Esculin

Morphological, biochemical, culture and physiological characterization of the actinobacterial isolates of Minnie Bay were performed as recommended by the International Streptomyces Project (ISP) which were described by Shirling and Gottileb [18 ]. Microscopic study was performed with cover slip culture and cellophane method [19 ]. Formation of aerial, substrate mycelium and spore arrangements on mycelium were monitored under a phase contrast microscope (Nikon ECLIPSE E600, USA) at 100× magnification. Culture characteristics such as growth, coloration of aerial and substrate mycelia, formation of soluble pigment were investigated in eight different media including SCA, nutrient agar, yeast malt agar (ISP-2), oat meal agar (ISP-3), inorganic salt agar (ISP-4), glycerol-asparagine agar (ISP-5), peptone yeast extract agar (ISP-6) and tyrosine agar (ISP-7) with the procedures as recommended by ISP. Biochemical characterization, namely, Gram’s reaction, MR-VP, H₂S production, nitrate reduction, oxidase, catalase, urease, starch, casein and gelatin hydrolysis, blood hemolysis, TSI, citrate utilization, esculin and hippurate hydrolysis was also performed as suggested by ISP. Physiological characterization such as, effect of pH (5–11), growth range in NaCl (5-30%) and survival at 50°C was also evaluated. Capability of the isolates to utilize various carbon sources was performed in ISP-2 agar medium with phenol red as indicator [20 ]. Carbon sources viz., fructose, lactose, starch, dextrose, rhamnose, mannitol, maltose, adonitol, arabinose and raffinose were used in this study. Identification of the isolates was made with reference to Bergey’s manual of Systematic Bacteriology [21 ] and Waksman [22 ].

Trichoderma reesei QM9414 (ATCC 26921), which is a general cellulase producer, and RUT-C30 (ATCC 56765), which is another cellulase high-producing strain that is less sensitive to glucose repression, were utilized as the control strains for the comparison of cellulase production. The strain SN1 is a hypercellulolytic variant isolated from our laboratory (Song et al., 2016 (link)) and used as the initial host for strain improvement. Escherichia coli DH5a (TransGen, Beijing, China) was used for vector construction and propagation. The pMD18-T cloning vector (Takara, Otsu, Japan) was purchased for TA cloning. The pTHB vector was used for bgl1 overexpression; this vector contained a T. reesei bgl1 expression cassette under the control of a modified four-copy cbh1 promoter, as described by Zhang et al. (2010) (link). The plasmid pAB4-1 contained the Aspergillus niger pyrG gene as a selection marker to encode orotidine-5′-phosphate decarboxylase (Hartl and Seiboth, 2005 (link)). All strains were grown and maintained on a potato dextrose agar plate (PDA) containing 20.0 g/L D-glucose and 20.0 g/L agar for 5–7 days at 30°C. The conidia were harvested, and 10⁶ conidia were inoculated in a 500 mL flask containing 150 mL minimal medium (MM; Penttilä et al., 1987 (link)). MM with 300 μg/mL hygromycin B and 1.5 mg/mL 5′-FOA (Sigma, USA) was applied as the selective medium to screen the uracil auxotroph transformants (Singh et al., 2015 (link)). An esculin plate containing 3 g/L of esculin, 10 g/L of sodium carboxymethy cellulose (CMC–Na), 0.5 g/L of ferric citrate, and 20 g/L agar was utilized to screen the strains showing β-glucosidase (BGL) activity. A CMC plate containing 10 g/L of CMC–Na, 1 g of yeast extract, and 20 g/L agar was utilized to screen the strains showing cellulase (EG) activity.

