Comparison of Taxonomic Resolutions of Various Typing Methods for Clostridium and Paraclostridium Species Isolated from Landfill Leachate

Abstract

Anaerobic bacteria and methanogenic archaea at municipal landfill dumping sites play a vital role in the landfill ecology, waste degradation, global warming and climate change. Although landfill works as a cheap way of solid waste management, unmanaged landfill plays a significant role in spreading pollutants and pathogens in natural ecosystems. The genera Clostridium and Paraclostridium are important groups of anaerobic microbes from a public and environmental health perspective. In the present study, we have isolated different species of Clostridium and Paraclostridium from landfill leachate and performed a comparative analysis to understand their role in landfill ecology and public health. Our data indicate that the anoxic zone of landfills acts as a breeding ground for different species of Clostridium and Paraclostridium, including pathogenic species like C. sporogenes and C. argentinense and leachate, plays a crucial role in the spread of Clostridium in soil and water ecosystem and acts as one of the primary sources of a cause of infection in field workers. We also reported that VITEK and MALDI-TOF/MS are not ideal ways to identify Clostridium at the species level, and 16S rRNA gene sequencing should be the method of choice. In the current study, we also demonstrated that members of the genus Clostridium showed better growth response on Brain Heart Infusion (BHI) and Gifu Anaerobic Medium (GAM), and they can be one of the alternatives to existing media for cultivation and physiological studies of Clostridium.

Introduction

Clostridium is a group of Gram-stain-positive, spore-forming, obligate anaerobes reported from different niches, including soil, wastewater, faecal samples, wounds and landfills [1, 2]. The formation of spores allows Clostridium to survive in harsh environments and retain its viability even after exposure to oxygen. Members of the genus Clostridium (C.), such as C. botulinum, C. difficile, C. perfringens, C. septicum, C. sordellii and C. tetani, are well known for their pathogenicity, clinical significance, and emerging antimicrobial resistance. There are two significant groups of pathogenic Clostridium based on myonecrosis or gas gangrene production. Pathogenicity of the strains is generally tested by hemolysis on the blood agar, using serological tests and study of antibiotic susceptibility patterns. Based on the hemolysis pattern, organisms are categorized as alpha, beta, and gamma hemolysin producers [3]. Based on laboratory animal data, it has been found that C. argentinense may or may not produce a neuroparalytic toxin responsible for botulism and can be neutralized by type G- botulinal antitoxin [4]. In addition, complete neurotoxin gene clusters were reported from the plasmid sequence of C. parabotulinum, C. sporogenes, and C. argentinense [5]. Like other bacteria, members of the genus Clostridium also produce biofilm to survive under antibiotics and other stress conditions [6, 7]. Several members of the genus Clostridium, including C. perfringens, C. sordellii, and C. tertium along with other anaerobic pathogens, have been reported from infected tissues like frozen brain tissue, diabetic foot wounds, aspirates, and septic arthritis [8–11]. Antibiotic resistance of C. difficile in the context of different antibiotics was well-reviewed by Spigaglia et al. (2018) [11]. In addition to their clinical significance and pathogenesis, members of the genus Clostridium play an important role in waste degradation and cycling of the materials in anoxic pockets of environments such as lower anoxic strata of landfills and anoxic landfill leachate sediment. It is found that Clostridium and other bacteria initially degrade dumped cellulosic materials at the landfills and make them accessible to further degradation [1, 12]. Hydrogen producer C. perfringens and vinyl chloride degrading C. saccarobutylicum have been isolated from landfill leachate sludge [13, 14]. In addition, several strains of cellulolytic Clostridium were isolated by Cainiez et al. (1993) from the municipal solid waste digester and used for biogas production [15].

