Folia Microbiol DOI 10.1007/s12223-015-0431-x
Bovine vaginal strain Kocuria kristinae and its characterization Eva Styková 1 & Radomíra Nemcová 2 & Soňa Gancarčíková 2 & Igor Valocký 1 & Andrea Lauková 3
Received: 30 April 2015 / Accepted: 15 October 2015 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015
Abstract Kocuria spp. are widely distributed in nature. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria. In general, limited knowledge exists concerning the properties associated with the representants of the genus Kocuria, Kocuria kristinae as well. Following our previous results, K. kristinae Kk2014 Biocenol™ (CCM 8628) was isolated from vagina of a healthy cow. Its taxonomical allottation was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system and phenotypic characteristics. Kk2014 strain showed strong adherence capability to the vaginal mucus, produced organic acids which can play a role in prevention of unsuitable contamination, and showed in vitro antagonistic/antimicrobial activity against strains Arcanobacterium pyogenes CCM 5753, Fusobacterium necrophorum CCM 5982, Streptococcus equi subsp. zooepidemicus CCM 7316, and Gardnerella vaginalis CCM 6221. Antimicrobial activity ranged from 100 to 200 AU/mL, up to 32 mm in size, respectively.
* Eva Styková [email protected] 1
Clinic of Horses, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice, Slovak Republic
2
Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice, Slovak Republic
3
Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovak Republic
Kocuria spp. are widely distributed in nature, and they are also found frequently as normal skin flora in human or other mammals (Stackebrandt et al. 1995; Szczerba 2003). On the other side, they were also isolated from the urinary tract infections (Ma et al. 2005) and recently from cholecystitis (Tewari et al. 2013). The species Kocuria kristinae is one of the representants of the genus Kocuria. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria (Savini et al. 2010). There is still limited knowledge concerning the different properties associated with the representants of the genus Kocuria, K. kristinae as well. Following our previous results related to beneficial bacteria from bovine vaginal swabs for their further use to prevent metritis and cow infertility (Styková et al. 2012, 2013, 2014), randomly grown colonies were picked up, taxonomically identified, and characterized in more detail. Among them, K. kristinae strain was detected. Up to date, the strains of K. kristinae are mostly referred as causative agents. In this study, the protective function and antimicrobial activity of K. kristinae strain are reported. The mucus plays an essential role in the reproductive process of the mammals. The primary function of reproductive tract mucus is its action as mechanical barrier against microbial infection of the uterus (Rutllant et al. 2005). The adherence ability of beneficial strains protects the host against the onset of pathogenic strains by their inhibition, by adhesion to surfaces, and by competition for nutrients. The antimicrobial substances produced by some strains also contribute to defense barrier (Tejero-Sariñena et al. 2012). In this study, identified Kocuria strain was tested to produce organic acids, to have adherence ability to vaginal mucus in different phases of the estrous cycle, and to show inhibitory activity against the indicator bacteria. Its antibiotic profile was also tested. This study would like to shed light on bovine K. kristinae.
Folia Microbiol
Material and methods Sample collection, isolation, and strain identification Two hundred forty-four vaginal swabs of 122 healthy heifers and cows were sampled from four localities in Slovakia. Sampling was provided with the agreement of local veterinary administration and farmers following the guides for animal handling. The vulvar area was washed with povidone-iodine and water. A disposable speculum was inserted into the vagina to swab the posterior area and placed into the Amies agar gel with charcoal (DispoLab, Copan Italia, Brescia, Italy). To isolate colonies, the standard microbiological method (ISO) was used; the samples were diluted in saline solution (pH 7.00). Appropriate dilutions were plated on the peptone yeast glucose (PYG) medium containing (v/w) 10 g of casein peptone, 5 g of glucose, 5 g of yeast extract, 5 g of natrium chloride, and 15 g of agar. Plates were incubated at 37 °C for 24 h. Together, 20 selected colonies were picked up and tested for their purity, and coccoid bacteria were phenotyped by BBL™ Crystal™ Gram-positive Identification System (Becton and Dickinson, Cockeysville, Maryland, USA) following the criteria according to Stackebrandt et al. (1995). Moreover, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based on protein Bfingerprints^ (MALDI-TOF, Bruker Daltonics 2008) was used. Lysates of bacterial cells were prepared according to the instruction of producer (Bruker Daltonics 2008) prior to identification. Taxonomic identification was evaluated on the base of highly probable species identification of MALDI-TOF identification system (value score 2.300–3.000, Bruker Daltonics 2008). Among selected bacteria, only one strain was allotted to the genus Kocuria; it is presented in this study; the other strains were stored for further processing. Preparation of mucus for adherence testing Preparation of mucus and adherence assay was provided as previously reported by Styková et al. (2012, 2013, 2014). The tested mucus was collected from the vagina of three slaughtered heifers and nine slaughtered cows with healthy reproductive tract by gently scraping the mucosa with a rubber spatula. Twelve mucus samples were prepared. The volume of the mucus sample was 5–10 mL from each animal depending on the phase of the estrous cycle. The phase of the reproductive cycle was determined by inspection of the ovaries. Glass beads were put into 50-mL test tubes (Nunc International, Roskilde, Denmark), and the mucus was prediluted with 0.15 mol/L phosphate-buffered saline (PBS; pH 7.2) in a 1:1 ratio. Test tubes were vigorously shaken on a shaker during 15–20 min at room temperature. Before use, the mucus was filtered through a glass filter
