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. Author manuscript; available in PMC: 2017 Dec 1.
Published in final edited form as: Arch Microbiol. 2016 Jul 23;198(10):1019–1026. doi: 10.1007/s00203-016-1272-y

Probiotic properties of Oxalobacter formigenes: an in vitro examination

Melissa L Ellis 1, Alexander E Dowell 1, Xingsheng Li 1, John Knight 1,✉
PMCID: PMC5083200  NIHMSID: NIHMS805518  PMID: 27449000

Abstract

Oxalobacter formigenes (O. formigenes) is a nonpathogenic, Gram-negative, obligate anaerobic bacterium that commonly inhabits the human gut and degrades oxalate as its major energy and carbon source. Results from a case-controlled study suggested that lack of O. formigenes colonization is a risk factor for recurrent calcium oxalate stone formation. Hence, O. formigenes colonization may prove to be an efficacious method for limiting calcium oxalate stone risk. However, challenges exist in the preparation of O. formigenes as a successful probiotic due to it being an anaerobe with fastidious growth requirements. Here we examine in vitro properties expected of a successful probiotic strain. The data show that the Group 1 O. formigenes strain OxCC13 is sensitive to pH < 5.0, persists in the absence of oxalate, is aerotolerant, and survives for long periods when freeze-dried or mixed with yogurt. These findings highlight the resilience of this O. formigenes strain to some processes and conditions associated with the manufacture, storage and distribution of probiotic strains.

Keywords: Oxalate, Oxalobacter formigenes, Intestine, Probiotic

Introduction

Oxalobacter formigenes is part of the microbiota in the large intestine of many humans and other mammalian species (Dawson et al. 1980; Allison et al. 1985; Daniel et al. 1987; Argenzio et al. 1988; Duncan et al. 2002; Miller et al. 2014). Recent evidence indicates a lack of colonization is a risk factor for calcium oxalate stone disease (Kaufman et al. 2008). Protection against calcium oxalate stone disease appears to be due to the oxalate degradation that occurs in the gut on low calcium diets (Jiang et al. 2011; Knight et al. 2013) with a possible further contribution from intestinal oxalate secretion (Hatch and Freel 2013). A review of worldwide data indicated that 38–77 % of a normal population and only 17 % of stone formers are colonized with O. formigenes (Kaufman et al. 2008), suggesting that colonization of calcium oxalate stone formers may be an efficacious method for limiting calcium oxalate stone risk.

Probiotic supplements that claim to contain O. formigenes are available for purchase over the Internet from ™PRO Lab, Ltd, and Sanzyme, Ltd. However, results from a recent analysis indicated that these supplements do not contain detectable O. formigenes, raising questions about the difficulty of manufacturing O. formigenes for probiotic use (Ellis et al. 2015). A recent randomized, double-blind, placebo-controlled multicenter study showed that ingestion of a lyophilized enteric coated capsulated preparation of O. formigenes, Oxabact®, currently not available for purchase, did not result in a significant reduction in urinary oxalate excretion (Hoppe et al. 2011), which the authors suggested could have been due to problems with bioavailability of the supplement or viability of O. formigenes in this formulation.

The main goal of this study was to examine the tolerance of the Group 1 O. formigenes strain OxCC13 to various processes and conditions associated with the manufacture, storage and distribution of probiotic products. The findings suggest that the O. formigenes OxCC13 has the capacity to survive in environments where oxygen is present and highlights the resilience of this strain to lyophilization and persistence in yogurt.

