I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?

The sudden emergence of antibiotic resistance in your Escherichia strain could be due to several mechanisms. One possibility is spontaneous mutation, where genetic changes confer resistance to Kanamycin and Spectinomycin. For example, mutations in the rRNA operon have been associated with spectinomycin resistance [3]. Horizontal gene transfer is another likely explanation; resistance genes from other bacteria can be acquired through plasmids, transposons, or integrons [1][5]. Plasmid-mediated resistance is well-documented in E. coli, with specific genes such as aphA1 and aadB conferring kanamycin resistance [4]. Additionally, selective pressure from sub-lethal antibiotic concentrations can favor the proliferation of resistant mutants over time [2]. Ensuring strict aseptic techniques and verifying antibiotic concentrations can help mitigate such issues.

Reference

[1] González-Zorn, B., Teshager, T., Casas, M., Porrero, M., Moreno, M., Courvalin, P., & Domínguez, L. (2005). armA and Aminoglycoside Resistance in Escherichia coli. Emerging Infectious Diseases, 11, 954 - 956.

[2] Mark, L., Sigmund, C., & Morgan, E. A. (1983). Spectinomycin resistance due to a mutation in an rRNA operon of Escherichia coli. Journal of Bacteriology, 155, 989 - 994.

[3] Seok, H., Jeon, J. H., choi, H. K., Choi, W. S., Park, D. W., Wi, Y. M., ko, K. S., & Peck, K. R. (2019). 611. Fosfomycin Resistance of Multidrug-Resistant Escherichia coli and Mechanisms of Fosfomycin Resistance. Open Forum Infectious Diseases, 6, S285 - S285.

[4] Ali, M. R. (2023). Phylogenetic Groups and Antibiotic Resistance Characteristics of Uropathogenic Escherichia coli. Journal of Communicable Diseases.

[5] Hong, T., Moland, E. S., Abdalhamid, B., Hanson, N., Wang, J., Sloan, C., Fabian, D., Farajallah, A., Levine, J., & Thomson, K. (2005). Escherichia coli: development of carbapenem resistance during therapy.. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, 40 10, e84-6 .

It certainly is possible for a strain to become resistant to multiple antibiotics at once from the acquisition of a resistance plasmid or other mobile element. However this is something you would expect to see in the wild and not with a laboratory strain assuming you are keeping it in pure culture.

You might want to double check the phenotype of the strain to be sure you didn't pick up a contaminant or mix it up with some other strain. If you have a frozen stock go back to the original.

Now if the frozen stock behaved one way earlier and differently now, and there is no contamination, then it may be something with the antibiotic plates.

Hi Karen,

You probably grew an E coli strain that had a plasmid in it during the incubation and made it competent cells and transferred the second plasmid including a different antibiotic gene. Did you isolate the plasmid from them? You can also find the gene they carry with T7 and SP6 primers by looking at the amplicon sizes? It would be best to use a new E coli from glycerol stocks? You can try using different antibiotic-containing media as well as the one that does not contain antibiotics in the incubation? You can test it this way.

I wish you luck.

Also see below about str resistancy in E coli.

Article The genetic background for streptomycin resistance in Escher...

Fatih Beris Michael J. Benedik Thank you for your answers, we did miniprep and didn't see any plasmids in the bacteria. Also we tried to culture the bacteria for about a week without any antibiotic, and they were still resistant. Moreover, we tested with Ampicillin too and they were also resistant to ampicillin. The Kanamycin resistant was lower than for the others.

The answer from Park Sowon seems to make sense for those results I imagine. I just arrived in this lab so I am not sure how they treated the bacteria. They just told me about a month ago bacteria were working fine, and suddenly they became resistant to every antibiotics.

We threw them away and used other bacteria now, but I am still curious if there is an explanation for that.