Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model.

The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.

Benzalkonium chloride and heavy metal resistance profiles of Listeria monocytogenes strains isolated from fish, fish products and food-producing factories in Poland.

Phenotypic and genotypic resistance to benzalkonium chloride (BC), cadmium and arsenic was tested (by susceptibility assays and molecular methods) in 287 Listeria monocytogenes strains isolated from fish and fish products, and food-producing factories in Poland. Overall, 40% of the isolates were resistant to BC, 56% to cadmium and 41% to arsenic (57% displayed resistance to more than one of the tested compounds). Among BC-resistant isolates, the most commonly detected resistance determinant was the qacH gene (83%). Three distinct types of cadA gene determining resistance to cadmium were detected, with the cadA1 variant predominant (88%), while most arsenic-resistant isolates (86%) harbored the arsA gene associated with a Tn554-like transposon (one strain harbored two copies of arsA in different arsenic resistance cassettes). 53% of all tested isolates contained plasmids (from 4 kb to > 90 kb in size), which were classified into 11 groups (p1-p11) based on their restriction patterns. Interestingly, 12 isolates harbored the small mobilizable pLMST6-like plasmid pLIS3 encoding multidrug efflux pump EmrC. Clustering analysis of PFGE patterns revealed that these isolates represent several diverse bacterial populations, which strongly suggests mobility of the pLMST6-like plasmids among L. monocytogenes strains and their role in dissemination of BC resistance.

Plasmidome of Listeria spp.-The repA-Family Business.

Bacteria of the genus Listeria (phylum Firmicutes) include both human and animal pathogens, as well as saprophytic strains. A common component of Listeria spp. genomes are plasmids, i.e., extrachromosomal replicons that contribute to gene flux in bacteria. This study provides an in-depth insight into the structure, diversity and evolution of plasmids occurring in Listeria strains inhabiting various environments under different anthropogenic pressures. Apart from the components of the conserved plasmid backbone (providing replication, stable maintenance and conjugational transfer functions), these replicons contain numerous adaptive genes possibly involved in: (i) resistance to antibiotics, heavy metals, metalloids and sanitizers, and (ii) responses to heat, oxidative, acid and high salinity stressors. Their genomes are also enriched by numerous transposable elements, which have influenced the plasmid architecture. The plasmidome of Listeria is dominated by a group of related replicons encoding the RepA replication initiation protein. Detailed comparative analyses provide valuable data on the level of conservation of these replicons and their role in shaping the structure of the Listeria pangenome, as well as their relationship to plasmids of other genera of Firmicutes, which demonstrates the range and direction of flow of genetic information in this important group of bacteria.

Genetic Carriers and Genomic Distribution of cadA6-A Novel Variant of a Cadmium Resistance Determinant Identified in Listeria spp.

Listeria monocytogenes is a pathogen responsible for severe cases of food poisoning. Listeria spp. strains occurring in soil and water environments may serve as a reservoir of resistance determinants for pathogenic L. monocytogenes strains. A large collection of Listeria spp. strains (155) isolated from natural, agricultural, and urban areas was screened for resistance to heavy metals and metalloids, and the presence of resistance determinants and extrachromosomal replicons. Of the tested strains, 35% were resistant to cadmium and 17% to arsenic. Sequence analysis of resistance plasmids isolated from strains of Listeria seeligeri and Listeria ivanovii, and the chromosome of L. seeligeri strain Sr73, identified a novel variant of the cadAC cadmium resistance efflux system, cadA6, that was functional in L. monocytogenes cells. The cadA6 cassette was detected in four Listeria species, including strains of L. monocytogenes, isolated from various countries and sources-environmental, food-associated, and clinical samples. This resistance cassette is harbored by four novel composite or non-composite transposons, which increases its potential for horizontal transmission. Since some cadAC cassettes may influence virulence and biofilm formation, it is important to monitor their presence in Listeria spp. strains inhabiting different environments.

Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures.