Methods

Culture conditions

Cultures of O. formigenes OxCC13 (BioProject, PRJNA32499) were grown at 37 °C in Schaedler’s broth (BD Biosciences) supplemented with 100 mM sodium oxalate and 10 mM sodium acetate (SBO) or in Schaedler’s broth without added oxalate (SB) in an anaerobic glass bottle or serum bottle with a rubber stopper and aluminum seal, as described previously (Ellis et al. 2015). SB is a rich broth containing pancreatic digestion of casein (8.1 g/l), peptic digestion of animal tissue (2.5 g/l), papaic digest of soybean meal (1.0 g/l), dextrose (5.82 g/l), yeast extract (5.0 g/l), sodium chloride (1.7 g/l), dipotassium phosphate (0.82 g/l), hemin (0.01 g/l), L-cystine (0.4 g/l) and TRIS (hydroxymethyl) aminomethane (3.0 g/l). Media was autoclaved to remove oxygen. Culturing O. formigenes in anaerobic SB supplemented with oxalate and acetate results in more consistent and rapid growth compared to anaerobic medium B (Ellis et al. 2015) [medium B (Allison et al. 1985) is an undefined medium with minerals, metals, cysteine, carbonate-buffering system, oxalate, acetate and 0.1 % yeast extract]. Cultures were grown to either log phase (OD595 = 0.04–0.06) or early stationary phase (OD595 = 0.10–0.12). For the determination of colony-forming units (CFUs) on solid plate medium, a variation of medium B was used that contained 40 mM sodium oxalate, 7 mM CaCl2, 0.5 % yeast extract, 0.1 % Na2CO3 and 2.0 % Bacto agar (OXMR). Plates were incubated in anaerobic containers using GasPak EZ Anaerobe Container System with Indicator (BD Biosciences) sachets at 37 °C until detectable numbers of CFUs were stable.

Survival without oxalate

Log-phase, 100-ml cultures were centrifuged at 6000×g for 10 min, washed once in 100 ml of anaerobic SB and recentrifuged. The cell pellet was resuspended in 1 ml anaerobic SB. SB contains less than 0.45 mg/l oxalate, as determined by ion chromatography (IC). Suspensions (0.5 ml, ~5 × 109 CFU) were immediately added to either 100 ml anaerobic SBO or anaerobic SB and incubated at 37 °C. CFUs were determined over 6 days.

Survival at various pH

Early stationary-phase, 100-ml cultures were centrifuged at 6000×g for 10 min, washed once in 100 ml of anaerobic SB and recentrifuged. The cell pellet was resuspended in 1 ml anaerobic SB. Suspensions (0.5 ml, ~5 × 109 CFU) were immediately added to 100 ml anaerobic SB at defined pH (adjusted with HCl or NaOH) and incubated at 37 °C. Samples of 100 μl were removed aseptically at specified time points, instantly diluted in 900 μl of SB to sharply stop the effect of the acidic environment, further diluted to an appropriate plating concentration and plated for CFU.

Tolerance of air

Log-phase cultures in SBO were incubated at 37 °C either statically (10 ml culture in a 14-ml polypropylene round-bottom tube with loosely fitted cap) or with shaking at 200 rpm (10 ml aliquot in a 125-ml baffled flask). Static, anaerobic cultures (10 ml culture in 14-ml tube) were used as controls. Oxalate concentration in the culture medium and CFUs were determined.

Survival in yogurt

Early stationary-phase cultures (100 ml) were centrifuged at 6000×g for 10 min, washed once in 1 ml of phosphate-buffered saline, pH 7.4 and recentrifuged. The cell pellet was resuspended in 1 ml of SBO (~ 1010 cells) and used to inoculate yogurt samples (35–38 g of Publix creamy blends vanilla low-fat yogurt with active yogurt cultures: Acidophilus, Bifidobacterium, L. Bulgaricus, S. Thermophilus and L. Casei.). The yogurt containing O. formigenes was stored at 4 °C, and CFU was determined over 4 weeks.

