Intraspecies diversity of dormant forms of Mycobacterium smegmatis

Mulyukin, A. L.; Kudykina, Yu. K.; Shleeva, Margarita O.; Anuchin, A. M.; Suzina, N. E.; Danilevich, V. N.; Дуда, В. И.; Kaprelyants, Arseny S.; Эль-Регистан, Г. И.

doi:10.1134/s0026261710040089

Cited by 11 publications

(5 citation statements)

References 23 publications

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“…Cells differed in shape, size, cell wall thickness, cell surface architecture and nucleoid density; features similar to those observed in other studies ( 2 , 29 ). Not observed in T. biformata non-dividing cells were large electron transparent inclusions that were reported in Mycobacterium smegmatis non-dividing cells ( 21 , 27 ).…”

Section: Resultsmentioning

confidence: 75%

Resuscitation Promoting Factor (Rpf) from Tomitella biformata AHU 1821T Promotes Growth and Resuscitates Non-Dividing Cells

et al. 2013

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Functional variation of Rpf, a growth factor found exclusively in Actinobacteria, is differentiated by its source and amino acid sequences. Only purified Rpf proteins from three species have been studied so far. To seek new Rpfs for use in future studies to understand their role in Actinobacteria, the objective of this study was to identify rpf gene homologs in Tomitella biformata AHU 1821T, a novel Actinobacteria isolated from permafrost ice wedge. Amplification using degenerate primers targeting the essential Rpf domain led to the discovery of a new rpf gene in T. biformata. Gene structure and the deduced Rpf domain amino acid sequence indicated that this rpf gene was not identical to previously studied Rpf. Phylogenetic analysis placed T. biformata Rpf in a monophyletic branch in the RpfB subfamily. The deduced amino acid sequence was 44.9% identical to RpfB in Mycobacterium tuberculosis, the closest functionally tested Rpf. The gene was cloned and expressed in Escherichia coli; the recombinant Rpf protein (rRpf) promoted the growth of dividing cells and resuscitated non-dividing cells of T. biformata. Compared to other studies, this Rpf was required at higher concentrations to promote its growth and to resuscitate itself from a non-dividing state. The resuscitation function was likely due to the highly conserved Rpf domain. This study provides evidence that a genetically unique but functional Rpf can be found in novel members of Actinobacteria and can lead to a better understanding of bacterial cytokines in this phylum.

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Section: Resultsmentioning

confidence: 75%

Resuscitation Promoting Factor (Rpf) from Tomitella biformata AHU 1821T Promotes Growth and Resuscitates Non-Dividing Cells

et al. 2013

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“…The major difference for the mycobacteria under study was the less profound decline in CFU numbers and the higher CFU/MPN ratio than in the case of M. smegmatis and M. tuberculosis [20,28,30]. On the whole, actually viable (despite their lost colony-forming ability), non-replicating M. abscessus cells that were cultivated under K + deficiency with decelerated respiratory activity and specific morphological traits can be regarded as an example of transitional dormancy, unlike resistant and differentiated resting cells produced under different and particular conditions [35].…”

Section: Discussionmentioning

confidence: 99%

Distinct Effects of Moxifloxacin and Bedaquiline on Growing and ‘Non-Culturable’ Mycobacterium abscessus

Mulyukin,

Recchia,

Kostrikina

et al. 2023

Microorganisms

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Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a ‘non-culturable’ state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The ‘non-culturable’ mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. ‘Non-culturable’ cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and ‘non-culturable’ M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.

