Isolation of Antibiotic-Resistant Bacteria from a Water Sample for Molecular Analysis

Abstract

Source: Ghadigaonkar, D. and Rath, A. Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes. J. Vis. Exp. (2023)

This video demonstrates the isolation and preparation of antibiotic-resistant bacteria from a water sample using selective media. The DNA is extracted from antibiotic-resistant bacteria and amplified to identify the bacterial antibiotic-resistant genes.

Protocol

1. Sample collection and processing

1. Sample collection
   1. Collect the appropriate volume of the water sample in sterile sample containers, ensuring that not more than 3/4 of the container is filled.
   2. Transport the samples to the laboratory under aseptic conditions as soon as possible after collection and immediately process them.
2. Sample processing
   1. Aseptically filter the water sample through a sterile muslin cloth to remove any particulate matter.
   2. Carry out appropriate serial dilutions of the filtered water for further analysis.

2. Estimation of the total bacterial load and the antibiotic-resistant bacteria count

1. Determination of the total bacterial load
   1. Suspend 18.12 g of Reasoner's 2A agar (R2A agar) modified powder in 1,000 mL of double-distilled water and dissolve the mixture by heating. Autoclave the dissolved mixture at 121 °C, 15 pounds per square inch (psi) for 20 min. Prepare R2A Agar Modified plates by pouring the appropriate quantity of the autoclaved mixture into sterile Petri plates (e.g., add approximately 20 mL of autoclaved sterile medium to a 90 mm sterile Petri plate).
   2. Once the medium solidifies, evenly spread 100 µL of the appropriate dilutions of the filtered water sample on the R2A Agar, Modified plate. Perform the experiment in duplicate.
   3. Incubate all the above plates at 35 - 37 °C for 48 h (vary the temperature and incubation time depending upon the media used for isolation).
   4. Express the total bacterial load in terms of colony-forming units per milliliter (CFU/mL) using equation (1):
2. Determination of the AR bacterial count
   1. Follow steps 2.1.1-2.1.4. However, instead of R2A Agar, Modified plates, use R2A Agar, Modified plates supplemented individually with five different antibiotics, namely cefotaxime (3 µg/mL), ciprofloxacin (0.5 µg/mL), erythromycin (20 µg/mL), kanamycin (15 µg/mL), and vancomycin (3 µg/mL).
   2. Add the antibiotics separately into tubes containing 20 mL of sterile molten R2A Agar, Modified (with the temperature of the molten R2A Agar, Modified at ≤40 °C) to achieve the final antibiotic concentration as mentioned in step 2.2.1.
   3. Swirl for even mixing and pour onto sterile Petri plates before the agar solidifies. Perform the experiment in duplicate.
   4. Incubate all the above plates at 35 - 37 °C for 48 h (the temperature and incubation time may vary if using a different media).
   5. For quality control and checking the efficacy of the antibiotics, spread 100 µL of bacterial suspensions of the Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 strains onto their respective antibiotic-containing R2A Agar, modified plates (ensure that the density of the fresh culture used for the inoculation is optical density, OD = 0.5 at 600 nm).
   6. Determine the antibiotic-resistant bacterial count in terms of CFU/mL as described in step 2.1.4.

3. Identification of culturable bacteria by 16S ribosomal RNA (rRNA) gene sequencing

1. Preparation of DNA template from the isolates for polymerase chain reaction (PCR)
   1. Using a sterile toothpick, take a single, isolated, pure colony of the isolate growing on a Petri plate. Suspend the bacterial colony in 100 µL of sterile double-distilled water in a sterile microcentrifuge tube and boil for 10 min.
   2. Centrifuge the suspension at 10,000 × g for 2 min to pellet the debris, and transfer the supernatant to a fresh sterile microcentrifuge tube for use as the crude DNA template.
2. Targeted PCR amplification of the V3 region of the 16S rRNA gene and sequencing
   1. Prepare 40 µL of the reaction mixture in a PCR tube for PCR amplification, as mentioned in Table 1.
      NOTE: The DNA preparation should be carried out on an ice block while minimizing the chance of contamination (wear gloves while handling the reagents, and clean the work surface thoroughly with 70% ethanol).
   2. Place the tube in the thermal block, and run the appropriate program in the PCR thermal cycler. See Table 2 for the standardized PCR cycling conditions and the primer information for the amplification of the V3 regions of the 16S rRNA genes.

Table 1: PCR mastermix constituents. The reaction mixture composition of various PCR reagents.

PCR reagents	Volume (μL)
2x Taq Master Mix	20
Forward primer (10 pico mole)	1.5
Reverse primer (10 pico mole)	1.5
Crude DNA template	1.5
Sterile molecular biology water	15.5
Total volume	40

Table 2: PCR cycle conditions for amplification of the 16S rRNA gene. The PCR cycle conditions required for 16S rRNA gene amplification are shown. The steps include an initial denaturation step, followed by 35 cycles of denaturation, annealing, and extension, and a final extension step.

Direction	Primer Sequence 5’ – 3’	PCR conditions	No. of cycles
Forward	GGAGGCAGCAGTAAGGAAT	94 °C for 10 min	1
		Denaturation at 94 °C for 30 s	35
		Annealing at 50 °C for 30 s
		Extension at 72 °C for 40 s
Reverse	CTACCGGGGTATCTAATCC	Final extension at 72 °C for 5 min	1

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2x PCR Taq mastermix	HiMedia	MBT061-50R	For making PCR reaction mixture
37 °C Incubator	GS-192, Gayatri Scientific	NA	For incubation of bacteria
Autoclave	Equitron	NA	Required for sterilization of media, glass plates, test tubes, etc
Bisafety B2 Cabinet	IMSET	IMSET BSC-Class II Type B2	Used for microbiological work like bacterial culturing, AST etc.
Cefotaxime antibiotic powder	HiMedia	TC352-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Centrifuge minispin	Eppendorf	Minispin Plus-5453	Used to pellet the debris during crude DNA preparation
Ciprofloxacin antibiotic powder	HiMedia	TC447-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Colorimeter	Quest	NA	For checking the OD of culture suspensions
Cooling shaker incubator	BTL41 Allied Scientific	NA	For incubation of media plates for culturing bacteria
Erythromycin antibiotic powder	HiMedia	CMS528-1G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Erythromycin antibiotic powder	HiMedia	TC024-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Kanamycin antibiotic powder	HiMedia	MB105-5G	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Luria Bertani broth	Himedia	M1245-500G	For enrichment of cultures
R2A Agar, modified	HiMedia	M1743	For preparation of media plates for isolation of total and antibiotic resistant (AR) bacterial load
Sodium chloride	HiMedia	TC046-500G	For preparation of 0.85% saline for serially diluting the water sample
Staphylococcus aureus subsp. aureus ATCC 29213	HiMedia	0365P	Used as a control while performing AST
Vancomycin antibiotic powder	HiMedia	CMS217	For preparation of antibiotic stock solution required during isolation of antibiotic resistant bacteria
Weighing balance	Mettler Toledo	ME204 Mettler Toledo	Used for weighing media powders, reagent powders etc.