Induction and Resuscitation of Viable but Nonculturable Corynebacterium diphtheriae

Abstract

Many pathogenic bacteria, including Escherichia coli and Vibrio cholerae, can become viable but nonculturable (VBNC) following exposure to specific stress conditions. Corynebacterium diphtheriae, a known human pathogen causing diphtheria, has not previously been shown to enter the VBNC state. Here, we report that C. diphtheriae can become VBNC when exposed to low temperatures. Morphological differences in culturable and VBNC C. diphtheriae were examined using scanning electron microscopy. Culturable cells presented with a typical rod-shape, whereas VBNC cells showed a distorted shape with an expanded center. Cells could be transitioned from VBNC to culturable following treatment with catalase. This was further evaluated via RNA sequence-based transcriptomic analysis and reverse-transcription quantitative PCR of culturable, VBNC, and resuscitated VBNC cells following catalase treatment. As expected, many genes showed different behavior by resuscitation. The expression of both the diphtheria toxin and the repressor of diphtheria toxin genes remained largely unchanged under all four conditions (culturable, VBNC, VBNC after the addition of catalase, and resuscitated cells). This is the first study to demonstrate that C. diphtheriae can enter a VBNC state and that it can be rescued from this state via the addition of catalase. This study helps to expand our general understanding of VBNC, the pathogenicity of VBNC C. diphtheriae, and its environmental survival strategy.

Antibiotic tolerance in environmentally stressedBacillus subtilis: physical barriers and induction of a viable but nonculturable state

“…Moreover, when VBNC cells were plated onto standard media supplemented with ROS mitigating agents like catalase or sodium pyruvate, the fraction of culturable cells increased significantly. Similar observations were made for other microorganisms (Wai et al 2000 , Masmoudi et al 2010 , Senoh et al 2015 , Hamabata et al 2021 ), showing that resuscitation of VBNC cells likely depends on their ability to neutralize highly ROS generated upon stress conditions. Notably, a recent study by Hong et al ( 2019 ) demonstrated that E. coli cells displayed a self-amplifying accumulation of toxic ROS postantibiotic treatment, which directly contributed to cell death.…”

Bovine serum albumin promotes reactivation of viable but non-culturableMycobacterium tuberculosisvia the activation of protein kinase-dependent cell division processes

“…4). We also examined catalase, a component of OADC supplementation, and its reactivation promoting effect toward VBNC Corynebacterium, Lactobacillus, Salmonella, Ralstonia and Vibrio (47–52); no restorative effects were observed (data not shown). These results suggest that the effect of albumin may be due to complex functions rather than its antioxidative property.…”

“…4). We also examined catalase, a component of OADC supplementation, and its resuscitation promoting effect toward VBNC Corynebacterium, Lactobacillus, Salmonella, Ralstonia and Vibrio (Kong et al, 2004(Kong et al, , 2014Senoh et al, 2015;Liu et al, 2017;Morishige et al, 2017;Hamabata et al, 2021); no resuscitative effects were observed (data not shown).…”

Transcriptional profiling of Xanthomonas campestris pv. campestris in viable but nonculturable state

“…Although the VBNC state is generally regarded as a survival strategy to against adverse conditions [4], the molecular mechanism is less reported. In previous reports, the transcriptomic approach was used to explore the mechanism of VBNC state in E. coli [18][19][20], Brettanomyces bruxellensis [21], V. alginolyticus [22], V. cholerae [23], Corynebacterium diphtheriae [24], P. syringae pv. syringae [25], Enterococcus faecalis [26], V. parahaemolyticus [27], and C. michiganensis [28].…”