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Investigating Putative Antibiotic Resistance Genes in Mycobacterium Abscessus

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Published: 23rd Sep 2019 in Biology

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Investigating putative antibiotic resistance genes in Mycobacterium abscessus

Mycobacterium abscessus can be defined as a group of; rapidly, growing, multi-drug resistant, non-tuberculosis mycobacterium (NTM) which are ubiquitous in soil and water. M. abscessus is capable of colonising the respiratory tract, and causes disease in certain groups of individuals, one of these groups is Caucasian females above 60 years of age. While most of them have no apparent underlying abnormality, 40% have predisposing conditions such as lung disease, lung transplantation, achalasia or recurrent vomiting (Jönsson et al, 2007). Another group at risk of M. abscessus colonisation are patients with Cystic Fibrosis. M. abscessus has been seen to cause pulmonary disease, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis. M. abscessus can be seen to be prevalent in respiratory specimens from patients with cystic fibrosis (Lee et al, 2015). A major problem with M. abscessus, is that the bacterium has acquired multi-drug resistance to commonly used antibiotics, and therefore limits the options of treatment for patients that present with M. abscessus infections. M. abscessus possess a variety of mechanisms that have led to the microorganism acquiring antibiotic resistance. They possess the ability to make enzymes which alter or destroy the antibiotic, the genome M. abscessus can be seen to encode several putative enzymes which can lead to the inactivation of antibiotics through either modifying/ degrading or lowering the affinity of the drug for its target (Luthra et al, 2018).  They also prevent antibiotics from getting into the cell through properties of their cell wall, they also pump (efflux) the antibiotic out of the cell.

M. abscessus β

– lactamases (BlaMab) can be seen to be primarily responsible for poor efficacy of β

– lactam antibiotics against M. abscessus. β

lactamases work by hydrolysing the β

– lactam ring and thereby inactivating them. In addition, to being able to degrade several β

– lactams, they are also not effectively inhibited by common β

– lactamase inhibitors such as: clavulanate, sulbactam and tazabactam. Within the study they produced a recombinant BlaMab which conferred high-level resistance to amoxicillin and ticarcillin, which were efficiently hydrolysed by the β

-lactamases (Soroka et al, 2013). This suggests that BlaMab plays a role in limiting the in vivo efficacy of β

-lactams and that β

– lactamase inhibitors are not effective. This raises a challenge for the use of β

-lactam antibiotcs in the treatment of M. abscessus and that research into drug combinations could be effective in reducing the resistance to β

– lactam antibiotics.

Furthermore, M. abscessus has been shown to produce a broad-spectrum β

– lactamase. It has been identified that the combination of β

-lactams with a BlaMab inhibitor may improve treatment efficacy. They studied the kinetics of BlaMab inactivation by avibactam, which is a non β

– lactam β

-lactamase inhibitor, in combination with ceftazidimine. They explored the role of BlaMab in β

-lactam resistance, by deleting the gene (MAB_2875) from the chromosome of M. abscessus CIP104356. Deleting this gene led to no β

– lactamase activity, indicating that BlaMab is the only β

-lactamase produced by M. abscessus CIP104356. Deleting BlaMab dramatically reduced the minimum inhibitory concentration (MICs) of penicillin and first/second/third generation cephalosporins (except ceftazidime) showing that BlaMab is the major determinant of high levels of resistance to penicillin and most cephalosporins in M. abscessus. The absence of activity of ceftazidimine and aztreonam against the mutation strain suggests that the transpeptidases of M. abscessus are not inhibited by these antibiotics (Dubée et al, 2014). This gave an insight into how a drug can efficiently inhibit BlaMab by the reversible formation of a covalent adduct. The usefulness of these inhibitors are still being tested and therefore their effectiveness may be limited as ceftazidime has a lack of activity in treatment of M. abscessus. However, the unique mode of action of this inhibitor, suggests that, the derivatives of this drug should be considered when trying to combat antibiotic resistance in the future. 

