Characterisation of the bacterial microbiota of the vagina of dairy cows and isolation of pediocin-producing Pediococcus acidilactici
© Wang et al.; licensee BioMed Central Ltd. 2013
Received: 2 March 2012
Accepted: 21 January 2013
Published: 29 January 2013
Uterine infections in dairy cows lower profitability of dairy operations. Infections of the reproductive tract are related to the overgrowth of pathogenic bacteria during the first three weeks after parturition. However, alterations in the vaginal microbiota composition in the first weeks after parturition remain poorly documented.
In this study, bacteria isolated from the vagina of healthy pregnant, and infected postpartum cows were characterised by random amplification of polymorphic DNA (RAPD) analysis and partial 16S ribosomal RNA (rDNA) gene sequencing. Populations of bacilli and lactic acid bacteria of the genera Enterococcus, Lactobacillus, and Pediococcus were present in both healthy and infected cows. Infected cows had a significant increase in the vaginal enteric bacteria population which consisted mainly of Escherichia coli. Three E. coli isolates harboured the gene coding for Shiga-like-toxin (SLT) I or II. Several isolates of the Pediococcus acidilactici were found to produce the bacteriocin pediocin AcH/PA-1. Quantitative PCR analyses of vaginal mucus samples collected from ten metritic cows before and after parturition confirmed the presence of the Lactobacillus group (Lactobacillus spp., Pediococcus spp., Leuconostoc spp., and Weissella spp.); Enterobacteriaceae, E. coli, and bacilli. The presence of the pediocin AcH/PA-1 structural gene and SLT genes were also confirmed with qPCR.
In conclusion, overgrowth of pathogenic bacteria, particularly E. coli, after parturition likely contributes to the development of metritis. Our microbiota analysis extends the information related to the composition of commensal bacteria in the bovine female reproductive tract and may facilitate the development of novel intervention strategies for prevention of uterine infections in dairy cows.
KeywordsLactic acid bacteria Dairy cow Metritis Pediocin PA-1/AcH Shiga-like toxin
Infection of the uterus has a significant impact on the profitability of the dairy industry because of lowered reproductive efficiency, decreased milk production, and increased costs associated with treatment and culling of animals due to infertility [1–3]. Uterine infections in dairy cows are associated with predisposing factors including calving difficulty, retained placenta, compromised immune status and parity, along with the overgrowth of pathogenic microorganisms in the reproductive tract . Immediately after calving, the dilated state of the cervix allows microorganisms from the environment, cow’s skin, and fecal material to enter through the vagina into the uterus and initiate inflammation of the endometrium, which is highly associated with infertility . Metritis associated bacteria have been classified as pathogens, potential pathogens, or opportunistic pathogens [6, 7]. Recognised uterine pathogens that are associated with severe endometrial inflammation and clinical endometritis include Escherichia coli, Arcanobacterium pyogenes, Fusobacterium necrophorum, Prevotella melaninogenica and Proteus species [6, 7]. Williams et al.  considered high cell counts of E. coli as the basis for the onset of uterine infection.
In a healthy female reproductive tract of humans, mice, or monkeys, lactobacilli are among the predominant organisms [9–11]. Vaginal lactobacilli inhibit the growth of genitourinary pathogenic micro-organisms through mechanisms of competitive exclusion of pathogens, stimulation of the host immune system, and production of specific antibacterial compounds such as acetic and lactic acids, hydrogen peroxide, and antimicrobial peptides [12, 13]. A contribution of bacteriocin production by vaginal probiotics to probiotic activity has not been demonstrated experimentally, but formation of the bacteriocin Abp118 by Lactobacillus salivarius UC118 conferred resistance to infection by Listeria monocytogenes in mice .
The microbial flora of a healthy bovine reproductive tract consists of a combination of aerobic, facultatively anaerobic, and obligately anaerobic microorganisms. Lactobacilli were found to be present in low numbers in the bovine vaginal microbiota ; additionally, Enterobacteriaceae are among the dominant populations . However, alterations in the vaginal microbiota composition in the first weeks after parturition, i.e. the time during which metritis develops, remain poorly documented. The aim of our study is to characterize the vaginal microbiota of both healthy pregnant and infected post-partum cows by culture-dependent analysis. In addition, retrospective culture independent quantitative PCR (qPCR) analysis was used to characterize the vaginal microbiota of metritic cows two weeks before and two weeks calving. Isolates were studied with regards to Shiga-like toxin and pediocin production.
