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Gallstones Containing Bacteria are Biofilms: Bacterial Slime Production and Ability to Form Pigment Solids Determines Infection Severity and Bacteremia

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Journal of Gastrointestinal Surgery Aims and scope

Abstract

Objective

Gallstone bacteria provide a reservoir for biliary infections. Slime production facilitates adherence, whereas β-glucuronidase and phospholipase generate colonization surface. These factors facilitate gallstone formation, but their influence on infection severity is unknown.

Methods

Two hundred ninety-two patients were studied. Gallstones, bile, and blood (as applicable) were cultured. Bacteria were tested for β-glucuronidase/phospholipase production and quantitative slime production. Infection severity was correlated with bacterial factors.

Results

Bacteria were present in 43% of cases, 13% with bacteremia. Severe infections correlated directly with β-glucuronidase/phospholipase (55% with vs 13% without, P < 0.0001), but inversely with slime production (55 vs 8%, slime <75 or >75, P = 0.008). Low slime production and β-glucuronidase/phospholipase production were additive: Severe infections were present in 76% with both, but 10% with either or none (P < 0.0001). β-Glucuronidase/phospholipase production facilitated bactibilia (86% with vs 62% without, P = 0.03). Slime production was 19 (±8) vs 50 (±10) for bacteria that did or did not cause bacteremia (P = 0.004). No bacteria with slime >75 demonstrated bacteremia.

Conclusions

Bacteria-laden gallstones are biofilms whose characteristics influence illness severity. Factors creating colonization surface (β-glucuronidase/phospholipase) facilitated bacteremia and severe infections; but abundant slime production, while facilitating colonization, inhibited detachment and cholangiovenous reflux. This shows how properties of the gallstone biofilm determine the severity of the associated illness.

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Acknowledgement

This work was supported by a VA Merit Review Grant.

Author information

Authors and Affiliations

  1. Department of Surgery (112), University of California San Francisco, 4150 Clement Street, San Francisco, CA, 94121, USA

    Lygia Stewart MD

  2. Surgical Service, San Francisco VA Medical Center, San Francisco, CA, USA

    Lygia Stewart MD

  3. Center for Immunochemistry, San Francisco VA Medical Center, San Francisco, CA, USA

    J. McLeod Griffiss MD & Gary A. Jarvis PhD

  4. Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA

    J. McLeod Griffiss MD & Gary A. Jarvis PhD

  5. 4 Koret Way, L-101, Box 0475, University of California San Francisco, San Francisco, CA, 94143, USA

    Lawrence W. Way MD

Authors
  1. Lygia Stewart MD
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  2. J. McLeod Griffiss MD
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  3. Gary A. Jarvis PhD
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  4. Lawrence W. Way MD
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Corresponding author

Correspondence to Lygia Stewart MD.

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DISCUSSION

Dr. D. Soybel (Boston, MA): I appreciate you sending me the manuscript to review, and I actually would like to commend the discussion in this paper. For anybody who thinks that we can beat bacteria at their own game simply with antibiotics, I think the discussion here actually was very enlightening.

There are two comments that I wanted to make and with questions. The first one is, you and of course Henry Pitt’s group have been interested in whether bacteria might find cholesterol versus pigment stones more hospitable, and both groups have shown that pigment stones are much more hospitable to bacteria than are cholesterol stones. In this study you lumped all stones together. But just for the sake of understanding the patient population, what were the percentages, among patients who actually had bacteria, for cholesterol versus pigment stones? Also do you have any thoughts about why cholesterol stones are less hospitable than pigment stones?

The second question is whether certain species of bacteria tend to lose the ability to make slime more than others? Are we looking at some sort of general environmental thing where essentially any bacteria could make slime or not make slime? What do you think is in the environment that is actually inducing them to stop making slime so that they can become more pathogenic?

Dr. Stewart: Thank you. Very good questions. With regards to the cholesterol-stone question, you can find bacteria in cholesterol stones that have a pigmented center about 35% of the time. We have an up-coming paper on this that shows that when the bacteria are in the center of a predominantly cholesterol stone, they are associated with less infectious manifestations.

But what is interesting, is that the bacteria in pigment and cholesterol stones make different amounts of pigment-forming factors, and bacteria preferentially stick to the surface of pigment stones. We studied this question some time ago by placing pigment and cholesterol stones into Tryptic-soy broth with 107 bacteria/cc. We found that the bacteria didn’t stick to the cholesterol stones even though there were 107 bacteria/cc, but they did stick to the pigment stones. Why is that? Well, go back to the biofilm research, and what you find is that positively charged ions are an attractor for negatively charged things (like bacteria). And if you look at a pigment stone, it is made of calcium salts: calcium bilirubinate, calcium palmitate, calcium phosphate, calcium carbonate. These calcium salts attract the bacteria, and this, by the way, is known across biofilm research in general. It doesn’t matter whether the biofilm is in a pipe or in a vein. Not only do the calcium salts in the pigment stone allow the bacteria to stick, but the bacteria facilitate pigment formation because they make beta-glucuronidase or pholpholipase. So it is a combination of things.

Now, you raise a very important issue of why do some bacteria make more slime than others. Is it the environment? Is it the bacteria? Can they actually lose their ability to make slime? What is the issue here? We know that intact host immune responses promote biofilm formation, as do factors like low nutrients, low oxygen, etc. And there is some evidence that bacterial species that are able to make abundant slime (like Pseudomonas) cause chronic infections in the intact host, but are more likely to cause severe illnesses in immunocompromised patients (burn, transplant, cancer patients, etc.). Why? Because the immune mechanism isn’t making that Pseudomonas go into the biofilm, change its genes and make the slime.

But in our study, all the bacteria were from the biliary tree. The environment of the biliary tree drives bacteria into biofilm formation because it has low nutrients, low oxygen, bile salts that are antibacterial, and IgA. So, there are several factors pushing those bacteria into the biofilm mode. We found that there was diversity among the bacteria, some made abundant slime while others didn’t. We have noted, in prior publications, that biliary bacteria make more slime than stool bacteria. So it seems, when you get right down to it, that both are important, the tendency of the bacteria to make slime and the environment both play a role.

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Stewart, L., Griffiss, J.M., Jarvis, G.A. et al. Gallstones Containing Bacteria are Biofilms: Bacterial Slime Production and Ability to Form Pigment Solids Determines Infection Severity and Bacteremia. J Gastrointest Surg 11, 977–984 (2007). https://doi.org/10.1007/s11605-007-0168-1

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