Furthermore, the impact of BlaMab on the activity of β

– lactams was assessed through a study which compared M. abscessus CIP104536 and its β

– lactamase deficient derivative, as well as using the β

– lactamase inhibitor avibactam. The study found that production of BlaMab limited the activity of imipenem. The combination of imipenem and amikacin was bactericidal against the M. abscessus mutant. Deletion of BlaMab extended the spectrum of β

– lactams active against M. abscessus to include the antibiotics: amoxicillin and ceftaroline. In the absence of BlaMab , amoxicillin could be seen as active as imipenem. These drugs were seen to be more active than ceftaroline and cefoxitin was the least active. Avibactam increased the intracellular activity of ceftaroline, but inhibition of BlaMab was only partial, as previously reported for amoxicillin (Lefebvre et al, 2016). By taking into consideration the results of this study, imipenem is superior to cefoxitin at clinically achievable drug concentrations and that inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drugs that could potentially be used to treat pulmonary infections associated with M. abscessus.

M. abscessus doesn’t just confer antibiotic resistance to β

-lactams. It has also been observed to have acquired resistance for aminoglycosides and macrolide, which is caused by mutations affecting genes encoding antibiotic targets. M. abscessus infections are seen to respond poorly to macrolides. Macrolide resistance can be conferred by a mutation in the 23S rRNA gene, leading to a base change at position 2058 or 2059 of the 23S rRNA gene. They also demonstrated that the expression of erm (41) in M. abscessus conferred resistance to macrolide antibiotics such as clarithromycin. Some isolates of M. abscessus have led to a complication of interpreting the susceptibility of macrolides and further to this it is unclear of the significance of erm (41) in M. abscessus as the presence provides an explanation for lack of efficacy of macrolide- based treatments but it is not known whether the presence of non-functional erm (41) would lead to a better outcome or whether non-functional alleles can acquire reversion mutations during treatment (Nash et al, 2009). Therefore, the results or findings of this study require further testing to ensure that the results are reliable and that the mechanisms described are correct. This was supported by a study carried out by Maurer et al (2012) that suggested that point mutations in 23S rRNA constitute the main mechanism for acquired high-level resistance in mycobacterial species which carry only a single rRNA gene copy. The association of 23S Rrna mutations with clarithromycin resistance in M. abscessus, that despite an inducible erm(41) there is a selective advantage for the acquisition of a 23S Rrna mutation, most probably due to either increased mutation associated antibiotic resistance or less resistance- associated biological cost. This therefore raises the question as to whether patients that carry a wild-type rrl strain with an inducible macrolide resistance determinant may still benefit from clarithromycin treatment (Maurer et al, 2012).

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M. abscessuspresents the issue of aminoglycoside resistance. Aminoglycosides can be seen to primarily target the 16S rRNA component of 30S ribosomal subunit. Bacterial resistance to aminoglycosides is conferred mainly by AG- modifying enzymes which are encoded by the genome M. abscessus. Through studying the genome of M. abscessus it was shown that 2’N acetyltransferase had a role in M. abscessus aminoglycoside resistance (Rominiski et al, 2017).

Furthermore, M. abscessus can be seen to acquire resistance to tetracycline antibiotics which is caused by a WhiB7 independent tetracycline activating monoxygenase, MabTetX (MAB_1496c). Deletion of MAB_1496c which encodes a putative Flavin adenonine dinucleotide (FAD) binding monoxygenase enhanced the susceptibility of M. abscessus towards tetracycline and doxycycline. Furthermore, they studied a complemented mutant MAB_ 1496c strain and this exhibited even high levels of resistance towards both antibiotics compared to the wild strain, which supports the idea that the MAB_1496c is primarily responsible for tetracycline resistance within M. abscessus (Rudra et al, 2018).

M.abscessus can be seen to exhibit intrinsic rifamycin resistance which is mediated by ADP-ribosyltransferase MAB_0591. Heterologous expression of MAB_0591 indicated that it is able to confer resistance to a susceptible host, however its role in innate rifampicin resistance in M. abscessus remained to be determined. Furthermore, M. abscessus MAB_0591 deletion mutant showed susceptibility to low rifampicin concentrations and rifampicin resistance was restored upon complementation (Rominiski et al, 2016).