Composition of microbiota in healthy and infected dairy cows: Isolation and identification of bacterial species
Qualitative characterization of the vaginal microbiota of dairy cows
% Identity to Type Strain(a)
Shiga -like Toxin Gene
Pediocin Immunity Gene
0.980 (Shigella flexneri)
0.976 (Shigella flexneri)
0.978 (Shigella flexneri)
Staphylococcus hominis subsp. hominis
0.987 (Shigella flexneri / Escherichia fergusonii)
0.980 (Shigella flexneri / Escherichia fergusonii)
0.973 (Escherichia fergusonii)
0.960 (Shigella flexneri)
Quantification of bacterial groups, SLT and pediocin structural genes
This study provides a comparison of the vaginal microbiota of healthy, pregnant dairy cows, and infected postpartum cows. In contrast to the stable commensal microbiota observed in humans and other mammals [9–11], total bacterial numbers in vaginal mucus were low and the composition of the bovine vaginal microbiota on species level was highly variable. Bacteria found within the microbiota are thus likely to be contaminants from the environment (Bacillus spp.), the cow’s skin (Staphylococcus spp.), or faecal material (E. coli, lactic acid bacteria), rather than representing a stable flora autochthonous to the reproductive tract. The lack of a competitive commensal vaginal microbiota may contribute to the susceptibility of dairy cows to bacterial overgrowth and metritis after parturition [8, 17]. Indeed, quantitative PCR demonstrated a substantial increase of bacterial numbers, particularly of Enterobacteriaceae and E. coli, in infected cows after parturition compared to samples from the same animals obtained pre-partum.
Overall, our data indicated that vaginal bacterial flora in cows affected by metritis was dominated by strains of E. coli, supporting previous observations . This study extends previous results [15, 16] by documenting changes of the vaginal microbiota in individual animals in the first two weeks after calving. Both the Enterobacteriaceae and E. coli showed marked increase in mucus samples collected from infected postpartum cows. The amplification of Shigella rDNA with E. coli species-specific primers is not surprising because Shigella spp. and E. coli are indistinguishable on the basis of rDNA sequences . In keeping with the recognition of Shigella spp. as human-adapted pathovar of E. coli, all isolates were identified as E. coli by biochemical tests. Culture-based analysis and qPCR demonstrated presence of shiga-like-toxin producing E. coli (STEC) in both healthy and infected animals. Three out of eleven E. coli isolates were found to carry genes coding for SLT-1 or SLT-II. Moreover, SLT-genes were consistently detected by qPCR in samples from metritic cows; STEC accounted for about 1 – 10% of the total E. coli population. SLT production causes diarrhoea in calves , but the role of STEC in the pathogenesis of metritis in adult animals warrants further clarification.
Bacilli are present in the environment and they frequently contaminate the bovine uterine lumen . However, pediococci have not yet been described as part of the bovine vaginal microbiota. The genus Pediococcus is closely related to the genus Lactobacillus. Pediococci produce antimicrobial compounds such as organic acids, hydrogen peroxide, and antimicrobial peptides such as pediocin AcH/PA-1 . Ped. acidilactici is a food fermenting organism  but was also isolated from the gastrointestinal tract of poultry, ducks, and sheep[22–24]. Pediocin AcH/PA-1 producing strains have been isolated from human infant faeces .
The synthesis of pediocin AcH/PA-1 was initially described for the strains Ped. acidilactici PAC1.0 and Ped. acidilactici H, but synthesis has also been observed in other Ped. acidilactici strains as well as Lactobacillus plantarum WHE92, Pediococcus parvulus ATO34, and ATO77 [26–28]. Pediocin AcH/PA-1 production is a plasmid-borne trait . The pediocin AcH/PA-1 operon consists of pediocin AcH/PA-1 gene (pedA/papA), a specific immunity gene (papB), and genes responsible for processing and secretion (papC and papD) . In keeping with prior reports on pediocin activity , pediocin was not active against E. coli, the dominant organisms in the vaginal microbiota of infected animals. Pediocin producing isolates characterized in this study harboured the pediocin AcH/PA-1 operon, and qPCR analysis consistently detected the operon in both prepartum and postpartum vaginal samples.