Furthermore, two clone libraries prepared from the cellulose-associated biomass and planktonic phase of the methanogenic landfill leachate bioreactor were dominated by the phylum Firmicutes, and the majority of them fall into one of five lineages of the Clostridium [16]. Amplification of landfill leachate DNA by specific primers of 16S rRNA gene showed that the C. thermocellum and C. leptum groups occurrence was prevalent, C. coccoides and C. lentocellum group was rarely found, and the C. botulinum group was not detected [1]. To understand the type of Clostridium present at Indian landfill sites and how landfill leachate plays a role in the spread of Clostridia from the landfill site to soil and freshwater bodies, in the present study, we performed a comparative analysis on eight different strains using phenotypic and molecular approaches. Our data indicate that Clostridium constitutes a substantial population of the anoxic lower part of landfills and plays an active role in cellulolytic waste degradation. In addition, we found the taxonomic resolution provided by VITEK-2 and MALDI-TOF/MS needs to be more satisfactory for the identification of Clostridium species, and we should only rely on 16S rRNA gene sequencing for typing of novel Clostridium, especially environmental isolates.

Materials and Methods

Sample Collection

Leachate was collected from the Moshi landfill site near Pune, India, as discussed previously [17]. The Geographical location and images of the sampling site are given in Fig. 1. In brief, serum bottles were washed, rinsed and autoclaved for collection of samples. Black-coloured leachate generated from the Moshi-landfill was collected in 500 mL sterile serum bottles pre-flushed with N2 gas. To minimize oxygen exposure and diffusion, bottles were filled with leachate up to the neck, put on the stopper and sealed by an aluminium crimp without allowing air bubbles. Collected leachate samples were transported to the laboratory on the ice blocks and kept at 4 °C in a cold room for further study and bacteria isolation.

Fig. 1.

Images of leachate collection sites included in this study

Media Preparation and Isolation of Anaerobes

All reagents, chemicals and media used were laboratory-grade and purchased from HiMedia (Mumbai, India). For the Isolation of anaerobes, tryptone soy agar (TSA) and half-strength leachate agar were prepared according to the protocol described by Ranade and Gadre [18]. Similarly, Reinforced Clostridium medium (RCM), Brain Heart Infusion (BHI) medium, Gifu Anaerobic Medium (GAM, HiMedia, M1801), Brucella agar base with vitamin K1, and tryptic soy agar (TSA) medium were prepared anaerobically. In brief, dissolved the appropriate amount of media component, adjusted pH and flushed the medium with inert nitrogen to make them anoxic. Anaerobic blood agar base medium was autoclaved and aseptically added with 5% sheep blood. Plates were poured inside the anaerobic chamber (Thermo Fisher Scientific, USA, model number 1025 (1029), running with 85% N2, 10% H₂ and 5% CO₂ and kept there for further use. The bacteria was isolated from leachate using serial dilution and spread plate method. Leachate was serially diluted in the phosphate-buffered saline prepared anoxically. Afterwards, 100 µL of serially diluted samples were spread on selected media plates and incubated anaerobically inside the anaerobic chamber (Thermo Fisher Scientific, USA), at 30 °C. Plates were observed every 24 h for a week. Morphologically distinct colonies were picked and purified by several re-streaking on similar media plates. Purified colonies were preserved for the long term in a deep freezer ( − 80 °C) by the protocol described by Prakash et al. [19].

Identification by 16S rRNA Gene Sequencing, Matrix-Assisted Laser

Desorption/Ionization-Time of Flight (MALDI-TOF/MS), VITEK and Phylogeny

The 16S rRNA gene sequence analysis identified isolated pure cultures. Genomic DNA was extracted and purified from freshly grown cultures using the cetyltrimethylammonium bromide (CTAB) method [20]. 16S rRNA gene was amplified using the bacterial universal primer pair 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') as described previously [21]. Sequencing was carried- out using BigDye® Terminator (Applied Biosystems) method using 27F, 704F, 907R, and 1492R primers. Raw sequence files were manually edited, and the DNASTARTM SeqManTM software package generated contigs. A similarity search with closely related type strains was conducted using the EzTaxon database [22]. The phylogeny of all the strains was constructed by using MEGA-X software [23]. Phylogenetically distinct Clostridium strains were taken further for comparative study. To assess the comparative resolving potential of MALDI-TOF/MS, VITEK-2 and 16S rRNA gene sequence of selected Clostridium, active (24 h grown) cultures were also identified using Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF/MS (Bruker) and VITEK-2 (BioMeriex) platform as per the protocol described previously by Prakash  et al. [19] and Joglekar et al. [21, 24]. For protein extraction, selected strains of Clostridium were grown on tryptic soy agar (TSA) plates using similar experimental conditions. For MALDI-TOF/MS typing, total cellular proteins were extracted using the formic acids method. At least two biological replicates were taken for the generation of protein spectra. Protein spectra were generated and visualized using the MALDI‐TOF MS Biotyper of (Bruker Daltonics) with software 3.1 (Brualtonics). The score values generated by the software were used to detect genus and species-level resolution.

