Subsequently, the resuspended bio-
ative abundance in at least one of the enrichment cultures,
mass was sonicated three times for 30 s at 20 kHz and at an ampli-
respectively.
tude of 20% using a Branson Digital Sonifier (Emerson Industrial
These data suggest that most probably diclofenac sodium inhib-
Automation); between two sonication steps the suspensions were
ited the growth of strain CBZ_1T which is not surprising because
vortexed for 30 s. After the ultrasound treatment (sonication), the
the antimicrobial activity of diclofenac sodium has already been
samples were centrifuged and the supernatants were filtered
reported earlier [66–68].
(0.45 lm cellulose acetate syringe filters) to round bottom evapo-
rating flasks (250 ml). The acetonitrile was evaporated under vac-
uum using an IKAÒ RV10 rotary evaporator (Sigma-Aldrich, Reconstruction of a Nocardioides affiliated bin genome from
Hungary) at maximum 40 °C and the residues were re-dissolved metagenomes, results of RPKM calculations
in 2.5 ml of mineral salts solution. The concentration of pharma-
ceuticals in the solution was determined using HPLC as described Genome-resolved metagenome analysis has been utilized for
above. the recovery of bacterial genomes from the most diverse environ-
ments and provided first genome representatives of uncultivable
microbes and insights into previously unexplored metabolic traits
Results and discussion
of the microbes [69]. Also, in our study, a high-quality metagen-
ome assembled genome, representative of a yet uncultivated bac-
Hereinafter, from the whole research regarding the microbiol-
terium was obtained and affiliated to the genus Nocardioides;
ogy of DIC, IBU and CBZ biodegradation we focus particularly only
completeness 100%, genome size 6.18 Mb, GC content 71.43%).
on those data that are relevant to the genus Nocardioides and
According to the calculated RPKM values, the Nocardioides spe-
Nocardioides sp. nov. strain CBZ_1T. Comprehensive evaluation
cies corresponding to the obtained bin genome was active in the
and presentation of enrichment cultures’ metagenome data, com-
carbamazepine and ibuprofen enrichments. Compared to the initial
plete results of bacterial isolation, identification and pharmaceuti-
state (RPKM values obtained for the starting biofilm sample), in the
cal biodegradation testing of other bacterial isolates are beyond the
carbamazepine and ibuprofen enrichments the obtained RPKM val-
scope of the present paper.
ues were up to 76 and 43 times higher, respectively. In contrast, in
the diclofenac sodium amended enrichment the activity of the bin
The relative abundance of the genus Nocardioides in the enrichment genome related Nocardioides sp. decreased reflected by the remark-
cultures as assessed by metagenome analysis able decrease of the RPKM value. In the case of the two- and three-
months old diclofenac sodium enrichments less than one tenth of
Read-based metagenomic data revealed that the genus Nocar- the initial RPKM value was obtained (Fig. 2).
dioides could have a substantial role in the IBU and CBZ amended The aforementioned findings were corroborated by the RPKM
enrichment cultures. Whereas in the initial biofilm community values obtained on the basis of the metatranscriptomic data of
the relative abundance of Nocardioides spp. was only 0.14%, in the three-months old enrichments. Also, on the basis of metatran-
the IBU and CBZ enrichments it increased to a maximum of 2.7% scriptomic data the Nocardioides species corresponding to the
(Fig. 1). Apart from Nocardioides spp., the IBU amended enrich- reconstructed bin genome was active in the ibuprofen and carba-
ments contained other genera such as Methyloversatilis (max. mazepine enrichments but not in diclofenac sodium. RPKM values
11.5%, 2nd month), Pseudomonas (12.1%, 3rd month), Methylibium in the ibuprofen (2.7) and carbamazepine (2.5) enrichments were
(2%, 1st month) and Rhodococcus (2%, 1st month); the CBZ 200 times higher than that of the diclofenac sodium enrichment
amended enrichments contained Pseudomonas (max. 4.1%, 3rd (0.01) (Fig. 2C).
month), Methyloversatilis (11.9%, 1st month) Azospirillum (4%, 2nd The results of RPKM value calculations also supported the inhi-
month), Rhodococcus (2.8%, 1st month), Variovorax (2%, 2nd month) bitory effect of diclofenac sodium on the growth of Nocardioides
and Pseudonocardia (1.5%, 3rd month). spp.
5
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

Fig. 2. RPKM values obtained in each enrichment based on metagenome (A-C) and metatranscriptome data (D). BF – initial biofilm; CBZ – carbamazepine enrichment; IBU –
ibuprofen enrichment; DIC – diclofenac sodium enrichment; one, two and three – number of months.

