Gut Pathogens
Assefa et al. Gut Pathog (2015) 7:35 DOI 10.1186/s13099-015-0082-0
Open Access
RESEARCH
Lactobacilli with probiotic potential in the prairie vole (Microtus ochrogaster) Senait Assefa1, Kathleen Ahles1, Simone Bigelow1, J. Thomas Curtis2 and Gerwald A. Köhler1*
Abstract Background: Recent research suggests integration of the intestinal microbiota in gut-brain communication which could lead to new approaches to treat neurological disorders. The highly social prairie voles are an excellent model system to study the effects of environmental factors on social behavior. For future studies on the role of probiotics in ameliorating disorders with social withdrawal symptoms, we report the characterization of intestinal Lactobacillus isolates with probiotic potential from voles. Methods and results: 30 bacterial strains were isolated from the vole intestine and found to be distinct but closely related to Lactobacillus johnsonii using 16S rRNA gene sequencing and Random Amplification of Polymorphic DNA fingerprinting. In vitro characterizations including acid and bile tolerance, antimicrobial effects, antibiotic susceptibility, and adherence to intestinal epithelial cells were performed to assess the probiotic potential of selected strains. Since previous studies revealed that mercury ingestion triggers social deficits in voles, mercury resistance of the probiotic candidates was evaluated which could be an important factor in preventing/treating these behavioral changes. Conclusions: This study demonstrates that lactobacilli with probiotic potential are present in the vole intestine. The Lactobacillus isolates identified in this study will provide a basis for the investigation of probiotic effects in the vole behavioral model system. Keywords: Lactobacillus johnsonii, Prairie vole, Probiotics, RAPD, Intestine, Mercury, Behavior Background Interest in the use of probiotic bacteria to enhance intestinal health in humans and animals has been growing in recent years. Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [1]. At the beginning of the 20th century, the Ukrainian bacteriologist Elie Metchnikoff [2] suggested that health benefits were associated with the ingestion of lactic acid bacteria such a Lactobacillus bulgaricus. At present, many intestinal probiotics belong to the genus Lactobacillus. Lactobacilli are aerotolerant gram-positive bacteria that form an important portion of the normal human microbiotas of the oral cavity [3], gastrointestinal tract [3, 4], and female genitourinary *Correspondence: [email protected] 1 Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, 1111 West 17th Street, Tulsa, OK 74107, USA Full list of author information is available at the end of the article
tract [5–7]. Of the more than 150 [8] known species of lactobacilli, the “acidophilus complex” has received particular attention because of the reported probiotic properties of some members of this subgroup [9]. An example is the species Lactobacillus johnsonii. Several studies reported that L. johnsonii strains isolated from the human intestine undergo processes that are thought to be beneficial to human health, particularly in the areas of immunomodulation, pathogen inhibition, and cell attachment [10, 11]. In addition, accumulating clinical and scientific evidence highlights the important role of probiotic lactobacilli in the bidirectional communication of the gut-brain-axis [12–14]. Studies in mice on L. rhamnosus JB-1 treatment have shown alteration in the central gamma–aminobutyric acid (GABA) expression and modulation of emotional behavior and depression [13]. At present, however, the mechanisms how probiotics such as L. johnsonii could affect brain function are unclear, but proposed mechanisms involve, e.g., the bacterial production of neurotransmitter precursors or of
© 2015 Assefa et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Assefa et al. Gut Pathog (2015) 7:35
chemical compounds that act as hormones or that stimulate vagal afferent pathways [13, 15, 16]. For the past two decades, prairie voles (Microtus ochrogaster) have been the dominant animal model in which to study the formation and maintenance of social affiliations [17, 18] and have been proposed as an important animal model in which to study disorders such as schizophrenia, autism, and the effects of traumatic brain injury, all of which negatively impact social functioning [19, 20]. Prairie vole social behavior has been well-characterized. Field studies show that prairie voles are highly social; pairs share a nest and parental duties and, in fact, both members of a pair often are found in the same trap [21]. In the laboratory, voles appear to avoid isolation by seeking out conspecifics, and in fact, voles suffer significant stress when isolated [22–26]. In contrast to more traditional laboratory animals, prairie vole social behaviors actually are remarkably similar to those of humans, even displaying characteristics such as long-term pair-bonding, care of offspring by both parents, and sharing of a nest even beyond the breeding season [27]. Further, autonomic responses in voles