 

M. abscessus has a particularly thick wall, which is composed of layers of complex hydrophobic molecules including; fatty acids, mycolic acids, lipoproteins, glycopeptidolipids, and has largely insoluble peptidoglycan (PG) and arabinogalactan layers. Two distinct colony morphotypes- rough and smooth can have an impact on antibiotic resistance with the bacterial cell (Story-Roller et al, 2018). The rough morphotypes tends to be associated with high rates of antimicrobial resistance, including against β

-lactams. However, these mechanisms are seen to be poorly understood in M. abscessus. The glycosylation of lipoproteins can limit the permeability of the cell wall and many antibiotics that could play a role in inhibiting PG synthesis. In addition, the cell walls porins present in M. abscessus are also partially responsible for β

– lactam resistance as they allow for the transport of small hydrophobic molecules across the membrane, that interact with targets within the cytoplasm to potentially activate expression of drug resistance genes (Story-Roller et al, 2018). Even though the cell wall is not essential for the expression of antibiotic resistance genes, it is still an important mechanism that should be considered as it could lead to the activation of these genes and the cell wall is important in the transport of antibiotics into M. abscessus.  

Active efflux mechanisms within M. abscessus can be seen to be one of the causative factors for acquiring antibiotic resistance. Efflux pump mechanisms, allow for the bacteria to protect itself against toxic molecules and to maintain homeostasis. M. abscessus encodes protein members of the major facilitator family ABC transporters and mycobacterial membrane protein large (MmpL) families. The ABC- type multidrug transporters use ATP energy to pump drugs out of the cell and can be classified either as importers or exporters, whereas the MmpL transporter family is involved in lipid transport to the membrane and encodes resistance, nodulation and cell division proteins, which are a family of multidrug resistance pumps that recognise and mediate the transport of a diverse group of compounds (cationic, anionic or neutral), including various drugs, metal and fatty acids. However, their role in this species has yet to be established. (Nessar et al, 2012).

It can be seen that M. abscessus has mutations in the MAB_2299c, which encodes a putative TetR transcriptional regulator. Mutants with these mutations are also cross-resistant to bedaquiline. MAB_2299c was found to bind to its target DNA, located upstream of the divergently oriented MAB_2300-MAB_2301 gene cluster which encodes MmpS/MmpL membrane proteins. Point mutations or deletion of MAB_2299c was associated with upregulation of the mmpS and mmpL transcripts and accounted for this cross resistance. However, when MAB_2300 and MAB_2301 were deleted in the MAB_2299c mutant strain this led to restored susceptibility to bedaquiline and clofazimine (Richard et al, 2018).

M. abscessus can be seen to have acquired resistance to a variety of antibiotics. Therefore, combinations of antibiotics are usually used in treatment of M. abscessus infections and are routinely co-administered based on the concept that this will minimise the spread of antibiotic resistance. However, this study suggests that exposure to clarithromycin, or likely any whiB7– inducing antibiotic, may antagonise the activities of amikacin (bacterial amiboglycoside) and other drugs. This raises important implications for the management of M. abscessus infection, both in cystic fibrosis (CF) and non-CF patients. It showed that this resistance was dependent of whiB7, which is a transcriptional activator of intrinsic antibiotic resistance that is induced by exposure to many different antibiotics. It can be seen that the deletion of whiB7 (MAB_3508c) resulted in increased sensitivity to a broad spectrum of antibiotics. WhiB7 is required for transcriptional activation of genes that confer resistance to three commonly used anti-M. abscessus drugs such as: clarithromycin, amikacin, and tigecycline. The whiB7– dependent gene that conferred macrolide resistance was identified as erm(41) (MAB_2297) which encodes a ribosomal methyltransferase. The whiB7-dependent gene contributing to amikacin resistance was eis2 (MAB_4532c), which can be seen to encode a Gcn5- related N-acetyltransferase (GNAT) (Pryjma et al, 2017).

Within the literature, there can be seen to be a wide range of research of different mechanisms within M. abscessus that lead to resistance against different antibiotics. By undertaking a project that focuses on the role of antibiotic resistance genes, which produce β

– lactamase activity, we are able to produce a recombinant protein to test for beta-lactamase activity in assays. By identifying genes and their mechanisms, we can reduce the effects of antibiotic resistance with M. abscessus, then we can work towards finding an antibiotic that works effectively and this will then positively impact on patient’s lives.