(Papírna Perštejn spol. s r.o. Keseg and Rathouský, Pernštejn, Czech Republic) using vacuum and then through the membrane filter (Millipore, 0.22 μm, 47 mm, Fisher Scientific Ltd., Dublin, Ireland). A vacuum filtration kit was used for large volumes of mucus. Syringe filters and manifold connected to a vacuum were used for small volumes of mucus. Mucus was further diluted after filtration with phosphate-buffered saline (PBS, pH 7.2) to 1:30 ratio. Homologicity of diluted mucus was experimentally verified in technical experiments in the Synergy™ 4 Multi-Mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA). The modified microtiter plate binding assay was used to test adherence ability of Kocuria strain (Štyriak and Ljungh 2003). Bacterial culture was adjusted to have the optical density (OD640 =0.5, Spekol EK, Jena, Germany) which was in accordance with cell concentration approximately 1 × 108 CFU/mL. Cell counts were controlled by plating of culture on PYG agar. Microtiter 96-well plates (Greiner ELISA 8 Well Strips, 350 μl, Flat Bottom, Medium Binding; Cruinn Diagnostics Ltd., Dublin, Ireland) were used. The absorbance values (A580 nm) were determined in the Synergy™ 4 MultiMode Microplate Reader (BioTek Instruments Inc., USA). By this method, bacteria are classified as strongly adherent (A580 nm ≥0.25), weakly adherent (A580 nm 0.15–0.24), or nonadherent (A580 nm
Bovine vaginal strain Kocuria kristinae and its characterization Eva Styková 1 & Radomíra Nemcová 2 & Soňa Gancarčíková 2 & Igor Valocký 1 & Andrea Lauková 3
Received: 30 April 2015 / Accepted: 15 October 2015 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015
Abstract Kocuria spp. are widely distributed in nature. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria. In general, limited knowledge exists concerning the properties associated with the representants of the genus Kocuria, Kocuria kristinae as well. Following our previous results, K. kristinae Kk2014 Biocenol™ (CCM 8628) was isolated from vagina of a healthy cow. Its taxonomical allottation was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) identification system and phenotypic characteristics. Kk2014 strain showed strong adherence capability to the vaginal mucus, produced organic acids which can play a role in prevention of unsuitable contamination, and showed in vitro antagonistic/antimicrobial activity against strains Arcanobacterium pyogenes CCM 5753, Fusobacterium necrophorum CCM 5982, Streptococcus equi subsp. zooepidemicus CCM 7316, and Gardnerella vaginalis CCM 6221. Antimicrobial activity ranged from 100 to 200 AU/mL, up to 32 mm in size, respectively.
* Eva Styková [email protected] 1
Clinic of Horses, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice, Slovak Republic
2
Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy, Komenského 73, 041 81 Košice, Slovak Republic
3
Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovak Republic
Kocuria spp. are widely distributed in nature, and they are also found frequently as normal skin flora in human or other mammals (Stackebrandt et al. 1995; Szczerba 2003). On the other side, they were also isolated from the urinary tract infections (Ma et al. 2005) and recently from cholecystitis (Tewari et al. 2013). The species Kocuria kristinae is one of the representants of the genus Kocuria. They are Gram-positive, coagulase-negative, coccoid bacteria belonging to the family Micrococcaceae, suborder Micrococcineae, order Actinomycetales, class Actinobacteria (Savini et al. 2010). There is still limited knowledge concerning the different properties associated with the representants of the genus Kocuria, K. kristinae as well. Following our previous results related to beneficial bacteria from bovine vaginal swabs for their further use to prevent metritis and cow infertility (Styková et al. 2012, 2013, 2014), randomly grown colonies were picked up, taxonomically identified, and characterized in more detail. Among them, K. kristinae strain was detected. Up to date, the strains of K. kristinae are mostly referred as causative agents. In this study, the protective function and antimicrobial activity of K. kristinae strain are reported. The mucus plays an essential role in the reproductive process of the mammals. The primary function of reproductive tract mucus is its action as mechanical barrier against microbial infection of the uterus (Rutllant et al. 2005). The adherence ability of beneficial strains protects the host against the onset of pathogenic strains by their inhibition, by adhesion to surfaces, and by competition for nutrients. The antimicrobial substances produced by some strains also contribute to defense barrier (Tejero-Sariñena et al. 2012). In this study, identified Kocuria strain was tested to produce organic acids, to have adherence ability to vaginal mucus in different phases of the estrous cycle, and to show inhibitory activity against the indicator bacteria. Its antibiotic profile was also tested. This study would like to shed light on bovine K. kristinae.