are more like those of humans than they are like those of other rodent species [28]. Importantly, both the behavioral repertoire and the physiology of voles are well documented (e.g., [27, 29–32], so there is a strong literature base upon which additional studies can rest. Given their social structure, prairie voles present an ideal animal model in which to study the of the role of the microbiota-gut-brain-behavior axis in mediating social affiliation and avoidance behaviors, mate choice, parental care and other complex social interactions. The primary objective of this study was to lay the groundwork for probiotic studies in voles by isolating Lactobacillus strains with high probiotic potential from the vole intestine. Host adaption is an important factor for probiosis. Therefore, we chose to isolate vole strains rather than using probiotics originating from humans or other animals. Lactobacilli were isolated using enrichment media and subsequently classified by 16S rRNA gene sequencing which also allowed for PCR-based analyses of Lactobacillus abundance in the vole intestine. Since orally administered probiotics must survive passage through the highly acidic stomach and withstand the adverse intestinal environment, the strains’ acid tolerance and bile resistance were determined. Further characteristics such as antimicrobial activities against fungi and bacteria as well as adhesion to intestinal epithelial cell lines were examined. In addition we included an assessment of the strains’ resistance to mercury chloride. There is evidence that probiotic bacteria could bind many toxic compounds such as aflatoxin B1 [33], cyanotoxins [34], cadmium and lead [35–37] from environmental samples. In this study, the probiotic candidate strains’ resistance to
Page 2 of 16
mercury chloride was also determined because research by Curtis and coworkers [38] revealed social withdrawal symptoms specifically in male voles upon inorganic mercury ingestion. Resistant strains might be more likely to survive mercury exposure and exert beneficial effects on an exposed host organism. All lactobacilli isolated from the vole intestine in this study were closely related to L. johnsonii and several of the isolated strains exhibited potential for probiotic properties.
Results For purposes of characterizing the baseline state of vole gut lactobacilli, we have used same-sex cage mates. This eliminates the potential confounds of stress responses associated with social isolation or endocrine responses associated with reproductive activation, mating, and parental behavior [22–26]. Further research will be needed to assess whether and how the microbiota might change in pair-bonded and/or parental animals. Although these are important questions, they are beyond the scope of this paper, and will be addressed in subsequent studies. Isolation of Lactobacillus strains from the prairie vole intestine
Plating of intestinal content from prairie voles on Lactobacillus enrichment media resulted in the selection of 30 bacterial isolates for further analysis. Sequence analysis of the respective PCR amplicons generated with the well-conserved 16S rRNA gene primers 8F and 1491R revealed distinct but closely related matches (e.g. 98 % at 100 % coverage) with database entries of the 16S rDNA of Lactobacillus johnsonii (Table 1; Additional file 1: Figure S1). The 16S rDNA sequences of strains PV012, PV021, and PV034 also were confirmed by genome sequencing results (see Additional file 1: Figure S1). Strain differentiation by RAPD analysis
Due to their close relatedness, a RAPD typing technique was employed to genetically type the 30 prairie vole Lactobacillus isolates. To systematically examine the genetic fingerprints of the different strains, a set of three previously published RAPD primers (272, 277, and 287; [39]) was evaluated for differentiation of the bacterial strains. Primer 272 (see Table 2) was chosen for further analyses because it delivered the best discriminatory power by reproducibly amplifying five or more random DNA fragments ranging in size from approximately 180 bp to 3000 bp (Fig. 1). Twenty-seven of the 30 isolates share common bands at 175, 375, 1200 and 1500 bp (Fig. 1). In this regard, the RAPD fingerprinting was able to cluster genetically identical strains as well as differentiate distinct strains among the isolates. For instance, multiple
Assefa et al. Gut Pathog (2015) 7:35
Page 3 of 16
Table 1 List of bacterial and fungal strains used in this study Species
Strains
Origin
References
Candida albicans
SC5314
Clinical isolate
[73]
Escherichia coli
NovaBlue Singles
EMD millipore
http://www.emdmillipore.com
Pseudomonas aeruginosa
PA01
Clinical isolate
[74]
Staphylococcus aureus
ATCC 25923
Clinical isolate
http://www.atcc.org
Lactobacillus spp.
PV010–PV019
Vole cecum
This study
Lactobacillus spp.
PV020–PV021
Vole small intestine
This study
Lactobacillus spp.
PV022–PV023
Vole cecum
This study
Lactobacillus spp.
PV024–PV027
Vole colon
This study
Lactobacillus spp.
PV028–PV031
Vole small intestine
This study
Lactobacillus spp.
PV032–PV035
Vole cecum
This study
Lactobacillus spp.