Screening of Media, Salinity, and pH for Optimum Growth

All the selected strains of Clostridium were streaked on RCM, BHI, GAM, TSA, and Brucella agar plates and incubated simultaneously at the same temperature to choose the medium for optimum growth. The growth pattern of all the strains was recorded on different media. Based on the better growth response of selected Clostridium, GAM medium was selected for physiological studies. For salinity and pH experiments, we prepared the GAM- broth with different salinity (0.17 to 0.85 M NaCl) and pH range (4–10) in Hungate tubes, inoculated inside an anaerobic glove box (Thermo Fisher Scientific, USA, model number 1025 (1029), and incubated in inbuilt incubator at 30 °C. We monitored the growth every 24 h up to three days. We conducted all the experiments using three biological replicates. Change in pH due to growth was also monitored during the experiments.

Study of Physiological Traits

All the physiological traits of selected Clostridium species were evaluated using an aseptic and anoxic method inside the anaerobic chamber (Thermo Fisher Scientific, USA) operated at zero PPM O2 with an environment of N2: H2: CO2 (85: 10: 5). Inoculum of the pure culture was prepared as per the instructions given by the API20E and APIZYM kit (BioMeriex). A minimum of three API20E strips were filled to the desired mark using an anoxic and aseptic approach. Sterile mineral oil was added in ADH, URE, LDC, ODC, and H2S marked couple. API strips were incubated at 37 °C for 24 h and observed the data. As mentioned in the instruction manual, we incubated the APIZYM- kits for six hours before taking the observation. After that, ZYM -A and ZYM- B solution was added. The change in colour of the wells was recorded and compared with the standard result table. In addition, the biofilm formation assay was carried out as per the protocol described by Charlebois et al. [25]. The seed inoculum of the selected strains was raised anoxically in a Hungate tube filled with 10.0 mM glucose-supplemented TSB. For each strain, 135 µL of broth was added in 96-well plates, followed by 15 µL of the seed culture. The inoculated plate was incubated inside the glove box for 96 h, and the growth was measured at 600 nm. The culture was then withdrawn from the wells, rinsed with distilled water, followed by phosphate-buffered saline, and air-dried. After that, 150 µL of 0.2% crystal violet was added to each well for 30 min. The extra stain was removed by washing it with distilled water followed by phosphate-buffered saline. Each well had the stain removed by adding 150 µL methanol and incubating for 30 min. The absorbance of the stain was taken at 570 nm [25–27]. Similarly, VITEK-2, APZyme and API kits data were also generated using three different strips and three independent biological replicates, and the observation taken was almost similar.

Data Availability

Strains are deposited in the National Centre for Microbial Resource (NCMR) with the following accession numbers: MCC 4633, MCC 4498, MCC 4657, MCC 4658, MCC 4659, MCC 4499, MCC 4382, and MCC 4383. 16S rRNA gene sequences are deposited in GenBank with the following accession numbers- OM737887, OM737888, OM737889, OM737890, OM737891, OM737892, OM737893, OM737894.

Result

The image of the landfill, GPS location of the sampling site and leachate are presented in Fig. 1. A total of 200 strains were isolated in this study using different media and conditions. Colonies with different morphology, colour, and size were picked and purified. Initially, all the strains were identified by 16S rRNA gene sequencing. Due to the ecological and clinical significance of Clostridium, a total of 8 strains of different species of the genus Clostridium isolated from this study were selected for the comparative analysis (Table 1). All eight strains showed rapid and luxuriant growth on BHI and GAM with similar growth patterns. Therefore, the Gifu Anaerobic Broth (GAM) medium was selected for further physiological studies. All cultures showed optimal growth up to 3% NaCl but a sudden decrease in Optical Density (OD) at 4% NaCl. No growth was observed at 5% NaCl. All strains showed positive growth in the pH range of 5 to 10, but growth inhibited below pH 5.

