Culture Methods

One of the current challenges in metagenomic studies and relying on sequence data is to determine whether the organisms you characterize are alive or not. Indeed, order just finding DNA of Staphylococcus aureus on a wooden bench or subway pole doesn’t mean the organism is alive.

Thus, sick to supplement our metagenomic profile of the subway system we performed two culture experiments. The first culture experiment was designed to directly compare sequence data against culture data. We would collect two swabs from the same surface, sales with one swab sequenced directly, and the other swab was first cultured then sequenced. This allowed us to compare the MetaPhlAn output and see how many organisms were found in both. Our data showed that the sequence dataset had greater number of taxa found than the culture set, with shared hits ranging from 7-13 organisms (See Figure 4 of the Paper). A set of cultures in this experiment also had tetracycline treatment, allowing us to perform a functional genetic analysis to survey for the presence of tetracycline-resistance genes. Indeed, we found high coverage to these tet genes (especially tetk) in the samples with tetracycline treatment. The second culture experiment dove deeper into the question of the presence of antibiotic resistance microorganisms. We collected more samples and plated them on various media including treatments of kanamycin, chloramphenicol, and ampicillin. The findings from this experiment further supported the first experiment that viable microorganisms can be cultured from the subway system. Moreover, the results suggested the presence of viable, antibiotic-resistant microorganisms. For more detailed results check out our full paper.

 The methodology of the second experiment wasn’t reported as in-depth as the first experiment in our paper. Thus, we’ve included more details regarding the second experiment below:

Bacteria were cultured in LB agar and then spread onto LB plates, after lawn growth, they used to select for singles. Single colonies were then plated onto AB plates and growth was assessed. Plates were incubated at 37 C. As a control, air samples were taken and cultured at every location. In all cases these did not yield growth. The non-selective plate done last when replica plating also serves as a control. The antibiotics were used according to the following concentrations:

• Kanamycin – 50ug/mL
• Chloramphenicol – 35ug/mL
• Ampicillin – 100ug/mL 

Finally, there was no quantitative confirmation of bacterial vs. non-bacterial organisms, although there was no observable fungal growth in the samples.

Further experiments are being done to dive deeper into the question of viability of microorganisms on the subway system as well as the presence of antibiotic-resistant bacteria.