Bacteriocin formation is increasingly recognized as an important trait of probiotic cultures . Studies on the isolation of bacteriocin-producing lactic acid bacteria from the human vagina demonstrated their antimicrobial activities against human vaginal pathogens [33, 34]. Bacteriocin-producing Lactobacillus strains inhibited vaginal pathogens including Gardnerella vaginalis and Pseudomonas aeroginosa. Although bovine vaginal microbiota have much lower total cell counts and lactobacilli populations in comparison to the human vaginal microbiota [16, 36], bacteriocin such as pediocin may influence the microbial ecology in the reproductive tract of dairy cattle if bacteriocin-producing lactic acid bacteria are administered in high numbers.
In conclusion, culture-dependent analysis of the bacterial vaginal microbiota of dairy cows, supported by qPCR analysis, allowed the characterization of the bovine vaginal microbiota of healthy pregnant and infected postpartum cows. Identification of pediocin-producing pediococci in the bovine vaginal microbiota may allow the development of novel prophylactic interventions against metritis by application of bacteriocin-producing probiotic bacteria into the vaginal tract of dairy cows.
In a first experiment, fifteen lactating Holstein dairy cows were used to characterize the vaginal microbiota of healthy pregnant and metritic postpartum cows. In a second experiment, ten animals were selected to characterize the vaginal microbiota of metritic cows two weeks before calving and two weeks after calving. Samples from these ten animals were selected retrospectively after diagnosis of metritis among a group of 40 dairy cows. All animals were maintained at the Dairy Research and Technology Centre of the University of Alberta. Metritis or uterine infections were diagnosed on the basis of criteria established by Sheldon et al. . Primarily, cows with watery reddish-brown, purulent, or mucopurulent discharges with fetid odour were considered to have metritis. Rectal temperatures of 39.5°C or higher and impaired general condition as expressed in a lowered feed intake or milk production were also taken into consideration for diagnosis. Ethics approval was obtained from the Animal care and Use Committee for Livestock of the Faculty of Agricultural, Life and Environmental Sciences (University of Alberta protocol #A5070-01).
For culture-dependent analyses in experiment 1, vaginal swab samples were obtained from seven healthy pregnant cows and eight infected post-partum cows. The vulvar area was thoroughly cleaned with water and then disinfected with 30% (vol/vol) iodine solution (Iosan, WestAgro, Saint Laurent, Canada) prior to sampling. A stainless steel vaginal speculum was gently inserted into the vagina, opened, and a long-handled sterile cotton swab was introduced to obtain a sample from the anterolateral vaginal wall. Each sample was collected in 4 mL of 0.1% (w/v) sterile peptone water with 0.85% (w/v) NaCl and 0.05% (w/v) L-cysteine-HCl x H2O. The cotton swab was moistened by immersion in the peptone water immediately before sampling. Owing to the low amount of mucus retrieved from healthy, pregnant cows, the weight of the mucus recovered was not recorded.
For culture-independent analyses in experiment 2, vaginal mucus samples were collected using syringes fitted with an approximately 30 cm long collection tube without the use of a vaginal speculum. The weight of mucus in each sample was determined by recording the total weight of each sample collection tube with 1 ml of peptone water before and after each mucus sample was collected. All samples were stored at temperatures between −20°C to −80°C.
Isolation of microorganisms
Ten-fold serially diluted samples were plated on Reinforced Clostridial Medium (RCM) with 5% animal blood, Endo agar (Difco, Sparks, USA), and modified MRS (mMRS) agar . Representative colonies from each type of plates and colony morphology were purified by repeated streak-plating until a uniform colony morphology was obtained. Isolates from mMRS and RCM with blood were streaked on mMRS agars whereas isolates from Endo plates were streaked on Luria Bertani (LB) agars. Frozen stock cultures of each isolate were prepared from a single colony and stored in 60% glycerol at −70°C.