PV036–PV039
Vole colon
This study
Lactobacillus johnsonii
ATCC 33200a
Human blood isolate
http://www.atcc.org
Lactobacillus reuteri
RC-14a
Human vaginal isolate
[75, 76]
Lactobacillus rhamnosus
LGGa
Human fecal isolate
[77]
PV prairie vole isolate a
Probiotic reference strain
Table 2 DNA oligonucleotide primers and hydrolysis probes used in this study Primer
5′-Sequence-3′
References
8F
AGAGTTTGATCM TGGCTCAG ACGGCTACCTTGTT ACGACTT
[65]
GACGGGCGGTGTGTRCA
[78]
1491R RAPD 272 1391R
AGCGGGCCAA
ATGGCTGTCGTCAGCTCGT GKUNI16STaqCCC VIC-AACGAGCGCAAC CC-MGB TGGAAACAGATG TaqLacF CTAATACCG CGTCCATTGTGGAAG TaqLacR ATTCCCT FAM-ACTGAGACACGGC GKLPV16STaq CC-MGB
GK1053F
[65] [39] Adapted from [79] This study [40, 41] Adapted from [40, 41] This study
strains such as PV010, PV014-PV019 or PV011, PV021, PV023, PV024, PV026, PV027, PV031, PV033, PV036, PV038 and PV039 were found to possess identical RAPD fingerprints suggesting that the isolates were identical or if genetic heterogeneity exists among these isolates, it could not be discriminated by RAPD. Overall, RAPD analysis of the 30 isolates revealed nine distinct clusters (Fig. 2). Notably, eight strains (PV012, PV013, PV020, PV029, PV030, PV032, PV034, and PV035) produced patterns with unique PCR bands (Fig. 2). RAPD bands at 1200, 650, 450, and 300 bp are shared with the human L. johnsonii ATCC 33200 strain by 23, 8, 11, and 12 isolates, respectively. In general, the RAPD fingerprinting analysis
was effective for rapid differentiation within the different isolates. L. rhamnosus GG was included as reference strain and showed almost no RAPD pattern similarities to the vole intestinal strains. Abundance of lactobacilli in the prairie vole GI tract
We conducted a comparative survey to estimate the amount of lactobacilli present in male and female vole GI tracts by 16S rRNA-based qPCR. Published Lactobacillus-specific 16S rRNA gene primers were adapted to ensure complementarity with the respective gene sequences of the 30 vole strains, i.e., primer TaqLacR (Table 2) differs in one base from the published oligonucleotide sequence [40, 41]. Additionally, hydrolysis probes were designed for Lactobacillus and broad-range bacterial (primers GK1053F-1391R; Table 2) qPCR assays. These assays allowed for determination of the relative abundance of Lactobacillus 16S rDNA copy numbers in DNA isolated from vole stomachs, proximal and distal small intestines, ceca, and colons (Fig. 3). Interestingly, this assay revealed very high levels of lactobacilli in the stomachs (up to 47 %) and to lesser extend (up to 10 %) in the small intestines of some animals (see Fig. 3). Other animals exhibited far lower Lactobacillus abundance in the upper GI tract. In the distal GI tract (cecum and colon), lactobacilli appear to be generally less prevalent, accounting for less than 1 % of the total 16S rRNA. No statistically significant differences (ANOVA) were found in Lactobacillus abundance between the tested males and females at the respective gastrointestinal sites. For purposes of characterizing the baseline state of vole gut microbiota, we have used same-sex cage mates. This
bp
LGG
PV031 PV032 PV033 PV034 PV035 PV036 PV037 PV038 PV039
M
PV030
PV020
M
PV021 PV022 PV023 PV024 PV025 PV026 PV027 PV028 PV029 Lj
PV017
Page 4 of 16
PV018 PV019 Lj
M
PV010 PV011 PV012 PV013 PV014 PV015 PV016
Assefa et al. Gut Pathog (2015) 7:35
3000 1500 1000 500 100
Fig. 1 Random amplified polymorphic DNA (RAPD) analysis of 30 prairie vole Lactobacillus isolates. Amplified fragment patterns for RAPD primer 272 (see text) are shown after electrophoresis on 1.5 % agarose gels. PV010-PV039: Prairie vole Lactobacillus strains PV010-PV039; Lj: L. johnsonii ATCC 33200; LGG: L. rhamnosus GG. M New England BioLabs 2-log DNA ladder
Fig. 2 Random amplified polymorphic DNA (RAPD) analysis of 30 prairie vole Lactobacillus isolates. Dendrogram of the cluster analysis of RAPD results using the DendroUPGMA program (see “Methods”). Putative clustering is indicated with roman numerals. Strains with the overall best performance in this study are boxed in red
Assefa et al. Gut Pathog (2015) 7:35
Page 5 of 16
% growth after incubation
a
150
100
50
7
3
2
1
0
Incubation pH for 2h
eliminates the potential confounds of stress responses associated with social isolation or endocrine responses associated with reproductive activation, mating, and parental behavior [6–10]. Further research will be needed to assess whether and how the microbiota might change in pair-bonded and/or parental animals. Although these are important questions, they are beyond the scope of this paper, and will be addressed in subsequent studies.