Using sequence read recruitment the reconstructed novel was N. humi DCY24T (Fig. 3B, Fig. S1). In addition, genome-based
Nocardioides sp. genome could be identified also in the metagen- phylogenetic studies have shown that the reconstructed Nocar-
ome of pharmaceutical amended liquid enrichment cultures where dioides sp. bin2 genome belongs to the proposed novel Nocardioides
pharmaceuticals were used as the sole source of carbon and species represented by isolate CBZ_1T; both, in silico dDDH and ANI
energy. The relative abundance of the novel Nocardioides sp. in values were well above the cut-off recommended for the demarca-
the trimethoprim amended enrichment was nearly 9% and in one tion of new species (Table 2).
of the carbamazepine enrichments it was 3% ([51]; Table S1). Compared to the closest relatives (23 Nocardioides spp.), on the
basis of the pan-genomic study the novel Nocardioides species har-
Isolation and phylogenetic characterization of Nocardioides sp. strain bors 1219 unique genomic islands (UGIs; Fig. S2, Supplementary
CBZ_1T file).
It must be highlighted that it was possible to bring into culture
The main subject of the study, the isolate Nocardioides sp. strain a metabolically relevant, new to science bacterial species most
CBZ_1T was obtained from the one-month-old carbamazepine probably involved in carbamazepine and ibuprofen degradation,
enrichment. A continuous stretch of 1403 bp of 16S rRNA gene of whose genome was first reconstructed by using genome-resolved
strain CBZ_1T was sequenced and taxonomic affiliation of the iso- metagenomics. To the best of our knowledge, in the literature there
late was determined. The closest 16S rRNA gene sequences are no other studies reporting the simultaneous isolation and gen-
belonged to N. kongjuensis DSM19082T (98.4%), N. daeguensis ome reconstruction of a novel bacterial species. The present study
JCM17460T (98.4%) and N. nitrophenolicus DSM15529T (98.2%) confirms the applicability of metagenome binning for the explo-
(Fig. 3A, Table 2). ration of the unknown bacterial diversity. Further evidence was
General features of the sequenced genome of strain CBZ_1T are obtained regarding the biological reality of reconstructed bacterial
summarized in Table 1. The genome was assembled into 25 con- genomes a topic thoroughly discussed also in the review of Sebutal
tigs. The size of the recovered genome was 6,303,422 bp with a [74].
G + C content of 71.43% that fell into the range of G + C contents
of the genus Nocardioides [70]. Based on available genomes, the Morphological characteristics of Nocardioides sp. CBZ_1T
ANI and in silico dDDH hybridization values between the type
strain CBZ_1T and the type strains of the closest relatives ranged Isolate CBZ_1T had Gram-positive, elongated coccoid cells mea-
from 83.3 to 85.8% and 29.3 to 33.6%, respectively (Table 2). The suring 0.8–1.2 lm in length and 0.4–0.6 lm in width. No motility
obtained ANI and dDDH values were lower than the 95–96% ANI was observed. Transmission electron microscopy of negatively
and 70% dDDH thresholds for species delineation [71,72].These stained cells showed no presence of flagella (Fig. 4). When grown
results indicated that strain CBZ_1T represents a distinct species on R2A agar, the isolate forms whitish, translucent, flat colonies
of the genus Nocardioides. with irregular margins.
Moreover, the generated phylogenomic trees based on 92 or
120 bacterial marker genes also supported the fact that Nocar- Chemotaxonomic characteristics
dioides sp. CBZ_1T is a new phyletic lineage within the genus Nocar-
dioides (Fig. 3B, Fig. S1). It has to be added that based on The major respiratory quinone of strain CBZ_1T was identified
phylogenomic studies the closest type strain to the novel species as tetrahydrogenated menaquinone with eight isoprene units,
6
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

