Does anyone know if there is a way to rescue Helicobacter pylori from VBNC state?
We grow H.Pylori from biopsy on selective agar in microaerophily (Campygen) at 37°C , it slowly grow as distinct colony in 48 hr but if cultured more than that enter VBNC, this is incovenient especially if antibiotic susceptibility is needed.
I heard about using brucella broth with scraped colonies from selective agars to rescue but my lab don't have that in home. There is other known ways to make H.pylori again?
Thank in advance.
It is possible to culture Helicobacter pylori from biopsy specimens, although it should be stated that this is not a routine procedure.Most of the many different techniques involved in diagnosis of H. pylori infection are performed in clinical microbiology laboratories. The best specimens to culture H. pylori are biopsy samples obtained during endoscopy. Care must be taken to insure that the patients did not receive antibiotics or antisecretory drugs, especially proton pump inhibitors (PPI). Although PPI have no direct antimicrobial effect at the concentration present in the gastric mucosa , they indirectly interfere with H. pylori distribution in the stomach by changing the pH of its bacterial niche, leading to its disappearance in the antrum. brucella broth with scraped colonies from selective agar is used to rescue H.pylori. Several virulence factors for gastric colonization, tissue damage, and survival have been identified in H. pylori . Flagella, urease, and adhesins are all essential factors for H. pylori to colonize the gastric mucosa. Mutants of H. pylori without flagella or without urease are unable to colonize the gastric mucosa in laboratory animal.
Dear Manlio, my suggestions are based on culture and recovery of H. pylori and related fastidious Helicobacter species but they may help. Firstly, if you can use a different means of creating a microaerobic atmosphere that contains 3-7% H2 this may help. A cabinet or the gas replacement method are suitable. An older method involved using the Oxoid BR38 sachet without catalyst - not sure if these are still available. Secondly, choose Brucella agar with caution- some formulations contain sodium metabisulphite and this is inhibitory to H. pylori. A better medium could be Brain Heart Infusion with 5-7% v/v blood. Good luck!
Once it happened to me, I had to rescue a Campylobacter from the previous colture, but it didn't grow. After several tryes (I know is not exacly the same) I tryed by incubation of all the colonies that were lefted, in BrainHeart Broth + MH, into a closed box putted into CO2 cabinet Over Night. The next day I inoculated 50microliter of the torbid broth on Campygen medium. It worked. And I was able to use the same torbidity to perform a Kirby Bauer test.
I know is a method a bit 'arranged, but you could always try and give it a chance.
Good luck!
Ramona.
That is a very interesting question. Our lab has a very good positive culture rate when using gastric biopsy samples. On the other hand we obtained only 3 positive cultures from 45 oropharyngeal biopsy samples even though we detected H.pylori from same samples with real-time PCR. We think that the reason for this discrepancy should be the non-culturable state of the bacterium. If anyone can rescue H. pylori from this state, maybe we will be able to get higher positive culture rate from oropharyngeal samples.
We tried for several years to resuscitate VBNC Hp, but were unsuccessful. I'm sure it is possible, however.
Hello Manlio! It is suggested in literature that free living amoeba could resuscitate VBNC bacteria, in which case H. pylori may be a candidate, in the lab. I haven't tried it though with your kind of organism but I have observed grow rate of Mycobacteria when co-cultured with A. polyphaga. Please try it and see if you would get results.
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