150
100
50
7
3
2
0
1
Fig. 3 Relative abundance of lactobacilli in the GI tract of prairie voles. As indicator for the amount of lactobacilli in the vole GI tract, qPCR assays using group-specific and universal primers in conjunction with hydrolysis probes (see Table 2) were conducted to determine the relative abundance of Lactobacillus rRNA gene copies in content samples from the vole stomach, proximal small intestine (PSI), distal small intestine (DSI), cecum, and colon. Percent abundance values for five female (ring symbols) and seven male animals (solid symbols) are depicted on a logarithmic scale. Individual animals are represented by a specific symbol-color combination. Experiments were performed at least in duplicate. The horizontal bars indicate the geometric means of the abundance at the indicated sites for the twelve animals
% growth after incubation
b
Incubation pH for 4h Fig. 4 Acid tolerance of vole intestinal Lactobacillus isolates. Lactobacilli were incubated for 2 h (a) and 4 h (b) at various pH levels (pH 1, pH 2, pH 3, and pH 7) in PBS. Subsequently, the bacteria were inoculated in MRS and growth was determined after 24 h by OD600nm measurement. Results are shown for the 17 most acid-tolerant Lactobacillus isolates as percent growth relative to growth after incubation at pH 7 (set to 100 %). L. johnsonii ATCC 33200 (green data points) and L. rhamnosus GG (red data points) were included as reference strains. While all strains tolerated prolonged incubation at pH 3 well, the depicted 17 strains survived pH 2 and some even pH 1. The reference strains appear to be more acid tolerant at pH 1 than the prairie vole strains. Data points are mean values from three experiments with duplicate measurements
Acid tolerance of isolated Lactobacillus strains
The 30 vole intestinal Lactobacillus strains were screened for tolerance to strongly acidic conditions. Nearly all strains survived an incubation period of 4 h at pH 3, but only 17 strains were able to exhibit greater than 50 % growth at this pH level (Fig. 4). Data of strains which did not perform well are not shown. Figure 4 shows percent growth calculated from acid resistance assays at pH 1–3 during various incubation periods. The results indicated that the 17 selected strains survived during the 4 h incubation with some reduction in growth (20–44 %) compared to the control at pH 7. Although greater than 50 % of growth was suppressed, 12 of 17 strains survived pH
2 and pH 1 during the 4 h incubation. In general, little or no growth occurred in strains PV010, PV019, PV022, and PV037 following a 2 h incubation at pH 1. The results show that the acid tolerance of the investigated strains was variable, but comparable to the probiotic reference strains L. johnsonii ATCC 33200 and L. rhamnosus GG (Fig. 4). Overall, L. johnsonii ATCC 33200 appeared to be the most acid resistant strain. Resistance to bile and the bile acid taurocholate
Intestinal survival requires resistance to the antimicrobial components of bile. Therefore, the strains’ susceptibility to
Assefa et al. Gut Pathog (2015) 7:35
Page 6 of 16
bile and the bile acid taurocholate was examined. Among the 30 isolated strains only 10 (PV011–PV014, PV017– PV019, PV021, PV024, and PV039) were resistant to high bile concentrations (0.5–8 %) within 24 h of exposure. Strain PV012 appeared to be the most resistant among these strains (Table 3) with an IC50 value of 4.2 % bile (comparable to human L. johnsonii ATCC 33200), whereas Table 3 H2O2 production and IC50 values of human and vole Lactobacillus strains for bovine bile, taurocholate, and HgCl2 Strain
H2Oa2
b
Bovine bile (%)b
Taurocholate (mmol/L)b
HgCl2 (mmol/L)
PV010 −
24 h
48 h
14
0.1 ± 0.01 0.1 ± 0.01
PV012 −
4.2 ± 0.3
>14
0.2 ± 0.01 0.2 ± 0.04
PV013 −
1.6 ± 0.1
>14
0.1 ± 0.03 0.1 ± 0.03
PV014 −
1.8 ± 0.8
>14
0.1 ± 0.03 0.1 ± 0.03
PV015 −
0.8 ± 0.4
>14
0.1 ± 0.03 0.2 ± 0.23
PV016 −
14
0.2 ± 0.07 0.2 ± 0.10
PV018 −
3.3 ± 0.7
>14
0.2 ± 0.07 0.2 ± 0.11
PV019 −
3.6 ± 0.7
>14
0.1 ± 0.03 0.2 ± 0.13
PV020 −
14
0.2 ± 0.22 0.2 ± 0.22
PV021 −
0.4 ± 0.3
14
0.1 ± 0.10 0.2 ± 0.22
PV022 −
0.2 ± 0.1
9 ± 2.3
0.2 ± 0.06 0.2 ± 0.24
PV023 −
0.2 ± 0.1
10 ± 3.5
0.1 ± 0.12 0.2 ± 0.15
PV024 −
1.4 ± 0.5
14
0.1 ± 0.05 0.1 ± 0.09
PV025 +
Assefa et al. Gut Pathog (2015) 7:35 DOI 10.1186/s13099-015-0082-0
Open Access
RESEARCH
Lactobacilli with probiotic potential in the prairie vole (Microtus ochrogaster) Senait Assefa1, Kathleen Ahles1, Simone Bigelow1, J. Thomas Curtis2 and Gerwald A. Köhler1*
