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It was 1981. Barry Marshall, an internist in Australia, began working with Robin Warren, a pathologist who, two years earlier, had discovered that the microbe Helicobacter pylori could overrun the gut. Marshall had been watching as his patients with ulcers slowly got worse and worse until they had their stomachs removed - or died. By biopsying his ulcer patients, and culturing those biopsies, Marshall found that not only could the ulcers be traced to H. pylori, but so could stomach cancers. Unfortunately, mainstream gastroenterologists refused to believe this, maintaining that stress was the sole cause. Marshall grew desperate - he couldn't come up with studies to prove the connection since H. pylori only affects primates (eliminating lab mice) and he was prohibited from experimenting on people. Finally, he decided that he would run the experiment on the only human patient he was ethically able to recruit - himself. He mixed some H. pylori from a patient's cultures into a broth, and drank it. He quickly developed gastritis, with vomiting, stinking breath, nausea, and fatigue. He biopsied his own gut, culturing H. pylori. And in 2005, he and Warren won the Nobel Prize for unequivocally proving that H. pylori causes ulcers and stomach cancer.
Helicobacter pylori is a Gram-negative, spiral shaped bacterium that exhibits rod shapes when cultured on solid medium. If cultured for long enough, coccoid shapes may appear. In vivo, it is found in the gastric mucous layer or adherent to the epithelial lining of the stomach. Approximately two-thirds of the world is infected with this microbe, though most never suffer any symptoms. According to the CDC, however, H. pylori causes 90% of duodenal ulcers and 80% of gastric ulcers. And people infected with H. pylori are 2 to 6 times more likely to develop stomach cancer. Unfortunately, we still know next to nothing about how this microbe is transmitted, nor why some patients show symptoms while others never do.
There are a variety of methods to diagnose H. pylori infection including a stool test, blood test, endoscopy, or a breath test. The breath test, a simple and painless way to test for H. pylori, was approved by the FDA in 1994 with the help of Barry Marshall. The patient breathes into a balloon-like apparatus and establishes a baseline carbon measurement. The patient then has to drink a beverage with urea enriched carbon. After about thirty minutes the patient breathes into a second balloon that will be sent for analysis. If the second balloon contains more carbon than the first balloon, then the patient is positive for H. pylori. This works because the urea-enriched carbon would be broken down by the secretion of urease from H. pylori, increasing the total amount of carbon released. Thankfully, there are many treatments for H. pylori infection, including a combination of antibiotics, proton pump inhibitors (i.e. prilosec, nexium), H-2 blockers (i.e. zantac, pepcid AC), and medication to protect the stomach lining. Unfortunately, H. pylori is a cunning bacteria that can evade antibiotics, including our best line of defense, the antibiotic Clarithromycin. H. pylori antibiotic resistance is continually evolving, and patients are requiring multiple rounds of treatment to clear the infection.
Given that this is a growing public health concern, it makes sense that this would be a well-studied organism. However, we're constantly learning about new facets of this microbe - some that have far-reaching effects.
They first needed to confirm that flagella were, in fact, present, a feat accomplished through scanning electron microscopy of a biofilm (Fig. 7A, SS1WT; the strands pointed out by the white arrows). This did, indeed, confirm the presence of flagella, and the researchers moved on to the next question - were flagella necessary for biofilm formation? To answer this question, the researchers developed a mutant strain of H. pylori that lacked flagella (ΔfliM), once again confirmed through scanning electron microscopy (Fig. 7A, SS1ΔfliM). When they cultured the mutant in an attempt to develop biofilms, they found significantly less biofilm mass (Fig. 7B), and within the microcolonies they did find, the flagella were completely lacking. This raised one further question - which is the important part in biofilm formation, the motility of the flagella or the flagella themselves? To answer this, the researchers once again developed a mutant strain - this time, one that had flagella but lacked motility (ΔmotB), created by disrupting the motor protein MotB (Fig. 7A, SS1ΔmotB). This strain produced significantly more biofilm mass than the non-flagellated mutant, but still much, much less than the wild-type version, which had both flagella and motility (Fig. 7B; showing the amount of biofilm formed by each version). These results indicated that the flagella themselves played an important role in biofilm formation and structure beyond their motility ability. To confirm that this finding applies to strains of H. pylori other than the SS1 strain, the researchers decided to perform the same experiments on another strain of H. pylori (G27), looking at wild-type biofilm formation in comparison to the G27ΔfliM mutant (no flagella) and the G27ΔmotB mutant (flagella, but no motility). They found the same results - normal biofilm formation in the wild-type version, partial biofilm formation in the mutant that lacked motility, but retained flagella, and weak biofilm formation in the non-flagellated mutant. The researchers suggested that, taken together, these data indicate that flagella play an important role in "promoting biofilm integrity by holding cells together and to the surface" (Hathroubi et al., 2018, p.10).
This study presents a new take on the role of flagella in biofilms. Flagella have been historically viewed in relation to biofilms as important only for bringing cells together initially, and assisting in later cell dispersion. If this long-held belief about flagella in biofilms is incorrect, what else might we be missing? And how might this new knowledge affect how we treat H. pylori infections?
About the Authors:
Tess Ahlers '19 is a biology major on the pre-nursing track, and a member of the hunt-seat equestrian team. She plans to go to nursing school after graduation, to eventually work as a nurse-midwife. When not working or riding, she enjoys reading, knitting, and excessive amounts of Netflix (all with a nice cup of tea)
Hannah Lavoie '19 is a Pre-Health Post Baccalaureate student at Mount Holyoke College. She currently works as an event planner. After finishing her program this semester, Hannah plans on applying to Physician Assistant programs. She enjoys spending time with family, playing with her dog, and traveling.
