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Ethidium Bromide-Degrading Bacteria from Laboratory Gel Electrophoresis Waste: History
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Subjects: Biotechnology & Applied Microbiology
Contributor: VIKRAM GANDHI

The bacterium Proteus terrae is a non-pathogenic and natural microflora of humans, but Morganella morganii is an opportunistic pathogen. These organisms belong to risk group II. Screening the sensitivity of these isolates to antibiotics revealed a sufficient number of antibiotics, which can be used to control them, if required. BR3 and BR4 exhibited resistance to individual antibiotics, ampicillin and vancomycin, whereas only BR3 was resistant to tetracycline. The current investigation, along with earlier reported work on these isolates, identifies BR3 as a useful isolate in the industrial application for the degradation of EtBr. Identical and related microorganisms, which are available in the culture collection repositories, can also be explored for such potential to formulate a microbial consortium for the bioremediation of ethidium bromide prior to its disposal. 

  • bioaccumulation
  • risk assessment
  • inhibition zone
  • biotransformation
  • 16S-rRNA
  • Proteus terrae
  • Morganella morganii
  • pathogenicity

1. Introduction

Ethidium bromide (EtBr) containing wastes are recommended to be decontaminated or treated before they are disposed. There are a number of ways these wastes can be treated or decontaminated, including treatment with bleach, incineration, processing of the solution through Rohm and Haas Amberlite XAD-16 resin, Fenton-like reaction using MNCs (magnetic nanocatalysts) and other methods and products [1][2][3][4][5][6]. However, some of these methods used agents, such as sodium nitrite and hypophosphorous acids, which are still harmful [1][7].
In addition to these traditional methods, bioremediation has been considered as an alternate method for detoxification of these xenobiotic compounds. There are different research groups working on EtBr biodegradation and have identified some EtBr-resistant and EtBr-degrading microbes, including Aeromonas hydrophila, Bacillus species, B. thuringiensis, Neisseria canis, N. subflava, N. macacae, Pseudomonas chlororaphis and P. putida from uncontaminated soil [7][8][9]. Two unidentified bacteria, BR3 and BR4, have also been reported from EtBr containing lab waste (agarose gel), which exhibited EtBr degradation and bioaccumulation, respectively [10]. A plan of a lab model for EtBr degradation using BR3 and BR4 was also proposed, which has to be explored and evaluated further [10]. However, aseptic operation and containment is one of the important aspects of laboratory-based or industrial applications of any organism or bioreactor, and hence determining the pathogenicity and control measures of pathogens becomes the important criteria for such applications or operations of a bioreactor [11]. There are different virulence-determining factors, including fimbriae, flagella, urease, IgA proteases, amino acid deaminases, invasiveness, hemolysins, capsular polysaccharide and LPS, which contribute to the pathogenicity of a bacterium depending upon its genus [12][13].

2. Current Insights

2.1. Antibiotic Resistance Assay and Gel Electrophoresis of Crude Lysate

The tested bacterial isolates revealed different susceptibilities to the tested antibiotics. Both isolates, BR3 and BR4, were resistant to ampicillin and vancomycin, but sensitive to gentamicin and kanamycin (Figure 1 and Figure 2, Table 1). BR3 was resistant to tetracycline but sensitive to chloramphenicol, whereas BR4 responded in the opposite manner as sensitive to tetracycline but intermediate to chloramphenicol (Table 1; Figure 2). No study reported the antibiotic profile of P. terrae (BR3) M. morganii (BR4) from laboratory gel electrophoresis waste. Generally, this has been reported from post-operative wound and urinary tract infections [14].
/media/item_content/202203/621eccdb7c5cdbiotech-11-00004-g002.pngFigure 1. Growth of isolates BR3 and BR4 on nutrient agar plates. (A) Nutrient agar plate without ampicillin. (B) Nutrient agar plate with ampicillin.
/media/item_content/202203/621eccfbe9bc9biotech-11-00004-g003.pngFigure 2. Antibiotic sensitivity profile of BR3 and BR4 on Mueller–Hinton agar plates. Letters on the discs represent antibiotics: C: chloramphenicol (30 µg/disc), V: vancomycin (30 µg/disc), T: tetracycline (30 µg/disc), K: kanamycin (30 µg/disc), and G: gentamicin (10 µg/disc). The inner diameter of the medium holding plate measures 86 mm.
Table 1. Antibiotic sensitivity profile of bacterial isolates BR3 and BR4.
Diameter (mm) of the Zone of Inhibition Following 24 h of Incubation
Bacterial Isolates Antibiotics
C V T K G
BR3 32 (S) 11 (R) 11 (R) 23 (S) 22 (S)
BR4 16 (I) <1 (R) 20 (S) 22 (S) 20 (S)
Letters in parentheses represent: R: resistant, S: sensitive, and I: intermediate. Reference standard inhibition zone sizes: C: chloramphenicol (R- ≤ 12, I-13-17, S- ≥ 18); V: vancomycin (R--, I--, S- ≥ 15); T: tetracycline (R- ≤ 14, I-15-18, S- ≥ 19); K: kanamycin (R- ≤ 13, I-14-17, S- ≥ 18); G: gentamicin (R- ≤ 12, I-13-14, S- ≥ 15). The reference inhibition zone diameter is given by the Antibiotic Sensitivity Teaching kit HTM002-15PR from HiMedia Laboratories Pvt. Ltd., India. Concentration of antibiotics: C: chloramphenicol (30 µg/disc), V: vancomycin (30 µg/disc), T: tetracycline (30 µg/disc), K: kanamycin (30 µg/disc) and G: gentamicin (10 µg/disc).
The gel electrophoresis of the crude lysate revealed the presence of a fluorescent band observed below the genomic/chromosomal DNA, but significantly above the usual position of the RNA band. The gel electrophoresis of the plasmid preparation using the kit confirmed the presence of plasmid band observed just above the molecular weight marker band of 15,000 bp (Figure 3). This plasmid probably bears the genes responsible for its resistance to antibiotics; however, it was not confirmed by plasmid curing as determined by Patil and Berde [9] in their studies. This plasmid may not be attributed to bear genes for EtBr degradation, as it was already revealed by a plasmid curing study that the EtBr-degrading trait is localized on chromosomal DNA [9].
/media/item_content/202203/621ecd1c56b27biotech-11-00004-g004.pngFigure 3. A total of 1.2% agarose gel electrophoresis of plasmid preparation from bacterial isolates BR3 (A) and BR4 (B). Lanes 1–3 represent the plasmid preparation from culture grown in 1: LB medium, 2: LB medium supplemented with ethidium bromide, and 3: LB medium supplemented with ampicillin. L represents the molecular weight marker; the molecular weight ranges from 100–15,000 bp.