Survival following lyophilization

Early stationary-phase cultures (600 ml) were centrifuged at 6000×g for 10 min, washed once with 100 ml of phosphate-buffered saline, pH 7.4 and recentrifuged. Each cell pellet was resuspended in 12 ml of Microbial Freeze Drying buffer (OPS Diagnostics, NJ, USA) and aliquoted into 1 ml samples in 5 ml, round-bottom, polystyrene tubes (Corning Inc., NY, USA). These samples were then snap-frozen in liquid nitrogen and lyophilized overnight using a FreeZone 1 Liter Benchtop Freeze Dry System (Labconco Inc., MO, USA). Lyophilized samples were stored at 4 °C in air and rehydrated with 1 ml of anaerobic SBO for 10 min at room temperature prior to plating for CFUs.

Oxalate and formate ion chromatography

Oxalate in culture media was quantified by ion chromatography (IC) using an AS22 2 mm column, as previously described (Ellis et al. 2015). Formate in culture media was quantified by IC using an AS11-HC-4 μm, 2 × 150 mm, hydroxide anion exchange column. A gradient of potassium hydroxide from 0.5 to 3 mM over 15 min at 30 °C and a flow rate of 0.38 ml/min was used to separate formate from other anions in culture medium.

Statistical analysis

All statistical analyses used SAS (version 9.4; SAS Institute Cary, NC, USA). Proc Mixed model was used for the effect of pH, starvation or air on CFU over time. Time effects on CFU with each test condition were further evaluated by repeated measures and Tukey multiple comparison where applicable. Data are expressed as mean ± SD. The criterion for statistical significance was P < 0.05. Experiments were repeated with three to four different cultures.

Results

The manufacture of O. formigenes for probiotic use has been difficult. This may be due to its sensitivity to oxygen and low pH, its strict requirement for oxalate for growth, and its intolerance to common processes associated with the manufacture, storage and distribution of probiotic products. To better understand the probiotic potential of O. formigenes, this in vitro study explores the survival of O. formigenes at low pH, in the absence of oxalate, in the presence of air, to lyophilization, and after mixing with yogurt.

O. formigenes survival without oxalate

To examine O. formigenes survival in the absence of oxalate, cells harvested from log phase were added to SB media not containing oxalate and CFU determined over time. Control incubations were performed with log-phase cells incubated in SBO media containing 100 mM oxalate. After 24-h incubation, viable cell counts decreased significantly (~2.5-fold) in SB lacking oxalate, while CFU in SBO cultures increased significantly (~3-fold) (P < 0.001, Fig. 1) due to the degradation of oxalate (Fig. 2). CFUs in SBO cultures were significantly higher at day 2 (P < 0.001), but not statistically different after 3- and 6-day incubation compared with SB cultures (P > 0.05, Fig. 1). After 6-day incubation, 0.2 and 0.8 % of the initial inoculum were viable in SBO and SB, respectively.

Fig. 1.

Fig. 1

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Survival of O. formigenes without oxalate. Values are mean total CFU ± SD. Filled square, Schaedler’s broth with added oxalate (100 mM). Open square, Schaedler’s broth lacking oxalate. **P < 0.01 between SBO and SB media at specific day

Fig. 2.

Fig. 2

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Oxalate and formate media levels over time in anaerobic O. formigenes cultures. Values are the mean ± SD. Filled circle, oxalate; filled square, formate

CFU decreased in SB between days 1 through 3, but this was not significant (P > 0.05). In contrast, CFU in SBO cultures decreased significantly each day between days 1 and 3 (P < 0.05). After six-day incubation of O. formigenes in SB, media pH had not significantly changed (initial pH 7.19 ± 0.01 vs. pH 7.26 ± 0.06 at Day 6). However, following 6-day incubation of O. formigenes in SBO media pH increased significantly from pH 7.06 ± 0.02 to 7.56 ± 0.06 (P = 0.003). An increase in media pH is to be expected as oxalate metabolism by O. formigenes results in consumption of protons (Kuhner et al. 1996). Oxalate and formate levels in SBO culture medium 24 h post O. formigenes inoculation were <25 μM and 96 mM, respectively (Fig. 2). Formate levels in media remained stable for the remaining 5-day culture period.