Table 1.

Identification by 16S rRNA gene sequence similarity, MALDI-TOF/ MS, and VITEK-2 instruments

Strain ID	Identification and GenBank no	Similarity (%)	Identification by MALDI	Similarity	Identification by VITEK-2	Sourceof isolation	Pathogenicity	Refs.
MLRT-1	C. sporogenes (JFBQ01000001)	99.3	C. sporogenes	2.1	P. bifermentans	Soil	Food spoilage; occasionally pathogenic	[41]
MLRT-2	C. sporogenes (JFBQ01000001)	99.4	C. sporogenes	1.8	P. bifermentans	Soil	Food spoilage; occasionally pathogenic	[41]
MLRT- 4	C. sporogenes (OQ931883)	99.7	C. sporogenes	2.1	P. bifermentans	Soil	Food spoilage; occasionally pathogenic	[41]
MLCol-4	C. argentinense (X68316)	98.9	C. subterminale	2.0	P. bifermentans	NoD	Formerly C. botulinum toxin type G	[4]
MLP-26	C. argentinense (X68316)	99.7	C. subterminale	1.8	P. bifermentans	NoD	Formerly C. botulinum toxin type G	[4]
MLCol-38	P. benzolyticum (LBBT01000182)	99.9	C. sp.	2.0	C. group	Marine sediment	NoD	[43]
MLRT- 3	P. benzolyticum (LBBT01000182)	100.0	NRI	1.6	ND	Marine sediment	NoD	[43]
MLP-25	C. thiosulfatireducens (AY024332)	99.8	NRI	1.4	C. group	Anaerobic sludge	NoD	[42]

For VITEK similarity index is not applicable, NRI Not reliable identification, ND Not detected, NoD No data

The result of comparative identification using 16S rRNA gene sequence comparison, MALDI- TOF/MS and VITEK-2 is presented in Table 1. Data obtained from a comparative study indicates that MALDI showed better agreement with 16S rRNA gene sequencing than VITEK- 2 (Table 1). 16S rRNA gene sequences > 1200 bp of all strains were obtained. Similarity search by BLAST using 16S rRNA gene sequence showed that almost all strains have 99% sequence similarity with existing type species. However, at the same time, they are phylogenetically and physiologically different from their type strains (Table 2 and Fig. 2). MALDI- TOF/MS accurately identified the cultures at the genus level, while some discrepancy was found in identification using the VITEK-2 instrument. Phylogenetic tree constructed using the neighbour-joining method gave almost similar tree topology. All the strains clustered with a close relative but at the same time showed substantial phylogenetic distance (Fig. 2) and indicated their novelty.

Table 2.

Differential phenotypic features of selected Clostridium and Paraclostridium Species isolated from landfill leachate

Features	Clostridium strains
	MLRT 1	MLRT 2	MLRT 4	MLCol 4	MLP 26	MLCol 38	MLRT 3	MLP 25
Colony size (mm)	> 5	> 5	1–2	> 5	2–3	2–5	2–5	1–2
Colony shape	Irregular, flat	Irregular, flat	Round convex	Irregular spreading	Round, raised	L-form, spreading	Round convex	Irregular spreading
Colony colour	Cream	Transpar ent cream	Cream	Cream	Cream	Cream	Cream	Cream
L-tryptophane (TRP)	–	–	–	–	–	W +	W +	W +
L-arginine (ADH)	–	–	–	–	–	+	–	–
Esculine ferric citrate (ESC)	+	W +	+	–	–	–	+	+
Alkaline phosphtase	–	–	–	+	+	2 +	3 +	–
Esterase (C4)	4 +	4 +	4 +	+	2 +	–	–	–
Esterase lipase (C8)	+	+	+	+	+	+	2 +	–
Lipase (C14)	–	–	–	–	–	–	–	–
Leucinearyl amidase	–	–	–	W +	4 +	3 +	4 +	–
Valinearyl amidase	–	–	–	+	+	–	–	+