Fig. 3. (A) Phylogenetic tree based on 16S rRNA gene sequences showing relationship between strain CBZ_1T and the closest relatives within the genus Nocardioides. The scale
of 1% nucleotide substitution rate and the bootstrap values with 10,000 re-samplings >50 are shown at branching points. (B) Phylogenetic tree inferred using UBCGs
(concatenated alignment of 92 core genes). Gene support indices (GSIs) are given at branching points. Bar, 0.1 substitution per position. For the construction of phylogenetic
trees, the 16S rRNA gene and whole genome sequence of Leifsonia naganoensis was used as an outgroup to root the trees. The accession numbers of the used sequences are
given in parentheses.

Table 1 The total hydrolysate (100 °C, 4 N HCl, 16 h) of the peptidogly-

General features of the genome of Nocardioides sp. strain CBZ_1T. can contained muramic acid (Mur) and the amino acids diaminopi-
Characteristic Value melic acid (Dpm), alanine (Ala), glycine (Gly) and glutamic acid.
checkM* Completeness (%) 98.5 (4 marker genes are missing)
Quantification of amino acids by GC/MS or N-heptafluorobutyric
checkM contamination (%) 0.8 (2 marker genes are duplicated) amino acid isobutylesters resulted in the following molar ratio:
Size (bp) 6,303,422 2.6 Ala, 1.4 Gly, 1.0 Glue, 1–0 Glu: 0.8 Dpm. The analysis of enan-
G + C content (mol%) 71.4 tiomers of the total hydrolysate showed the presence of LL-Dpm.
Total number of genes 6,407
The peptides Mur-Ala, Mur-Ala-Glu, Glu-Dpm, Gly-Ala and a minor
Number of protein coding sequences 6,328
Number of pseudogenes 2 amount of DPm-Gly-Ala-Dpm could be detected after hydrolysis
Average gene length (bp) 956 under milder conditions (4 N HCl, 45 min., 100 ℃). Based on the
rRNA (16S, 23S, 5S) 5 before mentioned the occurrence of the following peptidoglycan
tRNA genes 45 type was concluded: A3y LL-DpM-Gly.
GenBank accession number (NCBI) JAHTLF000000000
The cellular fatty acid profiles of the investigated strains are
*CheckM embedded in the MicroScope platform is an automated method for summarized in Table 3. The major components for strain CBZ_1T
assessing the quality of a microbial genome regarding completion and contami- were 10Me C18:0 (20.7%), C18:1 x9c (15.9%), C16:0 ISO (10.4%) C17:0
nation [73].
ISO (9.7%) and C17:1 x6c (8.0%).

MK8 H4 (98.8%). The major polar lipids were phosphatidylglycerol Physiological and biochemical characteristics
(PG) and diphosphatidylglycerol (DPG) (Fig. S3). Both the major
respiratory quinones and polar lipids are typical of Nocardioides Growth of strain CBZ_1T occurred in a pH range of 6–9 with an
spp. [75]. optimum at pH 8. The temperature range for growth was 15–37 °C
7
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

Table 2
Phylogenetic relatedness between strain CBZ_1T and the type strains of previously established Nocardioides species based on whole genome
sequences. Whole genome sequence accession numbers are given in parentheses.

Nocardioides sp. strain CBZ_1T

16S rRNA gene sequence similarity (%) ANI (%) dDDH (%) Mol%
G+C
N. kongjuensis DSM 19082T 98.4 83.3 29.7 72.1
(NZ_JACCBF000000000.1)
N. daeguensis JCM 17460T 98.4 85.5 30.1 74.9
(JAHUBO000000000)
N. nitrophenolicus DSM 15529T 98.2 85.1 29.3 71.4
(NZ_JAFBBY000000000.1)
N. humi DCY24T 96.5 85.8 33.6 71.4
(CP041146.1)
Nocardioides sp. bin2 -* 99.9 99.4 71.4
(JAJTIU000000000)

