The biofilm formation of Mycobacterium abscessus and Mycobacterium chimaera and its impact on antimicrobial susceptibility.

Abstract

Background The biofilm formation of nontuberculous mycobacteria (NTM) gained more and more attention in the past years. NTM biofilms were found in contaminated medical devices causing globel outbreaks and in the lung cavities of patients where they complicate treatment. These findings raised the attention on NTM biofilms.

Methods In the present study, a porous glass bead (PGB) biofilm cultivation model was adjusted to Mycobac- terium abescessus and Mycobacterium chimaera. The biofilms were cultivated in different media and the bacteria quantified using multiple experimental approaches (CFU, ATP and qPCR quantification). The biofilms were further analysed in structure and composition and their susceptibility towards dis- infectants and antibiotics was tested. Further, the proteome and transcriptome expression profiles of two M. chimaera isolates were analysed to identify biofilm specific genes.

Results All tested isolates formed surface attached biofilms, including rough M. abscessus isolates. Strong dif- ferences in the structures were found between the isolates also within one species and, in M. abscessus, even between different colony morphotypes of the same isolate. Moreover, it was found that the used medium had a fundamental influence on growth and structure of the cultivated biofilms, affecting for example the expression of a cording phenotype in smooth M. abscessus isolates. In all cases the biofilms showed an enhanced biomass of extracellular matrix (ECM) components when compared to the corresponding suspension. Most impressively it was found that the biofilm of the outbreak strain ZUERICH-1 consists of an extremely enhanced amount of protein compared to the other M. chimaera isolates. The differences in the composition of the biofilms were less isolate specific in M. abscessus than in M. chimaera. The proteome and the transcriptome analysis revealed the expression of biofilm specific genes (e.g. glmM, glgX, pepN ) including genes of the type VII secretion systems. Finally, it was found that that the biofilms provide enhanced tolerance to antibiotics and disinfectants with the exception of sodium hypochlorite. In this case, the suspension of all M. chimaera isolates were more tolerant than the corresponding biofilm. An interesting finding was, that the outbreak strain M. chimaera ZUERICH-1 presents an enhanced tolerance to peracetic acid (PAA) in compari- son to the other isolates. It is important to mention, that commonly recommended concentrations of disinfectants were not able eradicate the biofilms of several isolates tested.

Conclusion The PGB model is well suited for cultivation of different NTM biofilms and is highly applicable to various analysis methods. The biofilms of the tested species provide very different characteristics highly depending on the cultivation condititons. It was possible to identify biofilm specific genes, which could be a first step to define a basic NTM biofilm genotype. Finally it was found that antimicrobials commonly used to inactivate NTM fail to eradicate or reduce the biofilms. It will be necessary to adjust the common laboratory methods to include NTM biofilms.