Cultures showed negative results for potassium nitrate (NO₃), d-glucose (GLU), Urea (URE), 4-nitrophenyl-β-D-galactopyranoside (PNPG), l arabinose (ARA), d – mannose (MNE), d – mannitol (MAN), N—acetyl- glucosamine (NAG), d – maltose (MAL), potassium gluconate (GNT), capric acid (CAP), adipic acid (ADI), malic acid (MLT), trisodium citrate (CIT), phenylacetic acid (PAC), lipase (C 14), cystinearyl amidase, trypsin, α- chymotrypsin, α- galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β- glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase

No mucilage found around any colony

All selected strains showed positive test for gelatin (GEL) degradation (bovine origin), and acid phosphatase and naphthol-AS-BI- phosphohydrolase

Fig. 2.

The evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model. The tree with the highest log likelihood (− 4584.61) is shown. The percentage oftrees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 0.3969)). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 28 nucleotide sequences. There were a total of 1174 positions in the final dataset. Evolutionary analyses were conducted in MEGA11

Differential results from API20E and APZYME test kits and morphotyping are presented in Table 2. Strains showed positive tests for the enzymes esculine ferric citrate (ESC), gelatin, alkaline phosphatase, esterase, esterase- lipase, leucine arylamidase, acid phosphatase, and naphthol-as-bi-phosphohydrolase while all other tests were negative. Our biofilm formation data showed that all selected strains were negative for biofilm formation except MLRT1 (Fig. 3, 4).

Fig. 3.

Cyclic representation of role of landfills and landfill leachate in contamination of soil and water ecosystems

Fig. 4.

The bar graph represents the relative biofilm formation potential of selected Clostridium and Paraclostridium strains isolated from landfill leachate sites. A higher optical density value (OD) indicates higher biofilm formation potential

Discussion

Our data indicate that landfills harbour different species of Clostridium with ecological and public health potential. Similar to our observation, clone sequences from 17 landfill samples showed a close relationship with known cellulose-degrading Clostridium [1]. Furthermore, a culture-independent study from the Laogang landfill refuge showed a variable abundance of Clostridium in the initial methanogenic phase. In contrast, the stable phase showed a 4–fivefold higher abundance of Clostridium [28]. It suggests that Clostridium facilitates the process of landfill methanogenesis in assisting the initial phase of waste degradation. In addition, our metagenomic study of the same leachate samples indicates that landfill leachate constitutes a different species of Clostridium [40]. All the previous reports support that landfills constitute a diverse species of Clostridium. Previous data showed that the Gifu anaerobic medium is good for culturing anaerobic organisms, and our result supported previous observations. We also found quicker growth of Clostridium on Gifu (GAM) and suggest the use of GAM even in clinical microbiology laboratories for quick cultivation of Clostridium [29–31]. Isolated Clostridium strains showed positive results for producing important enzymes like esterase, lipase, phosphatase, and leucinearylamidase gelatinase. L-leucine arylamidase facilitates penetration of Candida albicans into the host tissue, making it pathogenic in human infection [32]. Acid phosphatase catalyzes the hydrolysis of phosphate esters in an acidic environment and plays an important role in the physiological process of the human being [33]. Esterase plays a major role in degrading natural materials and industrial pollutants, like cereal wastes, plastics, and other toxic chemicals. The applications of esterase and other enzymes in agriculture, food, and pharmaceutical industries are very well reported by and Singh [34] and Panda [35]. A study conducted on an anoxic gut-bacterial community of mealworms showed higher activities of enzymes β-galactosidase, acid phosphatase, β-glucuronidase, naphthol-AS-BI-phosphohydrolase, leucine arylamidase, and alkaline phosphatase and potential of plastic degradation [36, 37]. Positive tests for these enzymes in selected Clostridium from this study indicate that these enzymes assist the organism to survive in the anaerobic zone of landfill and assist in waste degradation at landfill sites. The role of Clostridium in waste management, degradation of pollutants, and production of enzymes is very well recognized. Strains isolated from landfills in this study can be used for biotechnological purposes, such as producing industrial enzymes or managing municipal solid waste [34]. Leachate is the main source of surface and groundwater contamination through mixing and subsurface recharge. Leachate has heavy loads of pollutants and pathogens and plays an important role in the spread of pathogens. This study was also focused on assessing the role of landfill leachate in the spread of pathogenic Clostridium in soil and water ecosystems. Multiple strains of C. sporogenes and C. argentenense were isolated from this study. It is initially considered that C. sporogenes are non-pathogenic and have been isolated as commensal organisms from the healthy human gut. Still, more studies with these organisms revealed their involvement in the pathogenesis of several diseases, including septic arthritis, gangrene, bacteremia and septicemia [38]. Kanaujia et al. reported non-traumatic gas-gangrene by C. sporogenes, which was rapidly progressing and lethal and needs rapid diagnosis and therapeutic intervention for better results [39]. Abusnina et al. reported lethal bacteremia caused by C. sporogenes from immunocompromised patients [38]. Similarly, C. argentinense was initially isolated from soil and considered non-pathogenic, but later studies demonstrated that it harbours the bont genes cluster required to produce botulinum neurotoxin [5]. Detection of multiple strains of C. sporogenes and C. argentenense, along with other members of the genus Clostridium (Table 1) from the current study, indicate that an anoxic zone of landfills acts as a breeding ground for a different group of Clostridium species due to the presence of ideal condition and nutrients. From landfills, they reach into liquid leachate and increase their quantity again. When untreated leachate is used in agricultural irrigation or is discharged as such in freshwater bodies, it plays a crucial role in the spread of Clostridium in soil and water ecosystems. It causes infection in field workers and other field populations. A cyclic representation of the role of landfill and landfill leachate in public and environmental health is given in Fig. 3. Thus, leachate treatment is imperative from public and environmental health perspectives before discarding them in freshwater ecosystems or using them for agricultural irrigation. Our findings also indicated that, except for one strain, all others had a moderate biofilm-forming potential, indicating that landfill leachate is not too harsh for their development and multiplication despite high levels of contaminants, salinity, and stress.

