Don't let the Bad Beer Bugs Bite

New insights into why some bacteria, but not others, spoil our beer

Many spoilage events in the beer industry are due to Lactic acid bacteria (LAB). While many bacteria are susceptible to hop extracts and do not grow well in beer, some strains have acquired resistance to hop-derived compounds.

Photo by CDC on Unsplash

Genetic and genomics studies have previously identified resistance genes (horA and horC) that confer resistance to hop extracts and promote growth in beer. While brewers and researchers routinely test and find hop resistance genes in commonly found LAB species, it is unclear whether they are present in rarer species. One way to learn about resistance mechanisms to hops is to identify new beer spoilage species (of the LAB kind) and define their resistance genes.

Isolation of a new bacterial strain, responsible for beer spoilage

A new study by Umegatani and authors (2021) sought to investigate new emerging and less common LAB species. The rationale for their work was that by cataloguing new species and identifying their resistance gene complements, brewers are better equipped to deal with beer infection and spoilage. Let's see what they found!

The authors focused on an infected pilsner-type beer and managed to isolate a bacterium deemed to be responsible for spoilage. The strain, Lactobacillus nagelii strain ABBC668, was isolated using common microbiology techniques, after which the authors sequenced the 16D rDNA region in the bacterial chromosome. 

The 16S rDNA region is highly conserved (present in most bacteria) but is variable due to random mutations that researchers use to discriminate between bacterial species or strains. When researchers compared the sequence of this particular strain to those of thousands of others (using a database and searching algorithm), they discovered that it was L. nagelii (The sequence was most closely related to one found in another known L. nagelii strain). Given that L. nagelii is not commonly known to spoil beer but slow fermentation of other beverages, the authors set out to genetically characterise this strain and learn more about its biology or properties.

First, the authors grew their strain (ABBC668 from here on) in beers containing or lacking hop extracts. Importantly, they used related strains not found in beers as controls to assess whether the ability of ABBC668 to spoil bacteria was due to its specific ability to grow in hopped beers. These experiments made it clear that ABBC668 but not the other two related strains could grow in beer with hops. Importantly, without hops present, all three strains grew well. These results indicate that the ability of ABBC668 to spoil beer is because it can grow in hopped beer. 

The question then became, what determines resistance to hops in ABBC668? For this, the authors decided to focus on known resistance genes (HorA and HorC) and ask whether these commonly found genes are present in ABBC668 and the other strains. Polymerase Chain Reaction (PCR) experiments in which researchers can specifically test for the presence/absence of gene sequences revealed that ABBC668 contained the HorA gene but lacked HorC. Critically, the other non-beer strains did not harbour any of these genes, thereby explaining resistance to hops in ABBC668 only. 

The last exciting experiment I will describe here pertains to stability of resistance genes. In bacteria, drug resistance often is encoded on mobile plasmids, which are small circular DNA molecules that can autonomously replicate and are not essential for survival. Because these plasmids need to reproduce at a cost to the bacterium, one can expect that conditions favour a loss of this plasmid in the absence of selective pressures (here hop compounds). 

The authors tested this notion by growing ABBC668 for multiple generations in media that lacked hop extracts. Subsequent PCR-based testing revealed that propagation of ABBC668 in the absence of hops led to the loss of HorA and the plasmid that carries it. These results illustrate that the HorA gene or plasmid is mobile, can work to confer resistance in bacteria, but its maintenance comes at a cost.

Why is this important? 

As the authors point out in their work, they were able to identify HorA using a simple PCR on their isolated strains. Given that HorA and HorC appear to be good markers for spoilage bacteria, diagnostic tests, able to detect these hop resistance genes would make a sensitive and straightforward means to identify spoilage potential in your beer. This is important as it would eliminate the need to isolate bacteria, which may not grow in artificial or formulated media.

I hope you enjoyed this read and have a great weekend!

Cheers!

Edgar, the Beerologist.