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“…In this study, we ascribe conditions needed for corynebacterial cells to transition to a state of dormancy, which was accompanied by: (1) the formation of stress-resistant forms intended for longterm survival that have been identified in many spore-forming and non-spore-forming bacteria (Zhang, 2004;Mulyukin et al, 2010Mulyukin et al, , 2014Lennon and Jones, 2011) and (2) the acquisition of an NC state during the prolonged incubation of post-stationary cultures, which was previously described for Micrococcus luteus , Rhodococcus rhodochrous (Shleeva et al, 2002) and mycobacteria (M. tuberculosis and M. smegmatis) (Shleeva et al, 2003(Shleeva et al, , 2004(Shleeva et al, , 2011(Shleeva et al, , 2015Kudykina et al, 2011). The dormant forms of mycobacteria, which form when the external medium undergoes gradual acidification, can be distinguished from active bacteria based on their distinct proteomic (Trutneva et al, 2018(Trutneva et al, , 2020 and metabolomic (Nikitushkin et al, 2020) profiles.…”

Section: Discussionmentioning

confidence: 99%

“…Further, dormant bacterial cells may develop "non-culturability" (NC) (a term which reflects the inability to form colonies on agar plates), which prevents the cells from being detected in vivo using routine plating procedures (Roszak and Colwell, 1987;Cellini et al, 1994;Porter et al, 1995;Kell et al, 1998;Barer and Harwood, 1999;Tholozan et al, 1999;Biketov et al, 2000;Currás et al, 2002;Shleeva et al, 2002;Mukamolova et al, 2003;Wood et al, 2005;Lee et al, 2007). For many non-sporulating gramnegative and gram-positive bacteria, including non-pathogenic relatives of pathogens, morphologically differentiated dormant forms have previously been described (Cellini et al, 1994;Currás et al, 2002;Shleeva et al, 2002Shleeva et al, , 2004Lee et al, 2010;Mulyukin et al, 2010). Studying the possible persistence and development of antibiotic resistance of other pathogenic and conditionally pathogenic bacteria, including representatives of the genus Corynebacterium is also relevant (Blokpoel et al, 2005;Tauch et al, 2005;Soriano et al, 2009;Olender, 2012).…”

Section: Introductionmentioning

confidence: 99%

Corynebacterium jeikeium Dormant Cell Formation and Photodynamic Inactivation

2020

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Pathogenic non-spore forming bacteria enter a dormant state under stressful conditions, which likely allows them to acquire resistance to various antibiotics. This work revealed the efficient formation of dormant “non-culturable” (NC) Corynebacterium jeikeium cells in stationary phase upon gradual acidification of the growth medium. Such cells were unable to form colonies and existed in a prolonged stationary phase. At an early stage of dormancy (approximately 14 days post-inoculation), dormant cells are able for resuscitation in liquid medium. However, those stored for long time in dormant state needed addition of supernatant taking from active C. jeikeium cultures for successful resuscitation. NC cells possessed low RNA synthesis and significant tolerance to antibiotics (rifampicin and vancomycin). They also accumulated free porphyrins, and 5-aminolevulinic acid addition enhanced free porphyrin accumulation which makes them potentially sensitive to photodynamic inactivation (PDI). PDI of dormant bacteria was accomplished by exposing cells to a 565 nm wavelength of light using a SOLIS-4C light-emitting diode for 60 min. This revealed that increased porphyrin concentrations were correlated with elevated PDI sensitivity. Results shown here demonstrate the potential utility of employing PDI to minimize levels of dormant, persistent corynebacteria and the C. jeikeium dormancy model developed here may be useful for finding new drugs and techniques for combatting persistent corynebacteria.

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Intraspecies diversity of dormant forms of Mycobacterium smegmatis

Cited by 11 publications

References 23 publications

Resuscitation Promoting Factor (Rpf) from <i>Tomitella biformata</i> AHU 1821<sup>T</sup> Promotes Growth and Resuscitates Non-Dividing Cells

Resuscitation Promoting Factor (Rpf) from <i>Tomitella biformata</i> AHU 1821<sup>T</sup> Promotes Growth and Resuscitates Non-Dividing Cells

Distinct Effects of Moxifloxacin and Bedaquiline on Growing and ‘Non-Culturable’ Mycobacterium abscessus

Corynebacterium jeikeium Dormant Cell Formation and Photodynamic Inactivation

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