The surface-enhanced Raman spectroscopy (SERS)-based analysis of bacteria suffers from the lack of a standard SERS detection protocol (type of substrates, excitation frequencies, and sampling methodologies) that could be employed throughout laboratories to produce repeatable and valuable spectral information. In this work, we have examined several factors influencing the spectrum and signal enhancement during SERS studies conducted on both Gram-negative and Gram-positive bacterial species: Escherichia coli and Bacillus subtilis, respectively. These factors can be grouped into those which are related to the structure and types of plasmonic systems used during SERS measurements and those that are associated with the culturing conditions, types of culture media, and method of biological sample preparation.

Correction to: Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures.

The authors would like to call the reader's attention to the fact that unfortunately following information was missing in the original article: "Evelin Witkowska is supported by the Foundation of Polish Science (FNP)."

Strain-level typing and identification of bacteria - a novel approach for SERS active plasmonic nanostructures.

One of the potential applications of surface-enhanced Raman spectroscopy (SERS) is the detection of biological compounds and microorganisms. Here we demonstrate that SERS coupled with principal component analysis (PCA) serves as a perfect method for determining the taxonomic affiliation of bacteria at the strain level. We demonstrate for the first time that it is possible to distinguish different genoserogroups within a single species, Listeria monocytogenes, which is one of the most virulent foodborne pathogens and in some cases contact with which may be fatal. We also postulate that it is possible to detect additional proteins in the L. monocytogenes cell envelope, which provide resistance to benzalkonium chloride and cadmium. A better understanding of this infectious agent could help in selecting the appropriate pharmaceutical product for enhanced treatment. Graphical abstract ᅟ.

The Prevalence of Campylobacter spp. in Polish Poultry Meat.

The prevalence, count and molecular identification of Campylobacter spp. in Polish poultry meat were analysed. 181 samples of meat from chicken (70), turkey (47), duck (54) and goose (10) were studied. Campylobacter spp. was found in 64% of meat samples. The highest prevalence of this pathogen was detected for duck meat. On average 80% of duck samples were contaminated with Campylobacter spp. The counts of Campylobacter spp. in positive samples remained under ten colony forming units per gram of product in 59% of poultry meat. C. jejuni was more frequently detected in poultry meat than C. coli.

Surface-enhanced Raman spectroscopy introduced into the International Standard Organization (ISO) regulations as an alternative method for detection and identification of pathogens in the food industry.

We show that surface-enhanced Raman spectroscopy (SERS) coupled with principal component analysis (PCA) can serve as a fast, reliable, and easy method for detection and identification of food-borne bacteria, namely Salmonella spp., Listeria monocytogenes, and Cronobacter spp., in different types of food matrices (salmon, eggs, powdered infant formula milk, mixed herbs, respectively). The main aim of this work was to introduce the SERS technique into three ISO (6579:2002; 11290-1:1996/A1:2004; 22964:2006) standard procedures required for detection of these bacteria in food. Our study demonstrates that the SERS technique is effective in distinguishing very closely related bacteria within a genus grown on solid and liquid media. The advantages of the proposed ISO-SERS method for bacteria identification include simplicity and reduced time of analysis, from almost 144 h required by standard methods to 48 h for the SERS-based approach. Additionally, PCA allows one to perform statistical classification of studied bacteria and to identify the spectrum of an unknown sample. Calculated first and second principal components (PC-1, PC-2) account for 96, 98, and 90% of total variance in the spectra and enable one to identify the Salmonella spp., L. monocytogenes, and Cronobacter spp., respectively. Moreover, the presented study demonstrates the excellent possibility for simultaneous detection of analyzed food-borne bacteria in one sample test (98% of PC-1 and PC-2) with a goal of splitting the data set into three separated clusters corresponding to the three studied bacteria species. The studies described in this paper suggest that SERS represents an alternative to standard microorganism diagnostic procedures. Graphical Abstract New approach of the SERS strategy for detection and identification of food-borne bacteria, namely S. enterica, L. monocytogenes, and C. sakazakii in selected food matrices.

