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bioRxiv · 10.64898/2026.03.16.712119

Heavy metal resistance promotes higher biomass formation in multidrug-resistant wastewater Escherichia coli isolates from Finland

Al-Mustapha, A. I.·Laukkanen-Ninios, R.·Lehto, K.-M.·Tammela, P.·Heikinheimo, A.·Cruz, C. D.
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Abstract

Antimicrobial resistance alone is a serious threat to global public health, but even more so when multidrug-resistant bacteria harbour heavy metal resistance genes, as these can drive co-selection of antibiotics and produce biofilms. The presence of such genes, combined with the bacterias ability to form biofilms, is strongly linked to treatment failure, persistent infections, and reduced therapeutic options. Here, we used the resazurin-crystal violet combination assay to screen a representative cohort of whole-genome sequenced Escherichia coli isolates (n=20) obtained from wastewater surveillance. The specific biofilm formation (SBF) index was used to grade the intensity of biofilm formation as strong, moderate, weak, and non-biofilm producers. Correlation analysis was used to test the association between the intensity of biofilm formation and genotypic features. The SBF index revealed that most of the wastewater E. coli isolates (n=13/20) were weak/non-biofilm producers, four isolates produced moderate biofilms, and three isolates (ST1434: A-O18ab:H55; ST401:A-H25; and ST399:A-O19:H12) produced strong biofilms. The diversity of virulence factors was similar in most of the isolates, except for the three isolates, which had fewer abundant virulence factors. The correlation analysis showed that there was no association between the expression of virulence genes and the formation of strong biofilms by the isolates (p > 0.05). Drug resistance profile was not correlated with higher biofilm production (p > 0.05), as 68.8% (n=11/16) of multi-drug resistant (MDR) and 50% (n=2/4) of non-MDR isolates had weak or no biofilm formation. Similarly, the SBF index was not associated with the number of plasmids in each of the E. coli genomes (p = 0.334). However, there was a positive association between the presence of two or more heavy metal resistance genes (HMRGs) and the strong biofilm formation in our isolates (p = 0.002). Our findings revealed the low occurrence of strong biofilm producers among wastewater E. coli isolates. Further studies are needed to evaluate the impact of the presence of HMRGs and their direct or indirect contribution to enhancing biofilm production and persistence in environmental reservoirs.

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Al-Mustapha, A. I., Laukkanen-Ninios, R., Lehto, K.-M., Tammela, P., Heikinheimo, A., Cruz, C. D.. 2026-03-18. Heavy metal resistance promotes higher biomass formation in multidrug-resistant wastewater Escherichia coli isolates from Finland. https://doi.org/10.64898/2026.03.16.712119

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