General molecular techniques
General DNA manipulations and agarose gel electrophoresis were performed as described by Sambrook et al.. Chromosomal DNA of isolated strains was extracted from 1 ml cultures using a DNeasy® Blood and Tissue Kit (Qiagen, Mississauga, Canada). Unless otherwise stated, PCR amplifications were performed in GeneAmp® PCR System 9700 (Applied Biosystems, Streetsville, Canada) by using Taq DNA polymerase and deoxynucleoside triphosphates (Invitrogen, Burlington, Canada). The PCR products were purified using the QIAquick PCR purification kit (Qiagen).
Random amplified polymorphic DNA-PCR (RAPD-PCR) analysis
Primers used in the study
Primer/Probe Sequence (5’ → 3’)
Annealing Temperature (°C)
†Lactobacillus –Pediococcus-Leuconostoc-Weissella (Lactobacillus group) (341 bp)
Lac1: AGC AGT AGG GAA TCT TCC A
Lab667r: CAC CGC TAC ACA TGG AG
†Enterococcus spp.(144 bp)
Ent-F: CCC TTA TTG TTA GTT GCC ATC ATT
Ent-R: ACT CGT TGT ACT TCC CAT TGT
†Enterobacteriaceae (195 bp)
Enterobac-F: CAT TGA CGT TAC CCG CAG AAG AAG C
Enterobac-R: CTC TAC GAG ACT CAA GCT TGC
†Staphylococcus spp. (370 bp)
TStaG422: GGC CGT GTT GAA CGT GGT CAA ATC
TStaG765: TIA CCA TTT CAG TAC CTT CTG GTA A
†Bacillus spp. (995 bp)
BacR: GTC ACC TTA GAG TGC CC
†E. coli (544 bp)
ECP79F: GAA GCT TGC TTC TTT GCT
ECP620R: GAG CCC GGG GAT TTC ACA T
†SLT-I (614 bp)
VT1 (SLTI-F): ACA CTG GAT GAT CTC AGT GG
VT2 (SLTI-R): CTG AAT CCC CCT CCA TTA TG
†SLT-II (779 bp)
VT3 (SLTII-F): CCA TGA CAA CGG ACA GCA GTT
VT4 (SLTII-R): CCT GTC AAC TGA GCA CTT T
16S rDNA Sequencing
616V: AGA GTT TGA TYM TGG CTC
630R: AAG GAG GTG GAT CCA RCC
Random Primer for RAPD
DAF4: CGG CAG CGC C
M13V: GTT TTC CCA GTC ACG ACG TTG
HDA1: ACT CCT ACG GGA GGC AGC AG
HDA2: GTA TTA CCG CGG CTG CTG GCA
HDA1 + GC: CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GGC ACG GGG GGA CTC CTA CGG GAG GCA GCA G
M13Forward (−20): GTA AAA CGA CGG CCA G
M13Reverse: CAG GAA ACA GCT ATG AC
†Pediocin Structural Gene pedA (100 bp)
pedA2RTF: GGC CAA TAT CAT TGG TGG TA
pedA2RTR: ATT GAT TAT GCA AGT GGT AGC C
TqM-pedA: FAM-ACT TGT GGC AAA CAT TCC TGC
TCT GTT GA-TAMRA
†Total Bacteria (727 bp)
TotalBac-F785: GGA TTA GAT ACC CTG GTA GTC
TotalBac-R1512r: TAC CTT GTT ACG ACT T
TaqMan 1400r Probe: 6-FAM-TGA CGG GCG GTG TGT ACA AGG C-TAMRA
Partial 16S ribosomal rRNA gene amplification and sequencing
Isolates differing in origin or RAPD pattern were identified by partial sequencing of 16S rRNA genes. PCR reaction was performed in a master mix with a final volume of 50 μL containing 1.5 U Taq DNA Polymerase (Invitrogen), 5 μL of 10X PCR Reaction Buffer (Invitrogen), 1.5 μL of 25 mmol L-1 MgCl2 (Invitrogen), 25 pmol of universal bacterial primers 616V and 630R (Table 2), 1 μL of 10 mmol L-1 dNTP, and 1 μL of template DNA. PCR product was electrophoresed in 1.0% (w/v) agarose gel, with a 2-log ladder (New England Biolabs). All sequencing data were obtained from sequencing services provided by Macrogen (Rockville, USA). The 16S rRNA gene sequences of isolates were compared with 16S rRNA gene sequences of type strains in the Ribosomal Project Database Project II (RDP-II; Michigan State University, East Lansing, USA, http://rdp.cme.msu.edu).