O. formigenes acid tolerance

Oxalobacter formigenes early stationary cultures were exposed to SB media at variable pH intervals in the absence of oxalate for 15 min. There was a significant change in O. formigenes viability with decreasing pH (P < 0.0001). Post hoc analysis showed that, relative to the pH 7.1 control, there was a significant decrease in viability at <pH 5.0. SB media at pH 4.0 and 3.0 brought about rapid death of O. formigenes, with only 3 %, and 0.12 % of O. formigenes remaining viable after 60 min, respectively (Fig. 3b). No viable O. formigenes remained after 2-h incubation in pH 3.0 SB media. An additional experiment with SBO containing 100 mM oxalate also showed no survival of O. formigenes after 2-h incubation at pH 3.0 (data not shown).

Fig. 3.

Fig. 3

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Survival of O. formigenes at various pH after 15 min (a), and over time at pH 3.0, pH 4.0 and pH 7.1 (b). Values are mean total CFU ± SD. Filled circle, pH 7.1; filled triangle, pH 4.0; open circle, pH 3.0. Significance relative to pH 7.1 shown by asterisk, where *P = 0.01; ***P = 0.0008; ****P < 0.0001

O. formigenes tolerance of air

Tolerance to air was examined with log-phase cells in SBO media containing 100 mM oxalate under both static and shaking conditions. Recovery of CFU decreased significantly in static and shaking cultures exposed to air compared to static, anaerobic control cultures at all time points (P < 0.05). Only 0.5 % of the initial CFU and no viable cells were recovered following incubation with shaking for 3 and 6 h in air, respectively (P < 0.01, Fig. 4). In contrast, 85 % of the initial CFU was recovered in static cultures after 24-h exposure to air (Fig. 4). Anaerobic control cultures grew 3.4-fold after 6-h incubation (P < 0.01) and remained stable for the remaining 18 h. No oxalate remained after 24-h incubation in both anaerobic control cultures and static aerobic cultures.

Fig. 4.

Fig. 4

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Tolerance of O. formigenes to air in broth culture. Values are mean total CFU ± SD. Filled square, static anaerobic control cultures. Open square, static cultures in air, filled triangle, shaking cultures in air. *P < 0.05, **P < 0.01, compared to anaerobic control cultures

O. formigenes survival in yogurt and following lyophilization

Survival in yogurt was examined by mixing freshly harvested cells from stationary-phase cultures into vanilla low-fat yogurt with active yogurt cultures. Survival of O. formigenes significantly dropped over 29-day incubation in yogurt (P < 0.01). After two-day incubation in yogurt, 14 % of the bacteria remained, and by day 29, only 0.01 % of the bacteria were viable (Fig. 5a).

Fig. 5.

Fig. 5

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Survival of O. formigenes in yogurt (a) and after lyophilization (b) at 4 °C in the presence of air. Values are mean ± SD. Survival in yogurt was examined after addition of freshly harvested cells from stationary phase. Impact of lyophilization was measured after lyophilization of stationary-phase cultures and rehydration with anaerobic SBO

Following lyophilization of stationary-phase cultures, CFU decreased one log to approximately 10 % of the original viable cell count or ~109 cells (P < 0.01, Fig. 5b). Lyophilized cells were stored in the presence of air at 4 °C and viability measured over 4 weeks. Under these conditions, CFU did not decrease over the 4-week test period (P > 0.05, Fig. 5b).

