Ayush Pathak

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Pathak

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Ayush

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0000-0002-2161-9561

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Publications 1 - 6 of 6

Ecology and evolution of antimicrobial production and resistance in microbial communities
Pathak, Ayush (2025)
Bacteria live in complex communities in nature with some species possessing antimicrobial production and resistance phenotypes or both. The antimicrobial production and resistance phenotypes can reciprocally select each other and coevolve. Furthermore, many of the antimicrobial resistance phenotypes can spread between species in complex communities via various horizontal resistance mechanisms due to anthropogenic and non-anthropogenic selection pressures. My PhD thesis explores some of these concepts in detail. In the first chapter, which also serves as the general introduction to my thesis, I highlight how many antimicrobial resistance mechanisms originate from those harboured by antimicrobial-producing bacteria for self-resistance to their own antimicrobials and to be resistant to the antimicrobials of their competitors. I discuss how natural antimicrobial production in some species of bacteria and fungi can induce selection pressures on competing species of bacteria to evolve novel resistance mechanisms and these mechanisms can spread to the clinical milieu. In addition, I discuss potential horizontal transfer mechanisms through which antimicrobial resistance genes can spread from actinobacteria to proteobacteria. Finally, I highlight that we must study the evolutionary dynamics of antimicrobial production in response to the evolution of antimicrobial resistance. This would allow us to find ways of replicating antimicrobial evolution in vitro utilising competition-based-coevolution between antimicrobial-producing and antimicrobial resistance organisms, enabling us to generate new antibiotics in vitro to combat bacterial infections. In the second chapter and third chapter, I have investigated how pOXA-48 plasmid-encoded antibiotic degrading resistance alters the composition of a six species community consisting of Escherichia coli, Staphylococcus aureus subsp. aureus, Salmonella enterica serovar Typhimurium, Enterococcus faecalis, Pseudomonas aeruginosa and Klebsiella pneumoniae subsp. Pneumoniae when the community is exposed to antibiotics piperacillin+tazobactam. We found that since pOXA-48 encodes an antibiotic-degrading beta-lactamase enzyme blaOXA-48, species not hosting the plasmid benefit from the antibiotic detoxification. Non-plasmid-carrying S. Typhimurium and P. aeruginosa benefit the most in communities from antibiotic detoxification due to their better survival and growth at relevant antibiotic concentrations. The plasmid was horizontally transferable between E. coli, S. Typhimurium and K. pneumoniae in pairwise conjugation assays but horizontal transfer was negligible in the community experiments potentially due to diluted transmission of the plasmid and increased plasmid costs due to interspecific competition. We also found that the species that benefited most from plasmid-induced antibiotic detoxification did not depend on the identity of the host carrying the plasmid. Conclusively, I show that antibiotic degrading plasmids must be acknowledged on the basis of their horizontal transfer potential and antibiotic detoxification potential in microbial communities in clinical as well as environmental contexts to understand better how microbial communities respond to antibiotic exposure. In the fourth chapter, I have developed a co-culture-based method that could potentially allow us to coevolve antibiotic production and resistance. Using Streptomyces species S. clavuligerus and S. violaceoruber and a common laboratory strain of Escherichia coli, I establish co-cultures on agar with Streptomyces species and E. coli. We find that such co-cultures can be used to evolve altered antibiotic production phenotypes when the co-cultured E. coli is resistant to the antibiotics produced by the Streptomyces in co-cultures. However, we found that it is unlikely that E. coli will evolve resistance to the antibiotics produced by Streptomyces in co-cultures. Such is the case because E. coli had much higher growth rates and carrying capacity abundances in co-cultures than the Streptomyces species. These differences in growth dynamics would potentially allow E. coli to rapidly grow to such high abundances that Streptomyces inhibited a very small fraction of E. coli cells at any point in time. Therefore, we propose an alternative way to evolve novel antibiotic production phenotypes in Streptomyces where we first evolve resistance to Streptomyces-produced antibiotics in E. coli, and then co-culture Streptomyces with the evolved E. coli. Overall, this chapter opens up new avenues investigating the evolution of antimnicrobial production and resistance in vitro using co-culture-based methods.
Resisting Antimicrobial Resistance: Lessons from Fungus Farming Ants
Pathak, Ayush; Kett, Steve; Marvasi, Massimiliano (2019)
Trends in Ecology & Evolution
Antifungals, arthropods and antifungal resistance prevention: lessons from ecological interactions
Kett, Steve; Pathak, Ayush; Turillazzi, Stefano; et al. (2021)
Proceedings of the Royal Society B: Biological Sciences
Arthropods can produce a wide range of antifungal compounds, including specialist proteins, cuticular products, venoms and haemolymphs. In spite of this, many arthropod taxa, particularly eusocial insects, make use of additional antifungal compounds derived from their mutualistic association with microbes. Because multiple taxa have evolved such mutualisms, it must be assumed that, under certain ecological circumstances, natural selection has favoured them over those relying upon endogenous antifungal compound production. Further, such associations have been shown to persist versus specific pathogenic fungal antagonists for more than 50 million years, suggesting that compounds employed have retained efficacy in spite of the pathogens' capacity to develop resistance. We provide a brief overview of antifungal compounds in the arthropods' armoury, proposing a conceptual model to suggest why their use remains so successful. Fundamental concepts embedded within such a model may suggest strategies by which to reduce the rise of antifungal resistance within the clinical milieu.