Identification of the Molecular Mechanism of Trimethoprim Resistance in Listeria monocytogenes.

Trimethoprim with sulfamethoxazole is a therapeutic agent combination used to treat infections caused by the facultative intracellular foodborne pathogen Listeria monocytogenes. The aim of this study was to assess the frequency of resistance of L. monocytogenes arising due to exposure to trimethoprim and subsequently investigate the molecular mechanisms of resistance. After exposure of a culture of L. monocytogenes ATCC 13932 to trimethoprim at 10-fold the minimal inhibitory concentration spontaneous resistant mutants were recovered, giving a frequency of resistance development of 6.85 ± 0.92 × 10-8. The isolates exhibited a 32-64-fold decrease in susceptibility compared with the parental strain. These results indicate the capacity of L. monocytogenes to develop low-level resistance toward trimethoprim after exposure to the drug. The trimethoprim resistance genes (dhfr) and their promoter regions from all trimethoprim-resistant isolates were amplified and sequenced, leading to the identification of four single amino acid substitutions (Met20-Val, Pro21-Leu, Thr46-Asn, Val95-Leu) and two double substitutions (Met20-Ile+Thr46-Asn and Thr46-Asn+Leu85-Phe) in DHFR. Of the identified mutations, the Thr46-Asn substitution has not been previously reported as the mechanism of resistance to trimethoprim in other bacteria; thus this substitution seems to be unique to L. monocytogenes. The expression of the mutated L. monocytogenes dhfr genes in Escherichia coli led to decreased susceptibility of the heterological host, therefore proving that the identified point mutations in dhfr serve as the molecular mechanism of acquired resistance of L. monocytogenes to trimethoprim.

Rifampicin- and Rifabutin-Resistant Listeria monocytogenes Strains Isolated from Food Products Carry Mutations in rpoB Gene.

The aim of this study was to investigate the mechanism of rifampicin resistance in Listeria monocytogenes strains isolated from different types of food and the impact of specific mutations in the rpoB gene on susceptibility to different antimicrobial agents and on fitness cost. Fifteen spontaneous rifampicin-resistant strains were selected. The DNA regions corresponding to clusters I-II, III, and N-terminal end of the rpoB gene of Escherichia coli were amplified and sequenced, leading to the identification of 10 different substitutions, nine of which (Ser466Pro, Gln470Lys Asp473Asn, Gly479Asp, His483Tyr/Arg/Asp, Arg486His, and Leu490Pro) were located in cluster I and one (Pro521Leu) in cluster II. From among these mutations, substitutions at positions 466, 470, 486, 490, and 521 have not been described for L. monocytogenes. Only substitutions at positions 470, 479, 483, and 486 lead to resistance to very high concentrations of rifampicin (minimum inhibitory concentration [MIC] ≥256 µg/mL) and rifabutin (MIC 128 µg/mL). Furthermore, mutations at positions 473, 490, and 521 had different effects on susceptibility to rifampicin compared to other bacterial species. A correlation between rifampicin resistance and susceptibility to a wide range of antimicrobials was determined. Substitutions in RpoB did not change the susceptibility of the mutants to different antimicrobials. The fitness of the mutants was assessed by paired competition experiments. Mutations at positions 470 and 479 were not associated with a reduction in fitness level. There was no correlation between the MIC of rifampicin and fitness cost. The risk of transmission of resistant strains through the food chain highlights the need for monitoring resistance, identifying mutant organisms, their genotypes, and their altered phenotypes to understand their dissemination.

Prevalence of Bacillus cereus in food products in Poland.