Identification of E. coliwith species-specific PCR and API 20E test system
Reference strains used in the study
Lactobacillus plantarum FUA3099
Positive control for RAPD with M13V primer
Shigella boydii ATCC4388
Negative control for species specific PCR of E. coli 16S rRNA gene
Shigella dysenteriae ATCC188
Shigella flexneri ATCC62
E. coli O157:H7 ATCC43888
Positive control for species specific PCR of E. coli 16S rRNA gene
E. coli O157:H7 ATCC43889
SLT-II positive control
E. coli O157:H7 ATCC43890
SLT-I positive control
Pediococcus acidilactici FUA3072
Bacteriocin-producing strain expressing the pediocin AcH/PA-1 operon
Listeria innocua ATCC33090
Indicator strains used in deferred inhibition assay for bacteriocins detection
Detection of bacteriocin production by Lactobacillus spp. and Pediococcusspp
Lactobacillus species and Pediococcus species were initially screened for production of pediocin AcH by PCR amplification of the pediocin AcH immunity gene. The gene amplification was performed with 50 μL reaction mixes that contained 1.5 U Taq DNA polymerase (Invitrogen), 5 μL of 10X PCR reaction buffer (Invitrogen), 1.5 μL of 25 mM MgCl2 (Invitrogen), 1 μL of 10 mM dNTP (Invitrogen), 2 μL of template DNA, 25 pmol of primers Pediocin-for (TCA ATA ATG GAG CTA TGG) and Pediocin-rev (ACC AGT CTC CAG AAT ATC TAA). Bacteriocin production by lactic acid bacteria was determined with bacteriocins screening medium as described . Overnight cultures of test strains were prepared in MRS broth that contained 2 g L-1 glucose. Test strains used in this study included Lactobacillus sakei FUA3089 as well as Ped. acidilactici FUA3138 and FUA3140. MRS plates with 2 g glucose L-1 were spotted with 3 μL of each overnight culture and the plates were incubated overnight under anaerobic conditions at 37°C. Ped. acidilactici FUA3072 was used as reference strain. Bacteriocin formation of this strain was previously characterized by sequencing of the pediocin operon, quantification of the expression of genes of the pediocin operon, and deferred inhibition assay (data not shown).
Cultures of indicator strains (Table 3) grown in overnight MRS broth with 2 g L-1 glucose were used to inoculate MRS soft agar at an inoculation level of 1% and the soft agar was overlayered over the MRS plates with test strains. Indicator strains included E. coli FUA1036, E. coli FUA1063, E. coli FUA1064, Listeria innocua ATCC33090, and Enterococcus facaelis FUA3141.
The deferred inhibition assay was repeated with the addition of 20 g L-1 proteinase K in 100 mmol L-1 Tris-Cl, pH 8.5, which was spotted adjacent to test strain colonies and plates were incubated for four hours at 55°C to maximize proteinase activity before overlayering was conducted.
Identification of library clones via sequencing
PCR-DGGE analysis was initially carried out characterise bovine vaginal microbiota by a culture-independent approach. The DNA concentration of samples from healthy cows, however, was below the detection limit of PCR-DGGE analysis and DGGE patterns could be obtained only for two samples from animals #2373 #2409 (data not shown). Total bacterial DNA was isolated from these two vaginal swab samples via both phenol chloroform extraction and Wizard MagneSil® Tfx™ System (Promega). Nested PCR was conducted to maximize DNA amplification by amplifying with 616V and 630R primers prior to amplification with HDA primers (Table 2). PCR products that were amplified with HDA primers were cloned into a pCR 2.1-TOPO vector using the TOPO TA Cloning® Kit (Invitrogen) according to manufacturer’s instructions. The Promega’s Wizard® Plus SV A clone library was constructed using PCR products that were amplified with HDA primers, which were then cloned into a pCR 2.1-TOPO vector, using the TOPO TA Cloning® Kit (Invitrogen) according to manufacturer’s instructions. The Promega’s Wizard® Plus SV Minipreps DNA Purification System was used for plasmid isolation. To confirm the cloning of the inserts, sequencing of the amplified insert was performed according to the Invitrogen TOPO TA Cloning® Kit manual.