Abstract Background: Recent research suggests integration of the intestinal microbiota in gut-brain communication which could lead to new approaches to treat neurological disorders. The highly social prairie voles are an excellent model system to study the effects of environmental factors on social behavior. For future studies on the role of probiotics in ameliorating disorders with social withdrawal symptoms, we report the characterization of intestinal Lactobacillus isolates with probiotic potential from voles. Methods and results: 30 bacterial strains were isolated from the vole intestine and found to be distinct but closely related to Lactobacillus johnsonii using 16S rRNA gene sequencing and Random Amplification of Polymorphic DNA fingerprinting. In vitro characterizations including acid and bile tolerance, antimicrobial effects, antibiotic susceptibility, and adherence to intestinal epithelial cells were performed to assess the probiotic potential of selected strains. Since previous studies revealed that mercury ingestion triggers social deficits in voles, mercury resistance of the probiotic candidates was evaluated which could be an important factor in preventing/treating these behavioral changes. Conclusions: This study demonstrates that lactobacilli with probiotic potential are present in the vole intestine. The Lactobacillus isolates identified in this study will provide a basis for the investigation of probiotic effects in the vole behavioral model system. Keywords: Lactobacillus johnsonii, Prairie vole, Probiotics, RAPD, Intestine, Mercury, Behavior Background Interest in the use of probiotic bacteria to enhance intestinal health in humans and animals has been growing in recent years. Probiotics are “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” [1]. At the beginning of the 20th century, the Ukrainian bacteriologist Elie Metchnikoff [2] suggested that health benefits were associated with the ingestion of lactic acid bacteria such a Lactobacillus bulgaricus. At present, many intestinal probiotics belong to the genus Lactobacillus. Lactobacilli are aerotolerant gram-positive bacteria that form an important portion of the normal human microbiotas of the oral cavity [3], gastrointestinal tract [3, 4], and female genitourinary *Correspondence: [email protected] 1 Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, 1111 West 17th Street, Tulsa, OK 74107, USA Full list of author information is available at the end of the article
tract [5–7]. Of the more than 150 [8] known species of lactobacilli, the “acidophilus complex” has received particular attention because of the reported probiotic properties of some members of this subgroup [9]. An example is the species Lactobacillus johnsonii. Several studies reported that L. johnsonii strains isolated from the human intestine undergo processes that are thought to be beneficial to human health, particularly in the areas of immunomodulation, pathogen inhibition, and cell attachment [10, 11]. In addition, accumulating clinical and scientific evidence highlights the important role of probiotic lactobacilli in the bidirectional communication of the gut-brain-axis [12–14]. Studies in mice on L. rhamnosus JB-1 treatment have shown alteration in the central gamma–aminobutyric acid (GABA) expression and modulation of emotional behavior and depression [13]. At present, however, the mechanisms how probiotics such as L. johnsonii could affect brain function are unclear, but proposed mechanisms involve, e.g., the bacterial production of neurotransmitter precursors or of
© 2015 Assefa et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Assefa et al. Gut Pathog (2015) 7:35
chemical compounds that act as hormones or that stimulate vagal afferent pathways [13, 15, 16]. For the past two decades, prairie voles (Microtus ochrogaster) have been the dominant animal model in which to study the formation and maintenance of social affiliations [17, 18] and have been proposed as an important animal model in which to study disorders such as schizophrenia, autism, and the effects of traumatic brain injury, all of which negatively impact social functioning [19, 20]. Prairie vole social behavior has been well-characterized. Field studies show that prairie voles are highly social; pairs share a nest and parental duties and, in fact, both members of a pair often are found in the same trap [21]. In the laboratory, voles appear to avoid isolation by seeking out conspecifics, and in fact, voles suffer significant stress when isolated [22–26]. In contrast to more traditional laboratory animals, prairie vole social behaviors actually are remarkably similar to those of humans, even displaying characteristics such as long-term pair-bonding, care of offspring by both parents, and sharing of a nest even beyond the breeding season [27]. Further, autonomic responses in voles are more like those of humans than they are like those of other rodent species [28]. Importantly, both the behavioral repertoire and the physiology of voles are well documented (e.g., [27, 29–32], so there is a strong literature base upon which additional studies can rest. Given their social