Antibiotic-degrading resistance changes bacterial community structure via species-specific responses
Pathak, Ayush; Angst, Daniel C.; Leon-Sampedro, Ricardo; et al. (2023)
The ISME Journal
Some bacterial resistance mechanisms degrade antibiotics, potentially protecting neighbouring susceptible cells from antibiotic exposure. We do not yet understand how such effects influence bacterial communities of more than two species, which are typical in nature. Here, we used experimental multispecies communities to test the effects of clinically important pOXA-48-plasmid-encoded resistance on community-level responses to antibiotics. We found that resistance in one community member reduced antibiotic inhibition of other species, but some benefitted more than others. Further experiments with supernatants and pure-culture growth assays showed the susceptible species profiting most from detoxification were those that grew best at degraded antibiotic concentrations (greater than zero, but lower than the starting concentration). This pattern was also observed on agar surfaces, and the same species also showed relatively high survival compared to most other species during the initial high-antibiotic phase. By contrast, we found no evidence of a role for higher-order interactions or horizontal plasmid transfer in community-level responses to detoxification in our experimental communities. Our findings suggest carriage of an antibiotic-degrading resistance mechanism by one species can drastically alter community-level responses to antibiotics, and the identities of the species that profit most from antibiotic detoxification are predicted by their intrinsic ability to survive and grow at changing antibiotic concentrations.
Antibiotic selective pressure in microcosms: Pollution influences the persistence of multidrug resistant Shigella flexneri 2a YSH6000 strain in polluted river water samples
Maruzani, Rugare; Pathak, Ayush; Ward, Malcolm; et al. (2020)
Environmental Technology & Innovation
Some urban rivers reach dangerous concentrations of residual antibiotics imposing a certain level of selective pressure on microorganisms to develop various antibiotic resistance mechanisms. In the current work, we have measured the persistence and growth of a multidrug resistant strain of Shigella flexneri 2a YSH6000 under a mock release of lethal concentration of oxytetracycline in microcosms of River Thames water. The water was sampled from upstream (lower levels of pollution) and downstream (higher levels of pollution) of London city centre. In our in-vitro microcosms, in the presence of 160 g/mL of oxytetracycline, growth of S. flexneri in the downstream sector was up to 2 log(cfu/mL) higher relative to the upstream sector. This difference in growth is a sum of undefined interactions of different chemicals with the antibiotic. We extrapolated the contribution of two abundant pollutants in downstream sector: iron and phenanthrene. In the presence of selection pressure, iron at a concentration of 6.49 mg/L was found to foster the growth of resistant bacteria while phenanthrene at concentration of 160 g/L reduced the growth of the resistant strain. In addition, label free proteomics analysis showed that there are 64 proteins that were differentially expressed by the bacteria exposed to the upstream section versus the downstream sector. In the presence of oxytetracycline, at concentration of 160 g/mL, the differences reduced to only a few proteins, demonstrating that environmental stress impacts protein synthesis. Such mock studies contribute to our knowledge of chemicals that reduce growth of resistant strains and aids in the identification of selective biomarkers.
Comparative genomics of Alexander Fleming’s original Penicillium isolate (IMI 15378) reveals sequence divergence of penicillin synthesis genes
Pathak, Ayush; Nowell, Reuben W.; Wilson, Christopher G.; et al. (2020)
Scientific Reports
Antibiotics were derived originally from wild organisms and therefore understanding how these compounds evolve among different lineages might help with the design of new antimicrobial drugs. We report the draft genome sequence of Alexander Fleming’s original fungal isolate behind the discovery of penicillin, now classified as Penicillium rubens Biourge (1923) (IMI 15378). We compare the structure of the genome and genes involved in penicillin synthesis with those in two ‘high producing’ industrial strains of P. rubens and the closely related species P. nalgiovense. The main effector genes for producing penicillin G (pcbAB, pcbC and penDE) show amino acid divergence between the Fleming strain and both industrial strains, whereas a suite of regulatory genes are conserved. Homologs of penicillin N effector genes cefD1 and cefD2 were also found and the latter displayed amino acid divergence between the Fleming strain and industrial strains. The draft assemblies contain several partial duplications of penicillin-pathway genes in all three P. rubens strains, to differing degrees, which we hypothesise might be involved in regulation of the pathway. The two industrial strains are identical in sequence across all effector and regulatory genes but differ in duplication of the pcbAB–pcbC–penDE complex and partial duplication of fragments of regulatory genes. We conclude that evolution in the wild encompassed both sequence changes of the effector genes and gene duplication, whereas human-mediated changes through mutagenesis and artificial selection led to duplication of the penicillin pathway genes.

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Ayush Pathak

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