INTRODUCTION AND OBJECTIVE: Bacillus cereus is a foodborne pathogen causing two main types of gastrointestinal diseases: emetic and diarrheal. The aim of this study was to investigate the prevalence of the Bacillus cereus group in ready-to-eat (RTE) food products available in retail in Poland. MATERIAL AND METHODS: Samples were collected by Sanitary and Epidemiological Stations within the framework of the national official control and monitoring sampling programme in Poland. In 2016-2020, a total of 45,358 food samples, such as: 'confectionery products and products with cream', as well as 'cereal grains and cereal and flour products', 'milk and milk products', 'sugar and others', 'meat offal and meat products', 'poultry offal and poultry products', 'eggs and egg products', 'fish, seafood and their preserves', 'vegetables' (including legumes), 'coffee, tea, cocoa, fruit, and herbal teas', 'delicatessen and culinary products', and 'foods for particular nutritional uses' were collected. RESULTS: The presence of the presumptive B. cereus group was monitored mainly in two categories of food products: 'confectionery products and products with uncooked cream' and 'confectionery products and products with heat-treated cream'. The number of samples disqualified due to presumptive B. cereus was 339 (0.75%). CONCLUSIONS: This study provides useful information regarding the contamination of RTE products with the B. cereus group, which may have implications for food safety.

Emergence of macrolide-resistant Campylobacter strains in chicken meat in Poland and the resistance mechanisms involved.

In this study, we investigated the molecular mechanisms involved in erythromycin resistance in the first resistant Campylobacter strains isolated from chicken meat in Poland, and analyzed their genetic relatedness. A total of 297 samples of raw chicken meat and giblets from retail trade in the Warsaw area collected between 2006 and 2009 were examined. Among 211 Campylobacter strains (52 C. jejuni and 159 C. coli), 10 C. coli isolates (4.7%) were resistant to erythromycin. All the C. jejuni strains were susceptible. Among the high-level macrolide-resistant isolates, two different point mutations within the domain V of the 23S rRNA gene were observed. Eight of the strains had adenine→guanine transitions at position 2075, two other isolates at position 2074. Sequence analysis of ribosomal proteins L4 (rplD) and L22 (rplV) indicated that ribosomal protein modifications did not contribute to macrolide resistance. A mutation in the inverted repeat in the cmeR and cmeABC intergenic region was found in a single resistant strain. The genetic relatedness of Campylobacter isolates showed that two resistant strains obtained from the same production plant in a 2-month interval were genetically identical. The risk of transmission of resistant strains via the food chain highlights the need for constant monitoring of resistance in Campylobacter isolates of human and animal hosts.

Bioanalytical insight into the life of microbial populations: A chemical monitoring of ureolytic bacteria growth.

In this publication an alternative approach to investigations of bacterial growth is proposed. Contrary to the conventional physical methods it is based on enzyme activity detection. The procedure for real-time and on-line monitoring of microbial ureolytic activity (applied as a model experimental biosystem) in the flow analysis format is presented. The developed fully-mechanized bioanalytical flow system is composed of solenoid micropumps and microvalves actuated by Arduino microcontroller. The photometric detection based on Nessler reaction is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system allows discrete assaying of microbial urease in the wide range of activity up to 5.4 U mL-1 with detection limit below 0.44 U mL-1, a high sensitivity in the linear range of response (up to 200 mV U-1 mL and relatively high throughput (9 detection per hour). The proposed differential procedure of measurements (i.e. a difference between peaks register for sample with and without external addition of urea is treated as an analytical signal) allows elimination of interfering effects from substrate and products of biocatalysed reaction as well as other components of medium used for microbial growth. The developed bioanalytical system was successfully applied for the control of growth of urease-positive bacteria strains (Proteus vulgaris, Klebsiella pneumoniae and Paracoccus yeei) including examination of effects from various microbial cultivation conditions like temperature, composition of culture medium and amount of substrate required for induction of bacterial enzymatic activity. The developed bioanalytical flow system can be applied for metabolic activity-based estimation of parameters of lag and log phases of microbial growth as well as for detection of decline phase.

Incidence and genetic variability of Listeria monocytogenes isolated from vegetables in Poland.