*no 16S rRNA gene was detected in the reconstructed Nocardiodes sp. bin2

with the optimum at 28 °C. NaCl tolerance ranged between 0 and

3%, with the optimum of 1%.
Physiological and biochemical characteristics, and enzymatic
activity of strain CBZ_1T compared to those of the related type
strains are shown in Tables 4 and 5. Strain CBZ_1T was oxidase neg-
ative and catalase positive, reduced nitrate to nitrite, produced H2S
from cysteine, did not produce indole and did not ferment glucose.
The isolate did not hydrolyze arginine, urea and starch, but did
hydrolyze aesculin, gelatin, casein and Tween 80. It was able to
assimilate only malic acid and D-glucose out of the 49 tested com-
pounds found in APIÒ 50CH test strips. According to the results of
the APIÒ ZYM, the strain showed alkaline phosphatase, esterase,
esterase lipase, lipase, leucine-, valine-, and cysteine arylamidase,
Fig. 4. Cell morphology of strain CBZ_1T observed by transmission electron
trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, b-
microscopy with negative-staining.
galactosidase, a-glucosidase and b-glucosidase, but not a-
chymotrypsin, a-galactosidase, b-glucunoridase, N-acetyl-b-
glucosaminidase and a-mannosidase activity (Table 5).

Table 3
Cellular fatty acid profiles of strain CBZ_1T and related species. Taxa: 1. strain CBZ_1T; 2. N. kongjuensis DSM 19082T; 3. N. nitrophenolicus DSM 15529T; 4. N. daeguensis JCM
17460T. Data are expressed as percentages of total fatty acids. -, Not detected. Fatty acids which were lower than 1.0% in all strains are not shown. All data are from the present
study.

Fatty acid 1 2 3 4
Saturated
C16:0 4.44 1.81 2.10 1.99
C17:0 1.03 0.59 1.09 0.56
C18:0 3.26 3.58 - 3.50
Unsaturated
C17:1 x6c 8.00 9.18 7.12 11.02
C17:1 x8c 2.17 2.04 2.24 2.11
C18:1 x7c 1.87 1.69 1.98 1.88
C18:1 x9c 15.90 16.52 19.63 19.59
Branched-chain fatty acids
iso-C15:0 3.38 1.67 2.65 1.56
iso-C16:0 10.42 22.27 11.98 23.72
iso-C16:1H 1.89 1.77 0.83 1.28
iso-C17:0 9.73 8.02 12.10 7.34
anteiso-C17:0 2.04 4.22 6.49 2.87
iso-C18:0 0.59 2.63 1.28 3.12
10-Methyl fatty acids
C16:0 2.18 1.60 1.63 1.30
C17:0 3.08 3.80 3.24 3.61
Hydroxy fatty acids
C16:0 2-OH 2.45 0.57 1.26 0.69
Summed featurea 4.48 1.89 2.53 1.94
TBSAb 10Me C18:0 20.72 13.45 18.96 9.09
a
Summed features represent groups of two or three fatty acids that could not be separated by gas–liquid chromatography with the MIDI system. Summed features: 3, C16:1
x7c/C15:0 ISO 2-OH.
b
Tuberculostearic acid.

8
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

Table 4
Physiological and phenotypic characteristics of strain CBZ_1T compared to the closely related species within the genus Nocardioides. Strains: 1. strain CBZ_1T; 2. N. kongjuensis
DSM 19082T; 3. N. nitrophenolicus DSM 15529T; 4. N. daeguensis JCM 17460T.

Characteristics 1 2 3 4
Reduction of nitrate to nitrite + -
+
H2S production + + + +
Oxidase

w +
Catalase + + + +
Indole production




Fermentation of glucose




Hydrolysis of
Arginine




DNA + + + +
Urea

+

Aesculin + + +

Gelatin + + + +
Casein + +
+
Tween 80 + + + +
Starch
+ + +
Assimilation of
D-glucose + + +

L-arginine




L-arabinose

w

D-mannose

w

D-mannitol
w w

N-acetyl-glucosamine
w w

D-maltose
w w

Potassium gluconate
w w

Capric acid




Adipic acid
w w w
Malic acid + w w w
Trisodium citrate
w w

Phenylacetic acid


w
Acid production
D-maltose




Mannose
w w

Ribose

+ +
D-fructose

+

D-glucose


w
Lactose




L-arabinose




Cellobiose




Xylose

+ +

Symbols: +, positive; –, negative; w, weak positive.