Discussion

Colonization of the intestine with the specialist oxalate degrading bacterium O. formigenes may prove to be an efficacious method for limiting calcium oxalate stone risk (Kaufman et al. 2008; Knight et al. 2013; Siener et al. 2013). The ability to colonize individuals lacking O. formigenes has previously been addressed by a study in which two healthy adults became colonized following the ingestion of O. formigenes (5 × 1010 O. formigenes cells, spread on a turkey sandwich, with a sodium oxalate load) and were still colonized 9 months later (Duncan et al. 2002). However, a study where O. formigenes was provided either in lyophilized form (~107 colony-forming units) or as a frozen cell paste (~1010 colony-forming units) to patients with Primary Hyperoxaluria resulted in none of the non-colonized patients remaining colonized 2 weeks after treatment (Hoppe et al. 2006). These studies suggest that differences in host traits and possibly the method by which the O. formigenes cells are prepared and delivered are important determinants for successful long-term colonization.

The goals of this in vitro study were to determine (1) the survival rates of the Group 1 O. formigenes strain OxCC13 following lyophilization or storage in yogurt, two processes commonly associated with the manufacture and distribution of probiotics, (2) its tolerance to air, (3) its survival at different pH and (4) its ability to persist in the absence of oxalate. The dependency of O. formigenes on oxalate for growth has the potential to cause dramatic shifts in its population and drive numbers to such low levels where persistence in the intestinal environment would become a significant challenge. However, results from the present study and our findings using ultra-low oxalate diets in a mouse model of O. formigenes (Li et al. 2015) suggest that this organism has the potential to persist for long periods in the absence of oxalate. The ability of O. formigenes to outcompete other bacteria for oxalate may allow it to persist when oxalate levels are very low. The recent publication of the genomic sequence of the O. formigenes strains HOxBLS and OxCC13 reveals a number of proteins that may prolong survival during nutrient starvation (reviewed by Knight et al. 2013). Prolonged starvation has been shown with certain bacteria to result in a “growth advantage in stationary phase,” or GASP, phenotype. The GASP phenotype is caused by stable mutations that confer an advantageous ability to persist during starvation, and it can either replace the parental population (Zambrano et al. 1993) or coexist with it (Rozen et al. 2009). Examining the GASP phenotype in O. formigenes is of interest as it may identify pathways critical to the survival of O. formigenes during periods of oxalate starvation.

Culture medium formate and oxalate measurements by IC indicated that 96 % of carbon from oxalate ends up in formate within 24 h, in keeping with an earlier report (Dawson et al. 1980). Of interest, was the finding that O. formigenes viable cell counts were not significantly different after 6-day incubation in SB media lacking oxalate, where formate levels were <2 mM (Fig. 2), compared to 6-day incubation in SBO media, where formate levels remained at ~100 mM from day 1 through day 6 (Fig. 2). These data suggest that the higher concentrations of media formate compared to SB cultures may not be a significant factor impacting viability under these experimental conditions, although the increased pH in SBO (pH 7.6) versus SB (pH 7.2) may have impacted these results.

