Comparison of Two Transmission Electron Microscopy Methods to Visualize Drug-Induced Alterations of Gram-Negative Bacterial Morphology

Nguyen, HoangKim; O’Donovan, Lisa A.; Venter, Henrietta; Russell, Cecilia C.; McCluskey, Adam; Page, Stephen W.; Trott, Darren J.; Ogunniyi, Abiodun D.

doi:10.3390/antibiotics10030307

Cited by 16 publications

(16 citation statements)

References 32 publications

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“…Although there is no clear evidence of membrane disruption, the leaflets of bacterial cell envelope cannot be distinguished. The same trend has already been observed for colistin (Nguyen et al, 2021) and Polymyxin B (Voget et al, 2015), two antimicrobials which also target the outer membrane.…”

Section: Effect Of Biocides On Cell Structure and Morphologysupporting

confidence: 79%

A Multi-Purpose Approach to the Mechanisms of Action of Two Biocides (Benzalkonium Chloride and Dibromonitrilopropionamide): Discussion of Pseudomonas fluorescens’ Viability and Death

2022

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Biocides are widely used in water treatment for microbiological control. The rise of antimicrobial resistance and the need to assure properly managed water systems require a better understanding of the mechanisms of action of biocides and of their impact on cell’s viability as a function of dosage concentrations. The present work addresses these two aspects regarding the biocides benzalkonium chloride (BAC) and dibromonitrilopropionamide (DBNPA)—two biocides commonly found in the water treatment industry. For that, the following parameters were studied: culturability, membrane integrity, metabolic activity, cellular energy, and the structure and morphology of cells. Also, to assess cell’s death, a reliable positive control, consisting of cells killed by autoclave (dead cells), was introduced. The results confirmed that BAC is a lytic biocide and DBNPA a moderate electrophilic one. Furthermore, the comparison between cells exposed to the biocides’ minimum bactericidal concentrations (MBCs) and autoclaved cells revealed that other viability parameters should be taken into consideration as “death indicators.” The present work also shows that only for the concentrations above the MBC the viability indicators reached values statistically similar to the ones observed for the autoclaved cells (considered to be definitively dead). Finally, the importance of considering the biocide mechanism of action in the definition of the viability parameter to use in the viable but non-culturable (VBNC) determination is discussed.

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Section: Effect Of Biocides On Cell Structure and Morphologysupporting

confidence: 79%

A Multi-Purpose Approach to the Mechanisms of Action of Two Biocides (Benzalkonium Chloride and Dibromonitrilopropionamide): Discussion of Pseudomonas fluorescens’ Viability and Death

2022

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“…Transmission electron microscopy (TEM) was used to look for changes in cell morphology of a selected bacterial strain (EC26) after treatment with the test agents. The procedure followed as described before with some modifications ( Nguyen et al, 2021 ). A single colony from an overnight culture was suspended in 10 ml LB broth (Invitrogen, United States), and incubated with shaking at 37°C overnight.…”

Section: Methodsmentioning

confidence: 99%

Discerning the role of polymyxin B nonapeptide in restoring the antibacterial activity of azithromycin against antibiotic-resistant Escherichia coli

et al. 2022

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Antimicrobial resistance is a global public health threat. Antibiotic development pipeline has few new drugs; therefore, using antibiotic adjuvants has been envisioned as a successful method to preserve existing medications to fight multidrug-resistant (MDR) pathogens. In this study, we investigated the synergistic effect of a polymyxin derivative known as polymyxin B nonapeptide (PMBN) with azithromycin (AZT). A total of 54 Escherichia coli strains were first characterized for macrolide resistance genes, and susceptibility to different antibiotics, including AZT. A subset of 24 strains was then selected for synergy testing by the checkerboard assay. PMBN was able to re-sensitize the bacteria to AZT, even in strains with high minimum inhibitory concentrations (MIC: 32 to ≥128 μg/ml) for AZT, and in strains resistant to the last resort drugs such as colistin and meropenem. The fractional inhibitory concentration index was lower than 0.5, demonstrating that PMBN and AZT combinations had a synergistic effect. The combinations worked efficiently in strains carrying mphA gene encoding macrolide phosphotransferase which can cause macrolide inactivation. However, the combinations were inactive in strains having an additional ermB gene encoding macrolide methylase which causes ribosomal drug target alteration. Killing kinetics study showed a significant reduction of bacterial growth after 6 h of treatment with complete killing achieved after 24 h. Transmission electron microscopy showed morphological alterations in the bacteria treated with PMBN alone or in combination with AZT, with evidence of damage to the outer membrane. These results suggested that PMBN acted by increasing the permeability of bacterial outer membrane to AZT, which was also evident using a fluorometric assay. Using multiple antimicrobial agents could therefore be a promising strategy in the eradication of MDR bacteria. PMBN is a good candidate for use with other antibiotics to potentiate their activity, but further studies are required in vivo. This will significantly contribute to resolving antimicrobial resistance crisis.

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“…As yet the drug target of these analogues remains undetermined. However, in our antibiotic studies with related compounds we observed that robenidine analogues affected the bacterial cell wall [31,32,36–39] . It is possible that a similar mechanism, against the trophozoite cell wall is in play.…”

Section: Discussionmentioning

confidence: 64%

Antigiardial Activity of Novel Guanidine Compounds

et al. 2022

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From four focused compound libraries based on the known anticoccidial agent robenidine, 44 compounds total were synthesised and screened for antigiardial activity. All active compounds were counter-screened for antibiotic and cytotoxic action. Of the analogues examined, 21 displayed IC 50 < 5 μM, seven with IC 50 < 1.0 μM. Most active were 2,2'-bis{ [4-(trifluoromethoxy)phenyl]methylene}carbonimidic dihydrazide hydrochloride (30), 2,2'-bis{[4-(trifluoromethylsulfanyl)phenyl]methylene}carbonimidic dihydrazide hydrochloride (32), and 2,2'-bis[(2-bromo-4,5-dimethoxyphenyl)methylene]carbonimidic dihydrazide hydrochloride ( 41) with IC 50 = 0.2 μM. The maximal observed activity was a 5 h IC 50 value of 0.2 μM for 41. The clinically used metronidazole was inactive at this timepoint at a concentration of 25 μM. Robenidine off-target effects at bacteria and cell line toxicity were removed. Analogue 41 was well tolerated in mice treated orally (100 mg/kg). Following 5 h treatment with 41, no Giardia regrowth was noted after 48 h.

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Comparison of Two Transmission Electron Microscopy Methods to Visualize Drug-Induced Alterations of Gram-Negative Bacterial Morphology

Cited by 16 publications

References 32 publications

A Multi-Purpose Approach to the Mechanisms of Action of Two Biocides (Benzalkonium Chloride and Dibromonitrilopropionamide): Discussion of Pseudomonas fluorescens’ Viability and Death

A Multi-Purpose Approach to the Mechanisms of Action of Two Biocides (Benzalkonium Chloride and Dibromonitrilopropionamide): Discussion of Pseudomonas fluorescens’ Viability and Death

Discerning the role of polymyxin B nonapeptide in restoring the antibacterial activity of azithromycin against antibiotic-resistant Escherichia coli

Antigiardial Activity of Novel Guanidine Compounds

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