Page 84 - AIH-1-2

Artificial Intelligence in Health Combating XDR-bacteria as we approach 2050

Instead, we emphasized peptide antibiotics, which are extracted with pure ethanol and centrifuged at 10,000 rpm
novel in their preparation and mode of action. 17 for 10 min to obtain a clear solution which was then dried
at room temperature.
2. Materials and methods
2.4. Biochemical and molecular biological assays
2.1. PubMed and database search
The methyl red assay principle is based on mixed acid
We conducted a search on PubMed (www.ncbi.nlm.nih. fermentation (acetic, lactic, and succinic) by certain
gov/pubmed) using the following terms: “MDR bacteria,” bacteria, resulting in a significant pH decrease in the
“nisin,” “salivaricin,” “phyto-drugs,” “herbal drugs,” and medium, dropping below 4.4. This pH change is indicated by
“MDR plasmids.” a color change of the pH indicator, methyl red (2-dimethyl-

2.2. Isolation of multi-drug-resistant bacteria 4-amino azobenzene-O-carboxylic acid), turning from
yellow when the pH is above 5.1 to red at pH 4.4.
We isolated MDR bacteria from samples obtained
from the Ganges River by plating 0.1 mL of water onto The Voges-Proskauer assay, named after two pioneering
LB+agar+50 µg/mL ampicillin or other antibiotics. microbiologists, is used to detect the formation of acetyl
Following incubation, single colonies were selected methyl carbinol by bacterial metabolism, a product of the
and tested for various drug sensitivities using different butylene glycol pathway. Organisms such as members
antibiotic-impregnated papers. Plasmids were isolated of the Klebsiella-Enterobacter-Hafnia-Serratia group
using the alkaline lysis method as described in Maniatis produce acetoin as the chief end product of glucose
et al. The polymerase chain reaction (PCR) was conducted metabolism and form smaller quantities of mixed acids. In
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using a standard PCR kit employing specific forward and the presence of atmospheric oxygen and 40% potassium
reverse primers targeting mdr genes, including bla, tet, hydroxide, acetoin is converted to diacetyl, which is then
acrA, mcr, and 16s rRNA genes for 30 cycles (95°C for 45 s, converted into a red complex under the catalytic action of
52°C for 1 min, and 72°C for 1.5 min). The sequencing of alpha-naphthol.
PCR fragments was performed at Xcelris Labs Ltd., India. Simmons citrate agar serves as an agar medium used
The primers used in this study are presented in Table 1. for the differentiation of Enterobacteriaceae based on the
utilization of citrate as the sole source of carbon. In the
2.3. Preparation of phytoextracts and purification of early 1920s, Koser developed a liquid medium formulation
NU2 and CU1 for the differentiation of fecal coliforms from the coliform
Phytoextracts were prepared by adding 5 mL of ethanol to group. Simmons later modified this formulation to
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1 g of semi-dry chopped root or bark in a 50 mL German- produce a solid medium that eliminated potential errors
made plastic tube and left overnight at room temperature when interpreting growth. When the bacteria metabolize
(25 – 30°C). Purification of NU2 and CU1 phytochemicals citrate, the ammonium salts are broken down to ammonia,
was carried out using preparative TLC (20 × 15 cm). The which increases alkalinity. This shift in pH turns the
CU1 band was visually identified and cut, while the NU2 bromthymol blue indicator in the medium from green to
band was cut under UV illumination. Silica band was blue above pH 7.6.

Table 1. Primers used in this study 14
Name Sequence of the primers Tm Size
P27F 5’-AGA GTT TGA TCC GAA CGC T-3’ 62°C 1.4 kb
P1392R 5’-TAC GGC TAC CTT GTT ACG ACT TCA-3’ 65°C
cmrF 5’-TTC GTT AGT CTG CCG TTG CT-3’ 56°C 323 bp
cmrR 5’-ATC GCT GGC AAA CAG GGT TA-3’ 57°C
tem-sF1U 5’-ATGATGAGCACYTTTAAAGT-3’ Y=C/T 56°C 312 bp
tem-sR1U 5’-TCATTCAGYTCCGKTTCCCA-3’Y=C/T; K=G/T 58°C
tetF 5’-CTT CGC TAC TTG GAG CCA CT-3’ 57°C 910 bp
tetR 5’-GCA GAC AAG GTA TAG GGC GG-3’ 57°C
acrAB-F 5’-ATG CTC TCA GGC AGC TTA GCC-3’ 59°C 1 kb
acrAB-R 5’-TGT CAC CAG CCA CTT ATC GCC-3’ 59°C
ctxF1U 5’-AACACMGCMGATAATTCACA-3’ M=A/C 59°C 586 bp

Volume 1 Issue 2 (2024) 78 doi: 10.36922/aih.2284