Folia Microbiol
Material and methods Sample collection, isolation, and strain identification Two hundred forty-four vaginal swabs of 122 healthy heifers and cows were sampled from four localities in Slovakia. Sampling was provided with the agreement of local veterinary administration and farmers following the guides for animal handling. The vulvar area was washed with povidone-iodine and water. A disposable speculum was inserted into the vagina to swab the posterior area and placed into the Amies agar gel with charcoal (DispoLab, Copan Italia, Brescia, Italy). To isolate colonies, the standard microbiological method (ISO) was used; the samples were diluted in saline solution (pH 7.00). Appropriate dilutions were plated on the peptone yeast glucose (PYG) medium containing (v/w) 10 g of casein peptone, 5 g of glucose, 5 g of yeast extract, 5 g of natrium chloride, and 15 g of agar. Plates were incubated at 37 °C for 24 h. Together, 20 selected colonies were picked up and tested for their purity, and coccoid bacteria were phenotyped by BBL™ Crystal™ Gram-positive Identification System (Becton and Dickinson, Cockeysville, Maryland, USA) following the criteria according to Stackebrandt et al. (1995). Moreover, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry based on protein Bfingerprints^ (MALDI-TOF, Bruker Daltonics 2008) was used. Lysates of bacterial cells were prepared according to the instruction of producer (Bruker Daltonics 2008) prior to identification. Taxonomic identification was evaluated on the base of highly probable species identification of MALDI-TOF identification system (value score 2.300–3.000, Bruker Daltonics 2008). Among selected bacteria, only one strain was allotted to the genus Kocuria; it is presented in this study; the other strains were stored for further processing. Preparation of mucus for adherence testing Preparation of mucus and adherence assay was provided as previously reported by Styková et al. (2012, 2013, 2014). The tested mucus was collected from the vagina of three slaughtered heifers and nine slaughtered cows with healthy reproductive tract by gently scraping the mucosa with a rubber spatula. Twelve mucus samples were prepared. The volume of the mucus sample was 5–10 mL from each animal depending on the phase of the estrous cycle. The phase of the reproductive cycle was determined by inspection of the ovaries. Glass beads were put into 50-mL test tubes (Nunc International, Roskilde, Denmark), and the mucus was prediluted with 0.15 mol/L phosphate-buffered saline (PBS; pH 7.2) in a 1:1 ratio. Test tubes were vigorously shaken on a shaker during 15–20 min at room temperature. Before use, the mucus was filtered through a glass filter
(Papírna Perštejn spol. s r.o. Keseg and Rathouský, Pernštejn, Czech Republic) using vacuum and then through the membrane filter (Millipore, 0.22 μm, 47 mm, Fisher Scientific Ltd., Dublin, Ireland). A vacuum filtration kit was used for large volumes of mucus. Syringe filters and manifold connected to a vacuum were used for small volumes of mucus. Mucus was further diluted after filtration with phosphate-buffered saline (PBS, pH 7.2) to 1:30 ratio. Homologicity of diluted mucus was experimentally verified in technical experiments in the Synergy™ 4 Multi-Mode Microplate Reader (BioTek Instruments Inc., Winooski, VT, USA). The modified microtiter plate binding assay was used to test adherence ability of Kocuria strain (Štyriak and Ljungh 2003). Bacterial culture was adjusted to have the optical density (OD640 =0.5, Spekol EK, Jena, Germany) which was in accordance with cell concentration approximately 1 × 108 CFU/mL. Cell counts were controlled by plating of culture on PYG agar. Microtiter 96-well plates (Greiner ELISA 8 Well Strips, 350 μl, Flat Bottom, Medium Binding; Cruinn Diagnostics Ltd., Dublin, Ireland) were used. The absorbance values (A580 nm) were determined in the Synergy™ 4 MultiMode Microplate Reader (BioTek Instruments Inc., USA). By this method, bacteria are classified as strongly adherent (A580 nm ≥0.25), weakly adherent (A580 nm 0.15–0.24), or nonadherent (A580 nm