Table 5
Enzymatic activity of strain CBZ_1T and the closest relatives as revealed by APIÒ ZYM test (bioMérieux SA, France). Strains: 1. strain CBZ_1T; 2. N. kongjuensis DSM 19082T; 3. N.
nitrophenolicus DSM 15529T; 4. N. daeguensis JCM 17460T.

Enzyme assay 1 2 3 4
Alkaline phosphatase + + + +
Esterase (C 4) + + (
a) + (
b) +
Esterase lipase (C 8) + + + +
Lipase (C 14) +



Leucine arylamidase + + + +
Valine arylamidase + + (
a) + +
Cystine arylamidase + + (
a) + +
Trypsin + + (
a) + +
a-chymotrypsin




Acid phosphatase + + + +
Naphthol-AS-BI-phosphohydrolase + + + +
a-galactosidase




b-galactosidase + w (
a) w (+b) w (+c)
b-glucunoridase




a-glucosidase + + + +
b-glucosidase + + (
a) w w (+c)
N-acetyl-b-glucosaminidase




a-mannosidase




a-fucosidase-fucosidase

Symbols: +, positive;
, negative; w, weak positive.

a
Data for the type strain of N. kongjuensis DSM 19082T from[76].
b
Data for the type strain of N. nitrophenolicus DSM 15529T from[21].
c
Data for the type strain of N. daeguensis JCM 17460T from[25].

9
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

Pharmaceutical biodegradation capacity of strain CBZ_1T cyclophosphamide); phenylpropionate dioxygenase involved in

aromatic compound metabolism, catechol 2,3-dioxygenase system
Contrary to our expectations, under the tested conditions strain involved in the ring fission of aromatic compounds could be iden-
CBZ_1T showed no remarkable carbamazepine biodegradation. tified (Supplementary file). A cytochrome P450 enzyme (CYP45)
Only a slight carbamazepine concentration reduction of 4%, was encoding gene – CypX (PDB:3A4Z) – was also identified as unique
detected after 7 weeks of incubation, in the presence of yeast for the novel Nocardioides species. This finding is interesting since
extract (0.3 g l
1) as co-substrate (data are not shown). These find- CYP45 enzymes are thought to be responsible for carbamazepine
ings contradict the results of metagenomic studies which showed biodegradation at least in the case of white-root fungi such as
that strain CBZ_1T was active also in the carbamazepine enrich- Trametes versicolor and Pleurotus ostreatus [83,84]. It may be spec-
ment. It can be speculated that most probably the new species ulated that this type of CYP45 encoding gene, unique amongst the
was not directly involved in the initial attack of carbamazepine analyzed 23 Nocardioides spp., might be responsible for the activity
but in the biotransformation of carbamazepine metabolites origi- and proliferation of the novel species in the carbamazepine
nating from the catabolic activity of other bacteria. It has to be amended enrichments.
noted that carbamazepine is an emerging contaminant that is
resistant to biodegradation. Only a few pure bacterial isolates, such
as Labrys portucalensis F11 and Streptomyces MIUG 4.89, have been Table 6
obtained so far capable of carbamazepine biodegradation [77,78]. Description of Nocardioides carbamazepini sp. nov.

A significant biodegradation was detected in the presence of Genus name Nocardiodies