 

References

 

  1. Dubée, V., Bernut, A., Cortes, M., Lesne, T., Dorchene, D., Lefebvre, A., Hugonnet, J., Gutmann, L., Mainardi, J., Herrmann, J., Gaillard, J., Kremer, L. & Arthur, M. (2014).  β -Lactamase inhibition by avibactam in Mycobacterium abscessus, Journal of Antimicrobial Chemotherapy.
  2. Lee, M., Sheng, W., Hung, C., Yu, C., Lee, L. & Hsueh, P. (2015). Mycobacterium abscessus Complex Infections in Humans, Emerging Infectious Diseases, 21(9).
  3. Luthra, S., Rominski, A. & Sander, P. (2018). The Role of Antibiotic-Target-Modifying and Antibiotic-Modifying Enzymes in Mycobacterium abscessus Drug Resistance, Frontiers in Microbiology, 9.
  4. Nash, K., Brown-Elliott, B. & Wallace, R. (2009). A Novel Gene, erm(41), Confers Inducible Macrolide Resistance to Clinical Isolates of Mycobacterium abscessus but Is Absent from Mycobacterium chelonae, Antimicrobial Agents and Chemotherapy, 53(4), pp. 1367-1376.
  5. Rominski, A., Selchow, P., Becker, K., Brülle, J. K., Dal Molin, M. & Sander, P. (2017). Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes. J. Antimicrob. Chemother. 72, pp. 2191–2200.
  6. Rudra, P., Hurst-Hess, K., Lappierre, P. & Ghosh, P. (2018). High levels of intrinsic tetracycline resistance in Mycobacterium abscessus are conferred by a tetracycline-modifying monooxygenase. Antimicrob. Agents Chemother.
  7. Soroka, D., Dubee, V., Soulier- Escrihuela, O., Cuinet, G., Hugonnet, J., Gutmann, L., Mainardi, J. & Arthur,M. (2013). Characterization of broad-spectrum Mycobacterium abscessus class A-lactamase, Journal of Antimicrobial Chemotherapy, 69(3), pp. 691-696.
  8. Story-Roller, E., Maggioncalda, E.C., Cohen, K.A. and Lamichhane, G. (2018). Mycobacterium abscessus and β-Lactams: Emerging Insights and Potential Opportunities, Frontiers in Microbiology, 9.
  9. Nessar, R., Cambau, E., Marc-Reyrat, J., Murray, A. & Gicquel, B. (2012). Mycobacterium abscessus: a new antibiotic nightmare, Journal of Antimicrobial Chemotherapy, 67(4), pp. 810–818.
  10. Rominski, A., Roditscheff, A., Selchow, P., Böttger, E. & Sander, P. (2016). Intrinsic rifamycin resistance of Mycobacterium abscessusis mediated by ADP-ribosyltransferase MAB_0591, Journal of Antimicrobial Chemotherapy, 72(2), pp.376-384.
  11. Jönsson, BE., Gilljam M., Lindblad, A., Ridell, M., Wold, AE. & Welinder-Olsson, C. (2007). Molecular epidemiology of Mycobacterium abscessus, with focus on cystic fibrosis, J Clin Microbiol.
  12. Richard, M., Gutiérrez, AV., Viljoen, A., Rodriguez-Rincon, D., Roquet-Baneres, F., Blaise, M., Everall, I., Parkhill, J., Floto, RA. & Kremer, L. (2019). Mutations in the MAB_2299c TetR regulator confer cross-resistance to clofazimine and bedaquiline in Mycobacterium abscessus. Antimicrob Agents Chemother.
  13. Maurer, FP., Rueegger, V., Ritter, C., Bloemberg, GV. & Boettger, EC. (2012). Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41). Journal of Antimicrobial Chemotherapy, 67, pp. 2606-2611.
  14. Pryjma, M., Burian, J., Kuchinski, K. & Thompson, CJ. (2017). Antagonism between front- line antibiotics clarithromycin and amikacin in the treatment of Mycobacterium abscessus infections is mediated by the whiB7 gene. Antimicrob Agents Chemother.
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