Gastric acidity is considered a major barrier to the survival of ingested bacteria in the gut (Bezkorovainy 2001). Bacteria, such as Escherichia coli, Salmonella enterica and Shigella flexeneri, are highly resistant to low pH and can survive for several hours at a pH as low as 2.5 in a stationary phase, mainly owing to various acid resistance mechanisms, such as proton efflux systems and synthesis of alkali products [reviewed in (Liu et al. 2015)]. Only one previous study has examined acid tolerance in O. formigenes (Duncan et al. 2002). Duncan et al. showed no decrease in viability and 60 % loss of viability after 2-h incubation at pH 3.0 and pH 2.0, respectively, of the Group 2 O. formigenes strain, VA1. In contrast, when performing the same tests with the Group 1 O. formigenes strain, HC1, only 1 and 0.1 % of the cells were viable after 2-h incubation at pH 3.0 and pH 2.0, respectively. However, they showed that despite the low acid resistance of Group 1 O. formigenes HC1, two healthy subjects were successfully colonized following ingestion of this strain (~5 × 1010 cells) when spread on a turkey sandwich suggesting that only small numbers of viable cells may have to reach the large intestine in order to colonize. In this present study, we defined the survival of O. formigenes Group 1 strain OxCC13 over a broad range of pH conditions present in the gastrointestinal tract and found this O. formigenes strain to be sensitive to pH < 5.0. These data also showed that O. formigenes Group 1 strain OxCC13 exhibits similar survival at pH 2.0 and 3.0 to that reported for Group 1 O. formigenes HC1 (Duncan et al. 2002). A review of the genomes of both O. formigenes strains, Group 1 OxCC13 and Group 2 HOx-BLS, for genes known to be involved in acid tolerance did not reveal any differences and showed that both strains contain genes associated with the glutaminase and Gad system and the multisubunit F1−F0-ATPase proton pump; two common mechanisms microorganisms employ to survive acidic environments (reviewed in Liu et al. 2015). Our recent proteomic study with Group 1 O. formigenes OxCC13 showed increased expression of L-glutaminase (OFBG_00245, 1.4-fold increase in stationary relative to log-phase growth, P = 0.12) and GadA (OFBG_00246, 2.3-fold increase in stationary relative to log-phase growth, P = 0.11), although these relative increases were not significant. These two proteins have been shown to be important in the Gad-dependent acid resistance system of numerous bacteria. For example, E. coli relies on L-glutamine for acid resistance, which is converted to L-glutamate by L-glutaminase with concomitant release of dissolved ammonia (Lu et al. 2013). O. formigenes may also require L-glutamine to tolerate low pH and warrants further investigation.

Cultures of growing bacteria will contain a small fraction of cells expressing a transient survival or “persister” phenotype. This is consistent with survival rates in this study with pH 3.0, which showed rapid cell death in the first 15 min followed by a slower rate of death. The production of specialized survivor cells has been shown to be dependent on growth stage. For example, it has been shown that the proportion of persister cells is higher in a stationary-phase population, and stationary-phase cultures are more resistant to killing by antibiotics (Leszczynska et al. 2013). Further studies examining O. formigenes persister cells may provide insight into the mechanisms by which O. formigenes survives following stress. The use of stationary-phase O. formigenes cultures, where persister cells may be more common, to colonize individuals lacking O. formigenes could result in more successful colonization rates and warrants further investigation.

Oxalobacter formigenes is considered a strict anaerobe and can only grow in the absence of oxygen. One previous study indicated O. formigenes can tolerate exposure to air (Duncan et al. 2002). While previous studies have not directly tested the aerotolerance of O. formigenes, this was readily observed in buffers gassed with 95 % O2/5 % CO2 where 14C-oxalate was completely degraded by the addition of viable O. formigenes cells at 37 °C (Hatch et al. 2006). Furthermore, Cornelius et al. demonstrated in rats that colonization with O. formigenes primarily occurs through horizontal transmission (Cornelius and Peck 2004), indicating that O. formigenes can tolerate exposure to air. In this study, we built on these initial observations and measured survival rates over time in both shaking and static O. formigenes cultures. Although aerated shaking conditions resulted in rapid death of O. formigenes, there was only a 15 % loss of viability following 24-h incubation under static aerobic conditions. Despite a loss of viability after 24 h, complete degradation of oxalate occurred under static aerobic conditions. The loss of oxalate could be due to both the slow equilibration of oxygen under such static aerobic conditions allowing cells still under anaerobic conditions to degrade oxalate, and lysis of cells releasing oxalate degrading proteins. Many studies have shown that anaerobic bacteria are not uniformly sensitive to oxygen. There is a broad range of oxygen tolerance from extreme sensitivity to those that are able to remain viable in the presence of oxygen for extended periods (Loesche 1969). It is believed that for some anaerobic bacteria, as in aerobic organisms, substantial protection against oxygen toxicity is afforded by the presence of superoxide dismutase and catalase (Hewitt and Morris 1975; Gregory et al. 1978; Rocha et al. 1996). Our recent mass spectrometry (MS)-based shotgun proteomics study of anaerobic O. formigenes cultures found superoxide dismutase to be expressed (Ellis et al. 2016), supporting the hypothesis that this protein may play a role in aerotolerance. However, the draft genomes of both O. formigenes strains do not contain a protein with similar sequence to other bacterial catalases.