The aim of the present study is to investigate the prevalence and genetic diversity of Listeria monocytogenes in various fresh and frozen vegetable products available in Poland. The samples were collected at retail market within the framework of national official control and monitoring program. In the years 2016-2019 a total of 49 samples out of 8712 collected vegetable samples were positive for L. monocytogenes. Our findings demonstrated that the occurrence of L. monocytogenes in various vegetable products was generally low, on average only 0.56% in the studied years. All isolates were susceptible to 11 antimicrobial agents: penicillin, ampicillin, meropenem, erythromycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, ciprofloxacin, chloramphenicol, gentamicin, vancomycin, and tetracycline. All of them harbored virulence-associated genes (inlA, inlC, and lmo2672), 82% harbored inlJ gene and few of them (22%) also possessed the llsX gene. The majority of collected isolates (65%) belonged to molecular serogroup 1/2a-3a, followed by 4ab-4b-4d-4e (33%), and only one to serogroup 1/2b-3b-7 (2%). Isolates yielded 18 different restriction profiles, revealing a large cluster of contamination linked to frozen corn (21 strains) and distributed in 3 pulsotypes. MLST analysis classified selected isolates into nine clonal complexes (CCs). The obtained results contribute to characterizing the diversity of L. monocytogenes isolated from various vegetable products in Poland and their impact on food safety and public health.

Genetic diversity of Listeria monocytogenes isolated from ready-to-eat food products in retail in Poland.

The study describes the characterization of Listeria monocytogenes isolated from the general 2017-2019 national official control and monitoring sampling program. A total of 60,928 of ready-to-eat (RTE) food products were collected in retail in Poland, while the number of L. monocytogenes contaminated samples was 67 (0.1%). The majority of the strains belonged to molecular serotype IVb followed by IIa, frequently associated with human listeriosis. Furthermore, 61.2% of the isolates were resistant at least to one of the tested antimicrobials: penicillin, ampicillin, meropenem, erythromycin, sulfamethoxazole-trimethoprim, amoxicillin-clavulanic acid, ciprofloxacin, chloramphenicol, gentamicin, vancomycin, tetracycline and rifampicin. Virulence genes inlA, inlC, inlJ and lmo2672 were detected in all of the isolates. In our study the llsX gene (encoding LLS) exhibited 11.6% positivity. The 32 strains were grouped into 12 clonal complexes (CCs) which belong to the major clones that are in circulation in Europe. Among them, seven strains with the cgMLST close relatedness (CC2) were isolated from diverse food sectors, underlining a large circulation of this clone in Poland, most likely from multiple introduction sources. Additionally, two RTE strains CC6 and one CC37 were identified as closely related by cgMLST to two publicly available genomes of clinical strains isolated in Poland in 2012-2013. These results indicate the large strain circulation and point to RTE food products as a potential source of human listeriosis. The present study provided data to capture the contamination status of L. monocytogenes in foods at the retail level in Poland and assess the potential risk of this pathogen for human safety.

Identification of the full set of Listeria monocytogenes penicillin-binding proteins and characterization of PBPD2 (Lmo2812).

BACKGROUND: Bacterial penicillin-binding proteins (PBPs) can be visualized by their ability to bind radiolabeled or fluorescent ß-lactam derivatives both whole cells and membrane/cell enriched fractions. Analysis of the Listeria monocytogenes genome sequence predicted ten genes coding for putative PBPs, but not all of their products have been detected in studies using radiolabeled antibiotics, thus hindering their characterization. Here we report the positive identification of the full set of L. monocytogenes PBPs and the characteristics of the hitherto undescribed PBPD2 (Lmo2812). RESULTS: Eight L. monocytogenes PBPs were identified by the binding of fluorescent ß-lactam antibiotic derivatives Boc-FL, Boc-650 and Amp-Alexa430 to proteins in whole cells or membrane/cell wall extracts. The gene encoding a ninth PBP (Lmo2812) was cloned and expressed in Escherichia coli as a His-tagged protein. The affinity purified recombinant protein had DD-carboxypeptidase activity and preferentially degraded low-molecular-weight substrates. L. monocytogenes mutants lacking the functional Lmo2812 enzyme were constructed and, compared to the wild-type, the cells were longer and slightly curved with bent ends.Protein Lmo1855, previously designated PBPD3, did not bind any of the antibiotic derivatives tested, similarly to the homologous enterococcal protein VanY. CONCLUSIONS: Nine out of the ten putative L. monocytogenes PBP genes were shown to encode proteins that bind derivatives of ß-lactam antibiotics, thus enabling their positive identification. PBPD2 (Lmo2812) was not visualized in whole cell extracts, most probably due to its low abundance, but it was shown to bind Boc-FL after recombinant overexpression and purification. Mutants lacking Lmo2812 and another low molecular mass (LMM) PBP, PBP5 (PBPD1)--both with DD-carboxypeptidase activity--displayed only slight morphological alterations, demonstrating that they are dispensable for cell survival and probably participate in the latter stages of peptidoglycan synthesis. Since Lmo2812 preferentially degrades low-molecular- mass substrates, this may indicate a role in cell wall turnover.

