Viable but Nonculturable Bacteria

By: Jvo Siegrist, Microbiology Focus Edition 1.4

Product Manager Microbiology…. ivo.siegrist@sial.com

In many specimens, more bacteria are present than we can detect with common cultural methods.

The expression “viable but nonculturable” (VNC) bacteria, describes cells that cannot normally be cultured. However this makes little sense, when one considers that the demonstration of culturability remains the best practically acceptable definition of viability. So a better explanation of the status of these bacteria would be “not immediately culturable”. In most cases the non-spore-forming bacteria is in a survival state (e.g., resting, dormancy, quiescence, or debilitation) and the metabolic pathways are still active but the organism are not growing. According to the latest VNC definition, VNC cells are regarded as viable and potentially replicative, but the methods required for resuscitation are beyond our current knowledge. With special media or with certain supplements it has been shown that it is possible to recover them. VNC bacteria have often undergone a treatment like heating, drying, setting under high osmotic pressure (high salt content) or contact with inhibiting chemicals. The end result of the treatment is sensitive cells or sub-lethally damaged cells, which can mean the loss of some ribosomes, damaged enzymes, cell membranes and other problems causing malfunctions in cells.

In the recent years species of Vibrio cholerae, E. coli, Campylobacter jejuni, Salmonella spp., Listeria monocytenenes and Yersinia enterocolitica have been reported to enter the viable but nonculturable (VNC) state [1-10].

Supplementing the pre-enrichment and enrichment broths with ferrioxamine E significantly improved the recovery of Salmonella, Cronobacter spp., Staphylococcus aureus and Yersinia enterocolitica from artificially or naturally contaminated foods [1-3]. A concentration of ferrioxamine E (available from Sigma, see Table 2) in the range of 5-200 ng/mL supports growth (see Table 1). Ferrioxamine E provides the essential micro-nutrient iron (III) to the organisms. This leads to a reduced lag-phase in the medium and reactivates damaged bacteria. The ferrioxamine E is often used in Buffered Peptone Water the medium recommended by the ISO-Norms for Enterobacteriacea (see Table 2). The motility of Salmonella is also improved which helps to improve the identification by semisolid selective motility media like MRSV, DIASSALM or SMS. It is recommended when isolating small quantities of cells from dried powders like tea, spices, dried fruits etc. Ferrioxamine E is semi-selective, as it does not improve growth of E. coli, Shigella, Proteus, Providencia and Morganella species.

Table 1. Recommended end concentration of Ferrioxamine E.

 

Table 2. Products to assist in the detection of VNC

Product Cat #
Ferrioxamine E 38266
Desferrioxamine B (Deferoxamine mesylate) D9533
Peptone Water, phosphate-buffered with Ferrioxamine E 67331
HiCrome™ MeReSa Agar 90923
MRSA Selective Supplement 51387

 

Another application is the fast and selective detection of methicillin-resistant Staphylococcus aureus (MRSA) where a combination of Ferrioxamine E and Desferrioxamine B is used in the enrichment media. Desferrioxamine B (available from Sigma, see Table 2) adsorbs iron traces and thus inhibits growth of concomitant microorganisms and Ferrioxamine E supports Staphylococcus aureus, which is able to utilize Ferrioxamine E for iron uptake.

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Materials

     

References

  1. S. Makino, et al., Does Enterohemorrhagic Escherichia coli O157:H7 Enter the Viable but Nonculturable State in Salted Salmon Roe?, Appl. Environ. Microbiol., 66(12): 5536–5539 (2000)
  2. I. Barcina, P. Lebaron, J. Vives-Rego, Survival of allochthonous bacteria in aquatic systems: a biological approach, FEMS Microbiol. Ecol., 23:1–9 (1997)
  3. J.C. Choa, S.J. Kim, Viable, but non-culturable, state of a green fluorescence protein-tagged environmental isolate of Salmonella typhi in groundwater and pond water, FEMS Microbiol. Lett., 170:257–264 (1999)
  4. R.R. Colwell, et al., Viable but non-culturable Vibrio cholerae and related pathogens in the environment: implications for release of genetically engineered microorganisms, Bio/Technology., 3:817–820 (1985)
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  8. H.-S. Xu, et al., Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment, Microb. Ecol., 8:313–323 (1982)
  9. V. Besnard, et al., Environmental and physico-chemical factors induce VBNC state in Listeria monocytogenes, Vet. Res. 33 (4) 359-370 (2002)
  10. A. Singh, G.A. McFeters. Survival and virulence of copper- and chlorinestressed Yersinia enterocolitica in experimentally infected mice. Appl Environ Microbiol., 53(8):1768–1774 (1987)
  11. R. Reissbrodt, et al., Resuscitation by ferrioxamine E of stressed Salmonella enterica serovar typhimurium from soil and water microcosms., Appl. Environ. Microbiol., 66(9):4128-30 (2000)
  12. R. Reissbrodt et al., Ferrioxamine E-supplemented pre-enrichment and enrichment media improve various isolation methods for Salmonella., Int. J. Food Microbiol., 29(1):81-91(1996)
  13. P. Pless, and R. Reissbrodt., Improvement of Salmonella detection on motility enrichment media by ferrioxamine E-supplementation of preenrichment culture., Int. J. Food Microbiol., 27:147-159 (1995)

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