structure, prairie voles present an ideal animal model in which to study the of the role of the microbiota-gut-brain-behavior axis in mediating social affiliation and avoidance behaviors, mate choice, parental care and other complex social interactions. The primary objective of this study was to lay the groundwork for probiotic studies in voles by isolating Lactobacillus strains with high probiotic potential from the vole intestine. Host adaption is an important factor for probiosis. Therefore, we chose to isolate vole strains rather than using probiotics originating from humans or other animals. Lactobacilli were isolated using enrichment media and subsequently classified by 16S rRNA gene sequencing which also allowed for PCR-based analyses of Lactobacillus abundance in the vole intestine. Since orally administered probiotics must survive passage through the highly acidic stomach and withstand the adverse intestinal environment, the strains’ acid tolerance and bile resistance were determined. Further characteristics such as antimicrobial activities against fungi and bacteria as well as adhesion to intestinal epithelial cell lines were examined. In addition we included an assessment of the strains’ resistance to mercury chloride. There is evidence that probiotic bacteria could bind many toxic compounds such as aflatoxin B1 [33], cyanotoxins [34], cadmium and lead [35–37] from environmental samples. In this study, the probiotic candidate strains’ resistance to
Page 2 of 16
mercury chloride was also determined because research by Curtis and coworkers [38] revealed social withdrawal symptoms specifically in male voles upon inorganic mercury ingestion. Resistant strains might be more likely to survive mercury exposure and exert beneficial effects on an exposed host organism. All lactobacilli isolated from the vole intestine in this study were closely related to L. johnsonii and several of the isolated strains exhibited potential for probiotic properties.
Results For purposes of characterizing the baseline state of vole gut lactobacilli, we have used same-sex cage mates. This eliminates the potential confounds of stress responses associated with social isolation or endocrine responses associated with reproductive activation, mating, and parental behavior [22–26]. Further research will be needed to assess whether and how the microbiota might change in pair-bonded and/or parental animals. Although these are important questions, they are beyond the scope of this paper, and will be addressed in subsequent studies. Isolation of Lactobacillus strains from the prairie vole intestine
Plating of intestinal content from prairie voles on Lactobacillus enrichment media resulted in the selection of 30 bacterial isolates for further analysis. Sequence analysis of the respective PCR amplicons generated with the well-conserved 16S rRNA gene primers 8F and 1491R revealed distinct but closely related matches (e.g. 98 % at 100 % coverage) with database entries of the 16S rDNA of Lactobacillus johnsonii (Table 1; Additional file 1: Figure S1). The 16S rDNA sequences of strains PV012, PV021, and PV034 also were confirmed by genome sequencing results (see Additional file 1: Figure S1). Strain differentiation by RAPD analysis
Due to their close relatedness, a RAPD typing technique was employed to genetically type the 30 prairie vole Lactobacillus isolates. To systematically examine the genetic fingerprints of the different strains, a set of three previously published RAPD primers (272, 277, and 287; [39]) was evaluated for differentiation of the bacterial strains. Primer 272 (see Table 2) was chosen for further analyses because it delivered the best discriminatory power by reproducibly amplifying five or more random DNA fragments ranging in size from approximately 180 bp to 3000 bp (Fig. 1). Twenty-seven of the 30 isolates share common bands at 175, 375, 1200 and 1500 bp (Fig. 1). In this regard, the RAPD fingerprinting was able to cluster genetically identical strains as well as differentiate distinct strains among the isolates. For instance, multiple
Assefa et al. Gut Pathog (2015) 7:35
Page 3 of 16
Table 1 List of bacterial and fungal strains used in this study Species
Strains
Origin
References
Candida albicans
SC5314
Clinical isolate
[73]
Escherichia coli
NovaBlue Singles
EMD millipore
http://www.emdmillipore.com
Pseudomonas aeruginosa
PA01
Clinical isolate
[74]
Staphylococcus aureus
ATCC 25923
Clinical isolate
http://www.atcc.org
Lactobacillus spp.
PV010–PV019
Vole cecum
This study
Lactobacillus spp.
PV020–PV021
Vole small intestine
This study
Lactobacillus spp.
PV022–PV023
Vole cecum
This study
Lactobacillus spp.
PV024–PV027
Vole colon
This study
Lactobacillus spp.
PV028–PV031
Vole small intestine
This study
Lactobacillus spp.
PV032–PV035
Vole cecum
This study
Lactobacillus spp.