Maintaining viability during processing and storage is a critical challenge for commercial production of bacteria for probiotic applications. Freeze drying is a common method for preservation of probiotics. A number of studies have examined the stability of freeze-dried bacteria (Miyamoto-Shinohara et al. 2008; Celik and O’Sullivan 2013) leading to the development of optimal microbial freeze-drying buffers, one of which was tested for this study. This present study demonstrated the resilience of O. formigenes OxCC13 to the freeze-drying process when rehydration of the lyophilized cells and subsequent culture conditions are optimal. A previous study showed that daily ingestion of lyophilized O. formigenes in enteric coated capsules (~107 CFU) effectively lowered urinary oxalate in four out of six primary hyperoxaluria patients (Hoppe et al. 2006). However, none of the non-colonized patients were colonized 2 weeks after treatment suggesting that other factors are required for successful long-term colonization. As the transit time through the gut is about 24 h and resuscitation of freeze-dried bacteria will not be optimal in the gut environment, further studies exploring the colonization potential of freeze-dried preparations of O. formigenes are needed.

Yogurt is a common food supplemented with probiotics, including Lactobacillus and Bifidobacterium species (Fisberg and Machado 2015). Several factors have been shown to impact the viability of probiotic bacteria in yogurt, including acid produced by yogurt bacteria and oxygen permeation through the package (Biavati et al. 1992; Shah 2000; Talwalkar and Kailasapathy 2004). In this study, we tested survival rates of O. formigenes in one brand of yogurt containing both Lactobacillus and Bifidobacterium probiotic strains. Despite the acidic environment of the yogurt (pH 4–4.5) and the presence of air, O. formigenes persisted for 4 weeks at 4 °C, and after 30 days, CFU was ~5 × 104 per gram yogurt. For therapeutic benefits, the minimum level of probiotic bacteria in yogurt has been suggested to be 105–106 viable cells per gram of product (Shah 2000). Further experiments optimizing pH and oxygen content of yogurt should significantly improve O. formigenes viability to within this recommended probiotic dose.

The results presented here only explored the tolerance of one strain of O. formigenes (Group 1 strain OxCC13) to various stress conditions. Duncan et al. (2002) showed a difference in response between a Group 1 and a Group 2 strain to bile acids, pH and air. Therefore, examining more carefully the response of different human O. formigenes strains to various environmental factors is of interest. A recent article provided interesting information regarding frequency of colonization with different O. formigenes strains (Barnett et al. 2015). Their whole genome sequencing results revealed 29 of 94 subjects (31 %) were positive for O. formigenes. All 29 O. formigenes-positive subjects were colonized with sequences represented by Group 1 strains, of whom 17 (59 %) were simultaneously colonized with organisms with sequences represented by Group 2 strains. These data suggest that either colonization with a Group 1 strain was obligatory for Group 2 colonization to occur, Group 1 strains were more readily detected, or possibly Group 1 strains are better “colonizers” than Group 2 strains. Examining whether colonization of the intestine with more than one strain confers greater colonization resilience and/or oxalate degradative capacity is also of interest in light of these data.

In summary, the present work suggests that the Group 1 O. formigenes strain OxCC13 is tolerant to some processes and conditions associated with the manufacture, storage and distribution of probiotic strains. This study also indicates that O. formigenes OxCC13 is sensitive to pH < 5.0 and persists in the presence of air and absence of oxalate, biological properties that warrant more detailed examination. Further work assessing O. formigenes strain differences and colonization potential of processed O. formigenes is needed.

Acknowledgments

This work was supported by National Institutes of Health (NIH) grant DK087967.

Footnotes

Compliance with ethical standards

Conflict of interest None.

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