Helicobacter pylori is a Gram-negative, spiral shaped bacterium that exhibits rod shapes when cultured on solid medium. If cultured for long enough, coccoid shapes may appear. In vivo, it is found in the gastric mucous layer or adherent to the epithelial lining of the stomach. Approximately two-thirds of the world is infected with this microbe, though most never suffer any symptoms. According to the CDC, however, H. pylori causes 90% of duodenal ulcers and 80% of gastric ulcers. And people infected with H. pylori are 2 to 6 times more likely to develop stomach cancer. Unfortunately, we still know next to nothing about how this microbe is transmitted, nor why some patients show symptoms while others never do.
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| (picture credit) |
There are a variety of methods to diagnose H. pylori infection including a stool test, blood test, endoscopy, or a breath test. The breath test, a simple and painless way to test for H. pylori, was approved by the FDA in 1994 with the help of Barry Marshall. The patient breathes into a balloon-like apparatus and establishes a baseline carbon measurement. The patient then has to drink a beverage with urea enriched carbon. After about thirty minutes the patient breathes into a second balloon that will be sent for analysis. If the second balloon contains more carbon than the first balloon, then the patient is positive for H. pylori. This works because the urea-enriched carbon would be broken down by the secretion of urease from H. pylori, increasing the total amount of carbon released. Thankfully, there are many treatments for H. pylori infection, including a combination of antibiotics, proton pump inhibitors (i.e. prilosec, nexium), H-2 blockers (i.e. zantac, pepcid AC), and medication to protect the stomach lining. Unfortunately, H. pylori is a cunning bacteria that can evade antibiotics, including our best line of defense, the antibiotic Clarithromycin. H. pylori antibiotic resistance is continually evolving, and patients are requiring multiple rounds of treatment to clear the infection.
Given that this is a growing public health concern, it makes sense that this would be a well-studied organism. However, we're constantly learning about new facets of this microbe - some that have far-reaching effects.
One study, published in 2018 by three researchers from UC Santa Cruz, details an example of such a discovery. The study looks at biofilm formation in one strain of H. pylori (SS1). This is important because biofilms are one way H. pylori is able to infiltrate and survive the stomach. They provide protection against the environment, as well as antibiotics, making H. pylori infections more difficult to treat. So understanding how biofilms work, and their components, is vital in gaining insight into more effective treatments for H. pylori infections. Some of the aspects of biofilm formation that the study looked at include which growth conditions are best, characterization of the biofilm, and a transcriptomic profiling of biofilm vs. planktonic cells (basically, which genes are being expressed in cells within a biofilm versus in cells that are motile, or wandering around). This last point brought up something interesting: in cells within the biofilm, a large number of the genes which encoded flagella (a microscopic appendage which enables many microbes to move around) were upregulated. This meant that their expression was increased, rather than the expected decrease given these cells no longer need to move - and thus shouldn’t need flagella. So the researchers decided to look further into this phenomenon.
They first needed to confirm that flagella were, in fact, present, a feat accomplished through scanning electron microscopy of a biofilm (Fig. 7A, SS1WT; the strands pointed out by the white arrows). This did, indeed, confirm the presence of flagella, and the researchers moved on to the next question - were flagella necessary for biofilm formation? To answer this question, the researchers developed a mutant strain of H. pylori that lacked flagella (ΔfliM), once again confirmed through scanning electron microscopy (Fig. 7A, SS1ΔfliM). When they cultured the mutant in an attempt to develop biofilms, they found significantly less biofilm mass (Fig. 7B), and within the microcolonies they did find, the flagella were completely lacking. This raised one further question - which is the important part in biofilm formation, the motility of the flagella or the flagella themselves? To answer this, the researchers once again developed a mutant strain - this time, one that had flagella but lacked motility (ΔmotB), created by disrupting the motor protein MotB (Fig. 7A, SS1ΔmotB). This strain produced significantly more biofilm mass than the non-flagellated mutant, but still much, much less than the wild-type version, which had both flagella and motility (Fig. 7B; showing the amount of biofilm formed by each version). These results indicated that the flagella themselves played an important role in biofilm formation and structure beyond their motility ability. To confirm that this finding applies to strains of H. pylori other than the SS1 strain, the researchers decided to perform the same experiments on another strain of H. pylori (G27), looking at wild-type biofilm formation in comparison to the G27ΔfliM mutant (no flagella) and the G27ΔmotB mutant (flagella, but no motility). They found the same results - normal biofilm formation in the wild-type version, partial biofilm formation in the mutant that lacked motility, but retained flagella, and weak biofilm formation in the non-flagellated mutant. The researchers suggested that, taken together, these data indicate that flagella play an important role in "promoting biofilm integrity by holding cells together and to the surface" (Hathroubi et al., 2018, p.10).
This study presents a new take on the role of flagella in biofilms. Flagella have been historically viewed in relation to biofilms as important only for bringing cells together initially, and assisting in later cell dispersion. If this long-held belief about flagella in biofilms is incorrect, what else might we be missing? And how might this new knowledge affect how we treat H. pylori infections?
About the Authors:
Tess Ahlers '19 is a biology major on the pre-nursing track, and a member of the hunt-seat equestrian team. She plans to go to nursing school after graduation, to eventually work as a nurse-midwife. When not working or riding, she enjoys reading, knitting, and excessive amounts of Netflix (all with a nice cup of tea)
Hannah Lavoie '19 is a Pre-Health Post Baccalaureate student at Mount Holyoke College. She currently works as an event planner. After finishing her program this semester, Hannah plans on applying to Physician Assistant programs. She enjoys spending time with family, playing with her dog, and traveling.
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