ibuprofen. After 7 weeks of incubation, in the presence of glucose Species name Nocardioides carbamazepini
(3 g l
1) as co-substrate, 70% of ibuprofen was eliminated from Specific epithet carbamazepini
Species status sp. nov.
the test solutions by strain CBZ_1T (Fig. 5). Analyses showed no
Species etymology car.ba.ma.ze.pi’ni. N.L. neut. n.
ibuprofen adsorption to the biomass. Although Nocardioides spp. carbamazepinum, carbamazepine; N.L. gen. n.
are well known due to their xenobiotic biodegradation capabilities carbamazepini, of carbamazepine
(dibenzofuran, nitro- and chlorophenols, carbendazim, pesticides, Description of the new Cells are Gram-positive, strictly aerobic, non-
taxon and diagnostic motile, the elongated coccoid cells are 0.8–1.2-
PAHs etc.) no Nocardioides strain has been reported so far capable
traits lm in length and 0.4–0.6 lm in width. The
of ibuprofen biodegradation [19–21,25–31]. It has to be added that colonies are flat and whitish, translucent with
ibuprofen degrading bacterial strains affiliating to other genera, irregular margins on R2A agar. Grows well at
e.g., Sphingobium, Sphingomonas, Patulibacter and Bacillus, do exist 15–37 °C with an optimum at 28 ℃. Grows at
in general [79–82]. No better or higher degradation rates could pH 6–9, with the optimum growth at pH 7–8.
Growth occurs in the absence of NaCl and in
be reached in the case of other experimental settings such as in
the presence of up to 3% (w/v) NaCl. Grows
the presence of other co-substrate or under lower co-substrate well in R2A, nutrient, LB and TSA broth,
concentrations. showing the best growth in R2A. Catalase
Based on whole-genome sequencing, gene annotations and positive and oxidase negative. Reduces nitrate
to nitrite, but does not reduce nitrate to
metabolic analyses it was found that isolate CBZ_1T encodes func-
dinitrogen gas. Tests for alkaline phosphatase,
tional genes involved in xenobiotics biodegradation including esterase (C 4), esterase lipase (C 8), lipase (C
pharmaceutical compounds (Table S2). Genes similar to 14), leucine arylamidase, valine arylamidase,
biodegradative genes involved in ibuprofen biodegradation (ipf- cystine arylamidase, trypsin, acid
DEFG), first identified in the case of Sphingomonas sp. Ibu-2 [80], phosphatase, naphthol-AS-BI-
phosphohydrolase, b-galactosidase, a-
could also be identified, however gene sequence similarities ran-
glucosidase, b-glucosidase and protease are
ged only between 33 and 50% (Table S2.) Among the identified positive; tests for a-chymotripsin, a-
UGIs, genes involved in xenobiotics biodegradation such as 1,2- galactosidase, b-glucuronidase, N-acetyl-b-
phenylacetyl-CoA epoxidase involved in phenylacetate degrada- glucosaminidase, a- mannosidase, a-
tion; acetyl-CoA acetyltransferase involved in benzoate and fucosidase, arginin dihydrolase, urease, indole
production, acidification of glucose are
butanoate degradation; 2-keto-4-pentenoate hydratase involved negative. Except assimilation of malic acid and
in benzoate, dioxin and xylenes degradation; aldehyde dehydroge- glucose, it is negative for assimilation of other
nase involved in chloroalkane and chloroalkene degradation; alco- carbon sources found in APIÒ 20NE and APIÒ
hol dehydrogenase involved in chloroalkane and chloroalkene 50CH test strips. MK8H4 is the predominant
respiratory quinone, the type of peptidoglycan
degradation; naphthalene degradation, metabolism of xenobiotics
is A3y LL-Dpm-Gly and the major cellular fatty
by cytochrome P450 pathway (trichloroethylene, felbamate, acid are TBSA 10Me C18:0 (20.7%), 18:1 w9c
(15.9%), 16:0 ISO (10.4%) 17:0 ISO (9.7%) and
17:1 w6c (8.0%).
Country of origin Hungary
Region of origin Central Region of Hungary
Date of isolation 19/01/2020
Source of isolation Biofilm bacterial community selectively
enriched on carbamazepine for 1 month in
mineral salts solution
Sampling date 18/12/2019
16S rRNA gene accession nr. MZ408918
Genome accession number JAHTLF000000000
Genome status Incomplete (draft)
Genome size 6,303 kbp
GC mol% 71.43
Number of strains in study 1
Information related to the Not applicable
Nagoya Protocol
Designation of the Type CBZ_1T
Strain
Strain Collection Numbers NCAIM B.0.2663 = LMG 32395
Fig. 5. The biodegradation rate of ibuprofen by Nocardioides sp. strain CBZ_1T.

10
T. Benedek, Márton Pápai, K. Gharieb et al. Systematic and Applied Microbiology 45 (2022) 126339

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