The milk samples were collected from four Holstein-Friesian dairy farms located in Serbia. The number of cows on the farms varied, ranging from twenty to three hundred. The samples were taken from lactating animals with clinical and subclinical mastitis, without other health problems. Clinical mastitis was diagnosed by clinical examination of udder, while subclinical mastitis was confirmed using somatic cell count in the milk samples.
Bacteriological testing was performed by taking aseptic milk samples from all animals (clinical and subclinical mastitis) during the morning milking. The samples were then taken in sterile tubes marked with an ID number of the cow and stored at 4 °C. Afterwards, the samples were processed at the Laboratory for Milk Hygiene at the Department of Veterinary Medicine, Faculty of Agriculture, University of Novi Sad. The samples were inoculated on 2% blood agar, using a platinum loop (0.01 mL), followed by incubation of the samples for 48 h at 37 °C. Biochemical and cultural characteristics of the grown microorganisms were taken into account during their determination. Isolation and identification of bacterial strain from milk samples was conducted using microbiological procedures for the diagnosis of udder infection published by National Mastitis Council. A loopful of milk sample was streaked on blood agar (Oxoid) and then subcultured on the following selective media: Mannitol Salt Agar, Edwards Agar, Salmonella-Shigella Agar, and MacConkey Agar. Then, plates were incubated aerobically at 37 °C for 24 h. After incubation, the plates were examined for colony morphology, pigmentation, and hemolytic characteristics at 24–48 h. Catalase test was applied for distinguishing between staphylococci and other Gram-positive cocci, mannitol fermentation test, coagulase test (either positive or negative), hemolytic pattern, and colony morphology. The isolates were confirmed by biochemical tests: oxidase activity, acid production (lactose sucrose and glucose fermentation), indole production, Voges–Proskauer, and hydrogen sulfide production. In addition, each strain was confirmed using Analytical Profile Index API-20 tests (API, bio Meraux, France). To isolate staphylococci, listed media were used: blood agar, nutrient agar, Ziehl–Neelsen, MSA, for E. coli isolation nutrient agar, MacConkey agar, and API 25 were used. For streptococci, Edwards agar and esculin were used. Of the phenotypic characteristics for staphylococci, the occurrence of α and β hemolysis and, for E. coli, there were pink colonies with precipitation. For streptococci determination, hydrolysis of esculin was used.

Samples were directly inoculated on Brilliance VRE agar (Oxoid, United Kingdom), as well as into bile aesculin azide broth (Liofilchem, Italy) supplemented with 6 μg/ml vancomycin (BEAV broth). The inoculated culture media were incubated aerobically at 37 °C and were examined for growth at 24 h and 48 h. The identification of the enterococci growing on Brilliance VRE agar was based on the observation of appropriately colored colonies – indigo to purple for E. faecium and light blue for E. faecalis. Growing colonies were transferred to a 5% Blood agar plate (BAP) and re-incubated for 24 h.
The positive BEAV broths that developed black color were subcultured to 5% BAP and chromID CPS Elite (bioMérieux, France) and incubated for an additional 24 h. All suspected VRE, isolated from Brilliance VRE agar and BEAV broth were identified using Vitek 2 Compact (bioMérieux, France). In cases of low-level discrimination between E. gallinarum and E. casseliflavus, motility and pigment production tests were also done.

Bacterial DNA was extracted from 248 isolates using a bacterial DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The extracted DNA was used as a PCR template for amplification; GPCN streptococci-like isolates were determined by 16S rDNA sequencing [41 (link)], where primer p27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and primer 1492r (5′-TACGGCTACCTTGTTACGACTT-3′) were used to amplify a 1460-bp product of the 16S rDNA gene. The PCR cycling conditions included an initial denaturation step at 95 °C for 3 min, followed by 35 cycles at 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 1 min, with a final extension step at 72 °C for 5 min. The PCR products were subjected to sequencing (Sanger sequencing by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) after verification on 1.2% agarose gel. The 16S rDNA sequences were compared with sequences deposited in the nucleotide database of the National Center for Biotechnology Information. Identification was deemed reliable if the values for sequence similarities were ≥99%. The biochemical reacting kit (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd., Qingdao, China) was used for biochemical testing. A total of 11 reagents (ribose, sucrose, lactose, liquid gelatin, sorbitol, maltose, esculin, galactose, VP, trehalose, and glucose) were fermented with isolates, following the manufacturer’s instructions. Briefly, isolates were seeded in the tubes that were subsequently cultured in an incubator at 37 °C for the required time; some of the reagents needed further operations and the colors were compared with negative tubes.

For analysis of gut microbiota, grams of feces were weighed, 9 ml of isotonic (0.9%) sodium chloride solution were added to a test tube, and the mixture was thoroughly rubbed until a homogeneous mass was formed. This created a 10^-1 dilution. Subsequently, a series of dilutions from 10^-2 to 10^-11 were prepared in the same way (Figure 2). Using sterile micropipettes, 10 μl was taken from each dilution and applied to nutrient media. For the isolation of enterobacteria, commercial nutrient media Endo agar and Bismuth sulfite agar were used; for Staphylococcus spp. - Mannitol Salt Agar; for Enterococcus spp. – Bile Esculin Agar; for Yeast – Sabouraud Agar. Iron sulfite agar (Wilson-Blair) was used for isolation of Clostridia, Sharpe agar for Lactobacillus spp., Bifidobacterium Selective Agar for Bifidobacterium spp., and Bacteroides Bile Esculin Agar for Bacteroides species. Identification of microorganisms was carried out according to the scheme (Figure 3) based on the Clinical Microbiology Procedures Handbook, Volume 1-3, 4th Edition (Dunn et al., 2016 (link)). For the convenience of presentation of the material and mathematical and statistical processing, decimal logarithms of the quantitative indicator of the grown colonies of microorganisms (lg CFU/g) were used, and the proportion of genera were also calculated.