Quantitative PCR was conducted with vaginal mucus samples collected from ten cows, using syringes fitted with an approximately 30 cm long collection tube. Samples from 10 animals that developed metritis after calving were randomly selected from samples of a larger cohort of animals. Total bacterial DNA was extracted using the Wizard MagneSil® Tfx™ System (Promega) and DNA concentrations were measured using the NanoDrop spectrophotometer system ND-1000, software version 3.3.0 (Thermo Fisher Scientific Inc., Wilmington, USA). All dagger-marked primer pairs that are listed in Table 2 were used in the preparation of standards and qPCR analyses. Standards were prepared using purified PCR products, which were serially diluted ten-fold. Diluted standards (10-3 to 10-8) were used to generate standard curves. TaqMan probes were used for the pedA gene and the total bacteria qPCR experiments. In both cases, each probe was labelled with 5’-FAM and 3’-TAMRA as fluorescent reporter dye and quencher respectively. The total reaction volume was set to 25 μL, which contained 12.5 μL TaqMan Universal PCR Master Mix (Applied Biosystems), 2.5 μL of template DNA extracted from vaginal mucus and 5 μmol L-1 of each primer (Table 2), and 0.2 μmol L-1 of the TaqMan probe. SYBR green assays were used for all remaining target-group primer pairs. The total reaction was also set at 25 μL containing 12.5 μL Fast SYBR Green Master Mix (Applied Biosystems), 1 μmol L-1 primer, and 1 μL DNA template. Amplification conditions generally followed an initial denaturation at 95°C for 5 min for 1 cycle; 40 cycles of denaturation at 95°C for 30 sec, annealing with listed annealing temperatures in Table 2 for 1 min, and extension at 72°C for 2 min. Quantitative PCR was executed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Reactions were performed in triplicates in MicroAmp Fast Optical 96-well reaction plates, sealed with MicroAmp Optical Adhesive Film (Applied Biosystems).
Results were analyzed using the general linear models procedure of SAS (Release 9.2, SAS Institute, Inc., Cary, NC, USA). The mathematical model used one animal as experimental unit and included the type of bacteria as the dependent variable and tested for differences in the least square means of log rDNA or DNA copy numbers for each target group between the two periods (i.e., pre-partum versus post-partum).
Gene accession numbers of 16S rRNA gene sequences obtained in this study
Sequences of 16S rRNA genes of isolates obtained in this study were deposited in GenBank® with the following accession numbers: FUA3086 (GQ222397), FUA3087 (GQ222398), FUA3088 (GQ222399), FUA3089 (GQ222408), FUA1167 (GQ205673), FUA1035 (GQ222390), FUA1037 (GQ222410), FUA3137 (GQ222393), FUA3140 (GQ222392), FUA3141 (GQ222407), FUA3226 (GQ222394), FUA3136 (GQ205672), FUA1062 (GQ222401), FUA2027 (GQ205674), FUA2028 (GQ222400), FUA3251 (GQ222395), FUA1046 (GQ222387), FUA3135 (GQ222404), FUA2023 (GQ205670), FUA2024 (GQ205671), FUA1036, (GQ222389), FUA3139 (GQ222406), FUA1063 (GQ222403), FUA3227 (GQ205669), FUA3138 (GQ222409), FUA1049 (GQ222388), FUA1070 (GQ222391), FUA1064 (GQ222405), FUA3180 (GQ222402), FUA2029 (GQ222396).
We acknowledge Judith van der Lelij and Marleen Roes for their excellent support and contribution to our research. The Alberta Livestock Industry Development Fund, Alberta Milk, and the Canada Research Chairs program are acknowledged for financial support.
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