2.2. Identification and Phylogenetic Status of Bacterial Isolates

The 16S-rRNA gene sequence was used for the identification of bacterial isolates and phylogenetic analysis. Nucleotide sequencing of the 16S-rRNA gene of isolates BR3 and BR4 resulted in 1034 and 963 nucleotide long sequences, respectively. The respective nucleotide sequences of BR3 and BR4 were searched in the EzBioCloud microbial identifier, which revealed a 99.90% and 99.48% identity match to a P. terrae N5/687 (LN680103)-type strain (T) and Morganella morganii subsp. morganii ATCC 25830 (AJ301681) (T), respectively.
The EzBioCloud microbial identifier was used for a 16S-rRNA gene sequence similarity search of bacterial isolates with type strain (T) microbial cultures. The phylogenetic trees of respective sequences are shown in Figure 4 and Figure 5. The phylogenetic tree shows a close clustering of BR3 with P. terrae, followed by P. terrae N5/687(T) and P. cibarius JS9 (T) (Figure 4) [15]. BR4 clustered with M. morganii subsp. morganii followed by M. morganii subsp. morganii ATCC 25830 (T) and M. morganii subsp. sibonii DSM 14850 (T) in its phylogenetic tree (Figure 5). The nucleotide sequences of BR3 and BR4 were submitted to GenBank under the accession numbers KY684830.1 and KY697117.1, respectively. Thus, the phylogenetic analysis is well supported by the researchers' earlier findings, which characterized these isolates as two distinct groups with quite distinct biochemical features, as well as quite distinct mechanisms for managing EtBr in its surroundings [10].
/media/item_content/202203/621ecd4104246biotech-11-00004-g005.pngFigure 4. Phylogenetic tree based on 16S-rRNA showing the clustering of BR3 with sequences retrieved form the EzBioCloud database. The tree was constructed in MEGAX by the neighbor-joining method with a 500-bootstrap value.
/media/item_content/202203/621ecd615c248biotech-11-00004-g006.pngFigure 5. Phylogenetic tree based on 16S-rRNA showing the clustering of BR4 with sequences retrieved form the EzBioCloud database. The tree was constructed in MEGAX by the neighbor-joining method with a 500 bootstrap value.

2.3. Pathogenicity Status of the Isolates

Isolates BR3 and BR4, which are identified as P. terrae and M. morganii, respectively, may be described as opportunistic pathogens and may be assigned to risk group II (moderate individual risk, limited community risk, and includes opportunistic pathogens) [16] in the light of earlier studies, where Proteus and Morganella, both bacterial genera, were revealed to cause skin wounds and urinary tract infections [12][13][17][18]. Additionally, M. morganii is an unusual opportunistic pathogen and results in a high mortality rate due to its virulence and increasing drug resistance [12][18], which corroborated the researchers' study that BR4 was found here to be resistant to the antibiotics ampicillin and vancomycin, thus adding to the pathogenic status of BR4. Out of seven of the reported species of Proteus, three species, myxofaciens, terrae, and cibarius, have no report of pathogenicity for humans [13][19][20]. Thus, the clustering of BR3 with P. terrae N5/687(T) revealed that isolate BR3 is a non-pathogenic bacterium. Although BR3 is resistant to some tested antibiotics, such as ampicillin, vancomycin, and tetracycline, it is sensitive to other tested antibiotics, such as kanamycin, gentamicin, and chloramphenicol. Thus, BR3 may be characterized as either non-pathogenic to humans or as an opportunistic pathogen causing mild infections of the skin or urinary tract. High EtBr bio-degradation efficiency [10], sensitivity to readily available antimicrobials and its mild severity or non-pathogenicity to humans [13][19][20], makes BR3 a suitable isolate for its use in industrial applications, when the utmost care is taken. On the other hand, the authors discourage the application of BR4 in EtBr biodegradation on an industrial scale, as it is an inefficient bio-degrader [10] with a high pathogenic status and increasing drug-resistant report.

This entry is adapted from the peer-reviewed paper 10.3390/biotech11010004

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