PV036–PV039
Vole colon
This study
Lactobacillus johnsonii
ATCC 33200a
Human blood isolate
http://www.atcc.org
Lactobacillus reuteri
RC-14a
Human vaginal isolate
[75, 76]
Lactobacillus rhamnosus
LGGa
Human fecal isolate
[77]
PV prairie vole isolate a
Probiotic reference strain
Table 2 DNA oligonucleotide primers and hydrolysis probes used in this study Primer
5′-Sequence-3′
References
8F
AGAGTTTGATCM TGGCTCAG ACGGCTACCTTGTT ACGACTT
[65]
GACGGGCGGTGTGTRCA
[78]
1491R RAPD 272 1391R
AGCGGGCCAA
ATGGCTGTCGTCAGCTCGT GKUNI16STaqCCC VIC-AACGAGCGCAAC CC-MGB TGGAAACAGATG TaqLacF CTAATACCG CGTCCATTGTGGAAG TaqLacR ATTCCCT FAM-ACTGAGACACGGC GKLPV16STaq CC-MGB
GK1053F
[65] [39] Adapted from [79] This study [40, 41] Adapted from [40, 41] This study
strains such as PV010, PV014-PV019 or PV011, PV021, PV023, PV024, PV026, PV027, PV031, PV033, PV036, PV038 and PV039 were found to possess identical RAPD fingerprints suggesting that the isolates were identical or if genetic heterogeneity exists among these isolates, it could not be discriminated by RAPD. Overall, RAPD analysis of the 30 isolates revealed nine distinct clusters (Fig. 2). Notably, eight strains (PV012, PV013, PV020, PV029, PV030, PV032, PV034, and PV035) produced patterns with unique PCR bands (Fig. 2). RAPD bands at 1200, 650, 450, and 300 bp are shared with the human L. johnsonii ATCC 33200 strain by 23, 8, 11, and 12 isolates, respectively. In general, the RAPD fingerprinting analysis
was effective for rapid differentiation within the different isolates. L. rhamnosus GG was included as reference strain and showed almost no RAPD pattern similarities to the vole intestinal strains. Abundance of lactobacilli in the prairie vole GI tract
We conducted a comparative survey to estimate the amount of lactobacilli present in male and female vole GI tracts by 16S rRNA-based qPCR. Published Lactobacillus-specific 16S rRNA gene primers were adapted to ensure complementarity with the respective gene sequences of the 30 vole strains, i.e., primer TaqLacR (Table 2) differs in one base from the published oligonucleotide sequence [40, 41]. Additionally, hydrolysis probes were designed for Lactobacillus and broad-range bacterial (primers GK1053F-1391R; Table 2) qPCR assays. These assays allowed for determination of the relative abundance of Lactobacillus 16S rDNA copy numbers in DNA isolated from vole stomachs, proximal and distal small intestines, ceca, and colons (Fig. 3). Interestingly, this assay revealed very high levels of lactobacilli in the stomachs (up to 47 %) and to lesser extend (up to 10 %) in the small intestines of some animals (see Fig. 3). Other animals exhibited far lower Lactobacillus abundance in the upper GI tract. In the distal GI tract (cecum and colon), lactobacilli appear to be generally less prevalent, accounting for less than 1 % of the total 16S rRNA. No statistically significant differences (ANOVA) were found in Lactobacillus abundance between the tested males and females at the respective gastrointestinal sites. For purposes of characterizing the baseline state of vole gut microbiota, we have used same-sex cage mates. This
bp
LGG
PV031 PV032 PV033 PV034 PV035 PV036 PV037 PV038 PV039
M
PV030
PV020
M
PV021 PV022 PV023 PV024 PV025 PV026 PV027 PV028 PV029 Lj
PV017
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PV018 PV019 Lj
M
PV010 PV011 PV012 PV013 PV014 PV015 PV016
Assefa et al. Gut Pathog (2015) 7:35
3000 1500 1000 500 100
Fig. 1 Random amplified polymorphic DNA (RAPD) analysis of 30 prairie vole Lactobacillus isolates. Amplified fragment patterns for RAPD primer 272 (see text) are shown after electrophoresis on 1.5 % agarose gels. PV010-PV039: Prairie vole Lactobacillus strains PV010-PV039; Lj: L. johnsonii ATCC 33200; LGG: L. rhamnosus GG. M New England BioLabs 2-log DNA ladder
Fig. 2 Random amplified polymorphic DNA (RAPD) analysis of 30 prairie vole Lactobacillus isolates. Dendrogram of the cluster analysis of RAPD results using the DendroUPGMA program (see “Methods”). Putative clustering is indicated with roman numerals. Strains with the overall best performance in this study are boxed in red
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% growth after incubation
a
150
100
50
7
3
2
1
0
Incubation pH for 2h
eliminates the potential confounds of stress responses associated with social isolation or endocrine responses associated with reproductive activation, mating, and parental behavior [6–10]. Further research will be needed to assess whether and how the microbiota might change in pair-bonded and/or parental animals. Although these are important questions, they are beyond the scope of this paper, and will be addressed in subsequent studies.
