Our method answers key questions, such as what is the overall load of antibiotic resistant bacteria in the water sample? What is the identity of these bacteria and what are different types of antibiotic resistant traits and genes they are carrying? The advantage of our protocol is that it can detect even bacteria that cannot be grown in the lab, which constitute a significant proportion of the total bacteria in the water sample.
Hence, theoretically, no bacterium or its resistant traits are left out. This combinatorial technique involving both culture-based and culture-independent techniques can be replicated and customized for any sample obtained from sewage, pharmaceutical or hospital effluence, clinical and nonclinical settings, and more. Along with Devika, demonstrating the experimental procedures will be Harshali Shinde and Maitri Mishra, both senior research fellows from my laboratory.
To begin, process the water sample aseptically by filtering it through a sterile muslin cloth to remove any particulate matter. Then, serially dilute the filtered water sample for further analysis. To determine the total bacterial load, place 100 microliters of the appropriate dilutions of the filtered water sample on the solidified R2A agar plates and duplicates, and spread evenly.
Incubate all the sample spread plates at 35 to 37 degrees Celsius for 48 hours. After the incubation period, count the colonies and express the total bacterial load in colony forming units per milliliter. To determine the antibiotic resistant bacterial count, add the antibiotics separately into tubes containing 20 milliliters of sterile molten R2A agar modified approximately at 40 degrees Celsius.
Swirl to mix evenly and pour onto sterile Petri plates before the agar solidifies. For quality control and to check the antibiotics'efficacy, spread 100 microliters of bacterial suspension onto their respective antibiotic containing R2A agar modified plates. Incubate the plates at 35 to 37 degrees Celsius for 48 hours, and then determine the bacterial count.
To prepare the DNA template from the isolates for PCR, using a sterile toothpick, take a single isolated pure colony of the isolate growing on a Petri plate. Suspend the bacterial colony in 100 microliters of sterile double distilled water in a micro centrifuge tube and boil it in a boiling water bath or a dry bath for 10 minutes at 100 degrees Celsius. Centrifuge the suspension at 10, 000 g for two minutes to pellet the debris and transfer the supernatant to a fresh sterile microcentrifuge tube to use as the crude DNA template.
To amplify the V3 region of the 16S rRNA gene by PCR, prepare 40 microliters of the reaction mixture in a PCR tube. Place the tube in the thermal block, and run the appropriate program in the PCR thermal cycler. For resolving and visualizing the PCR amplicons, mix 10 microliters of the amplified PCR product in two microliters of 6x gel loading buffer, and load this mixture into the wells of a 1.5%agarose gel along with a 100 base paired DNA ladder for estimating the size of the amplicons.
Carry out electrophoresis of the gel in the TAE tank buffer at 80 to 100 volts until the tracking dye runs 3/4 of the gel. Then visualize the amplicon bands under a UV transilluminator. To prepare the inoculum, aseptically inoculate a single isolated purified antibiotic resistance colony using a sterile loop in two milliliters of Luria-Bertani, or LB, broth without any antibiotic and incubate at 37 degrees Celsius at 80 rpm overnight.
The following day, add 100 to 150 microliters of the overnight grown culture to two milliliters of fresh non-selective LB broth and incubate for two to four hours until the optical density at 600 nanometers reaches 0.4 to 0.5. Dilute the freshly grown culture suspension using sterile 0.85%saline solution and mix gently to distribute the cells evenly to reach a culture density equivalent to 0.5 McFarland standard. Within 15 minutes of dilution, dip a sterile cotton swab into the inoculum and remove the excess suspension to avoid over inoculation of the plates.
Then spread the culture evenly on the prepared Mueller Hinton agar plate, starting from the top and going back and forth from edge to edge. Then rotate the plate by 60 degrees, and start swabbing again. To place the antibiotic discs, use a flame sterilized forceps and aseptically transfer the antibiotic discs onto the inoculated agar plates.
Press the discs gently to ensure complete level of contact with the agar. Place the appropriate number of antibiotic discs on the agar plate by considering the organism the antibiotic used and the plate size to avoid overlapping the inhibition zones. Within 15 minutes of the antibiotic disc application, invert the plates and incubate them at 37 degrees Celsius overnight.
The following day, measure the zone of inhibition diameter in millimeters and interpret it according to the breakpoint values given by EUCAST. The total bacterial load in the water sample was found to be 3.0 x 10 to the ninth CFU/mL, and the antibiotic resistant bacterial load was found to be high. The antibiotic resistance cultural isolates sequenced belonged to the Enterobacteriaceae family with most being Escherichia coli and Klebsiella pneumoniae.
In addition, rare opportunistic bacteria were also identified. The antibiotic susceptibility testing by disc diffusion method showed a Multiple Antibiotic Resistance index of approximately 0.2 for eight isolates, indicating a high degree of resistance. However, Comamonas and Arthrobacter species did not show resistance to any of the tested antibiotics.
The culturable isolates revealed the presence of antibiotic resistance genes encoding the factors against beta-lactams, trimethoprim, and aminoglycosides. Metagenomic DNA analysis for total bacterial diversity led to the identification of 50 phyla, indicating the high bacterial diversity where Proteobacteria was the most dominant phylum, consisting of the Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria classes. At the order level, Burkholderiales was the most prevalent, belonging to the Betaproteobacteria class.
At the general level, Pseudomonas, Acinetobacter, Pedobacter, Prosthecobacter, Limnohabitans, Flavobacterium, and Comamonas were some of the abundant bacteria. The metagenomic DNA analysis also revealed more antibiotic resistance genes. In this method, the serial dilution step is critical as any error can lead to a wrong interpretation of the total CFU and the antibiotic resistant bacterial load.
Also, the diameter of the zone of inhibition should be measured carefully to correctly interpret if the isolate is resistant or sensitive. Using these protocols, besides water sample, any other sample, be it environmental, food, or clinical, can be studied. Moreover, besides bacteria, other microbial populations in any ecosystem can also be studied using similar approaches.