[Antibiotic resistance of bacteria Campylobacter sp]. / Opornosc bakterii Campylobacter sp. na antybiotyki i chemioterapeutyki.

Campylobacter is recognized as a major cause of human acute bacterial enteritis. The incidence of human Campylobacter infection has increased markedly in both developed and developing countries and, more significantly, so has rapid emergence of antibiotic-resistant Campylobacter strains. It is caused by improper applying antibiotics in treating people and too frequent applying these substances in the animal husbandry. In this review, the patterns of emerging resistance to the antimicrobial agents useful in treatment of the disease are presented and the mechanisms of resistance to these drugs in Campylobacter spp. are discussed.

Novel targeted therapies of T cell lymphomas.

T cell lymphomas (TCL) comprise a heterogeneous group of non-Hodgkin lymphomas (NHL) that often present at an advanced stage at the time of diagnosis and that most commonly have an aggressive clinical course. Treatment in the front-line setting is most often cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) or CHOP-like regimens, which are effective in B cell lymphomas, but in TCL are associated with a high failure rate and frequent relapses. Furthermore, in contrast to B cell NHL, in which substantial clinical progress has been made with the introduction of monoclonal antibodies, no comparable advances have been seen in TCL. To change this situation and improve the prognosis in TCL, new gene-targeted therapies must be developed. This is now possible due to enormous progress that has been made in the last years in the understanding of the biology and molecular pathogenesis of TCL, which enables the implementation of the research findings in clinical practice. In this review, we present new therapies and current clinical and preclinical trials on targeted treatments for TCL using histone deacetylase inhibitors (HDACi), antibodies, chimeric antigen receptor T cells (CARTs), phosphatidylinositol 3-kinase inhibitors (PI3Ki), anaplastic lymphoma kinase inhibitors (ALKi), and antibiotics, used alone or in combinations. The recent clinical success of ALKi and conjugated anti-CD30 antibody (brentuximab-vedotin) suggests that novel therapies for TCL can significantly improve outcomes when properly targeted.

Nanoplasmonic sensor for foodborne pathogens detection. Towards development of ISO-SERS methodology for taxonomic affiliation of Campylobacter spp.

According to EU summary report on zoonoses, zoonotic agents and food-borne outbreaks in 2017, Campylobacter was the most commonly reported gastrointestinal bacterial pathogen in humans in the EU. Unfortunately, the standard methods for the detection of thermotolerant Campylobacter spp. in foods are time-consuming. Additionally, the qualified staff is obligatory. For this reason, new methods of pathogens detection are needed. The present work demonstrates that surface-enhanced Raman scattering (SERS) is a reliable and fast method for detection of Campylobacter spp. in food samples. The proposed method combines the SERS measurements performed on an Ag/Si substrate with two initial steps of the ISO standard procedure. Finally, the principal component analysis (PCA) allows for statistical classification of the studied bacteria. By applying the proposed ISO-SERS-PCA method in the case of Campylobacter bacteria the total detection time may be reduced from 7 to 8 days required by ISO method to 3 to 4 days in the case of SERS-based approach.