150
100
50
7
3
2
0
1
Fig. 3 Relative abundance of lactobacilli in the GI tract of prairie voles. As indicator for the amount of lactobacilli in the vole GI tract, qPCR assays using group-specific and universal primers in conjunction with hydrolysis probes (see Table 2) were conducted to determine the relative abundance of Lactobacillus rRNA gene copies in content samples from the vole stomach, proximal small intestine (PSI), distal small intestine (DSI), cecum, and colon. Percent abundance values for five female (ring symbols) and seven male animals (solid symbols) are depicted on a logarithmic scale. Individual animals are represented by a specific symbol-color combination. Experiments were performed at least in duplicate. The horizontal bars indicate the geometric means of the abundance at the indicated sites for the twelve animals
% growth after incubation
b
Incubation pH for 4h Fig. 4 Acid tolerance of vole intestinal Lactobacillus isolates. Lactobacilli were incubated for 2 h (a) and 4 h (b) at various pH levels (pH 1, pH 2, pH 3, and pH 7) in PBS. Subsequently, the bacteria were inoculated in MRS and growth was determined after 24 h by OD600nm measurement. Results are shown for the 17 most acid-tolerant Lactobacillus isolates as percent growth relative to growth after incubation at pH 7 (set to 100 %). L. johnsonii ATCC 33200 (green data points) and L. rhamnosus GG (red data points) were included as reference strains. While all strains tolerated prolonged incubation at pH 3 well, the depicted 17 strains survived pH 2 and some even pH 1. The reference strains appear to be more acid tolerant at pH 1 than the prairie vole strains. Data points are mean values from three experiments with duplicate measurements
Acid tolerance of isolated Lactobacillus strains
The 30 vole intestinal Lactobacillus strains were screened for tolerance to strongly acidic conditions. Nearly all strains survived an incubation period of 4 h at pH 3, but only 17 strains were able to exhibit greater than 50 % growth at this pH level (Fig. 4). Data of strains which did not perform well are not shown. Figure 4 shows percent growth calculated from acid resistance assays at pH 1–3 during various incubation periods. The results indicated that the 17 selected strains survived during the 4 h incubation with some reduction in growth (20–44 %) compared to the control at pH 7. Although greater than 50 % of growth was suppressed, 12 of 17 strains survived pH
2 and pH 1 during the 4 h incubation. In general, little or no growth occurred in strains PV010, PV019, PV022, and PV037 following a 2 h incubation at pH 1. The results show that the acid tolerance of the investigated strains was variable, but comparable to the probiotic reference strains L. johnsonii ATCC 33200 and L. rhamnosus GG (Fig. 4). Overall, L. johnsonii ATCC 33200 appeared to be the most acid resistant strain. Resistance to bile and the bile acid taurocholate
Intestinal survival requires resistance to the antimicrobial components of bile. Therefore, the strains’ susceptibility to
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bile and the bile acid taurocholate was examined. Among the 30 isolated strains only 10 (PV011–PV014, PV017– PV019, PV021, PV024, and PV039) were resistant to high bile concentrations (0.5–8 %) within 24 h of exposure. Strain PV012 appeared to be the most resistant among these strains (Table 3) with an IC50 value of 4.2 % bile (comparable to human L. johnsonii ATCC 33200), whereas Table 3 H2O2 production and IC50 values of human and vole Lactobacillus strains for bovine bile, taurocholate, and HgCl2 Strain
H2Oa2
b
Bovine bile (%)b
Taurocholate (mmol/L)b
HgCl2 (mmol/L)
PV010 −
24 h
48 h
14
0.1 ± 0.01 0.1 ± 0.01
PV012 −
4.2 ± 0.3
>14
0.2 ± 0.01 0.2 ± 0.04
PV013 −
1.6 ± 0.1
>14
0.1 ± 0.03 0.1 ± 0.03
PV014 −
1.8 ± 0.8
>14
0.1 ± 0.03 0.1 ± 0.03
PV015 −
0.8 ± 0.4
>14
0.1 ± 0.03 0.2 ± 0.23
PV016 −
14
0.2 ± 0.07 0.2 ± 0.10
PV018 −
3.3 ± 0.7
>14
0.2 ± 0.07 0.2 ± 0.11
PV019 −
3.6 ± 0.7
>14
0.1 ± 0.03 0.2 ± 0.13
PV020 −
14
0.2 ± 0.22 0.2 ± 0.22
PV021 −
0.4 ± 0.3
14
0.1 ± 0.10 0.2 ± 0.22
PV022 −
0.2 ± 0.1
9 ± 2.3
0.2 ± 0.06 0.2 ± 0.24
PV023 −
0.2 ± 0.1
10 ± 3.5
0.1 ± 0.12 0.2 ± 0.15
PV024 −
1.4 ± 0.5
14
0.1 ± 0.05 0.1 ± 0.09
PV025 +