Identification based on MALDI-TOF/MS and VITEK-2 platforms relies on the database provided in the respective systems. According to the available taxonomic records, the Clostridium comprises > 159 species with validly published names, while Paraclostridium comprises two species with validly published names. However, the database used in MALDI-TOF/MS and VITEK platforms cannot distinguish all the validly published species of Clostridium and Paraclostridium species due to a lack of timely updates and entries of novel taxa in the existing database. Week database is one of the major limitations of MALDI-TOF/MS and VITEK-2-based identification and non-accurate typing. Researchers from the non-taxonomic field need to be made aware of this fact, wasting time and money and getting non-reliable identification. It is also noticeable here that some Clostridium species are indistinguishable based on 16S rRNA gene sequence analysis alone and need further clarity using polyphasic approaches. For instance, strain MLRT1 (OM737887) showed relatively high pairwise sequence similarity to Clostridium sporogenes DSM 795 T (99.38%), Clostridium botulinum ATCC 25763 T (99.30%) and Clostridium combesii ATCC 25545 T (99.30%), and make it indistinguishable and indicate need polyphasic approach of taxonomy for further resolution.

In summary, we performed a comparative study using 8-different strains from 5-different species of Clostridium isolated from landfill leachate. Morphotypic, molecular and physiological data indicate they are phylogenetically and physiologically different. They could be the novel species of the genus Clostridium but need further in-depth characterization to resolve their taxonomic status better. VITEK-2 and MALDI-TOF/MS are emerging tools in clinical diagnosis, but typing using these platforms sometimes gives inaccurate result and need an updated database for better resolution of Clostridium. We also proposed that landfills act as a breeding ground for Clostridium species, and leachate play an active role in their transmission in soil and water ecosystem. This is the first report on Clostridium and Paraclostridium isolates from leachate, which can provide valuable inputs on the role of landfill Clostridium in public and environmental health, including AMR and route of transmission from landfill to the natural ecosystem.

Abbreviations

C.

Clostridium

P.

Paraclostridium

MALDI‐TOF/MS

Matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry

ADH

Arginine dihydrolase

URE

Urease

LDC

Lysine decarboxylase

ODC

Ornithine decarboxylase

VITEK

VITEK® 2 COMPACT microbial detection system