TY - JOUR
AU - Judith, Molina-Garza, Zinnia
AB - Abstract This study identified marine microorganisms from Mexican coasts that had antimicrobial activity against Staphylococcus aureus and Vibrio parahaemolyticus, which are known worldwide to be food-poisoning agents. Representative specimens of algae, saline sediment, crustaceans and mollusks were collected. Of the 42 tested strains, 15 inhibited these pathogens. Bacillus and Virgibacillus strains were identified by 16S rRNA gene sequencing. The strains with the highest inhibitory activity against S. aureus were PCRS1-07 (B. aerius), BLCG-05 and GUO-01 (B. pumilus). The strains GUHC-04, BLCG-05, GUHC-03 (B. altitudinis) and BLBSe-05 (B. oryzicola) showed higher antimicrobial activity against V. parahaemolyticus. Biofilm production by all strains was moderate, but B. altitudinis produced a stronger biofilm. This is the first study to isolate B. aerius, B. oryzicola, B. safensis, B. boroniphilus, B. altitudinis and V. senegalensis from marine ecosystems in Mexico as well as the first study to report their inhibitory effects against both S. aureus and V. parahaemolyticus. Bioactivity of spent media from the antagonistic strains cultured as biofilm also demonstrated high antimicrobial activity. The active compounds of the antagonists are currently being studied and tested. Marine ecosystems have the highest bacterial diversity associated with invertebrates and seaweed; however, this bacterial diversity has not been well-studied on Mexican coasts. marine microorganisms, Mexican coasts, Bacillus, 16S rRNA gene sequences, antimicrobial activity, biofilm INTRODUCTION Many human, veterinary and agricultural diseases are caused primarily by emerging multidrug-resistant bacteria, which are becoming an increasingly serious threat. This resistance occurs naturally via different mechanisms including gene mutations or acquisitions by transformation or plasmid infection (Morens, Folkers and Fauci 2004). However, the misuse and overuse of antimicrobial drugs (i.e. antibiotics, antifungals, antivirals, antimalarials and anthelmintics) accelerate this process (WHO 2018), which is detrimental to public health and increases the mortality rates of infectious diseases (Amabile-Cuevas 2010). In hospitals, and even in the community, multidrug-resistant pathogens occur (Solórzano and Miranda 2012), and estimates suggest that antimicrobial resistance leads to ∼2050 000 cases and 23 000 deaths per year in the USA (CDC 2013). Emerging multidrug-resistant pathogenic microorganisms, such as the ESKAPE group (Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas and Enterobacter), nosocomial bacteria (Stenotrophomonas maltophilia, Salmonella) and Vibrio species (Pendleton, Gorman and Gilmore 2013), represent serious threats to global health (Zavala-Norzagaray et al.2015). Staphylococcus aureus causes food poisoning and other infections, but the resistant ones cause serious antibiotic-resistant hospital infections. Evidence indicates that S. aureus might be involved in sudden infant death syndrome (SIDS) (Zorgani et al.1999), which is the predominant cause of death between one week and one year of life in industrialized countries (Blackwell and Weir 1999). There is no nationwide evaluation data available in Mexico, but a 14.9% prevalence of methicillin-resistant S. aureus (MRSA) was found in 659 isolates from infected patients at the Hospital de Pediatría del Centro Médico Nacional Siglo XXI, Instituto Mexicano del Seguro Social (IMSS), between 1997 and 2003 (Velazquez-Meza et al.2004). A 35% prevalence of MRSA in 483 isolates from hospitalized patients and carriers was reported from August 2009 to February 2011 at the Hospital de Pediatría, Centro Médico Nacional de Occidente, IMSS, Guadalajara, Jalisco (Villaseñor-Martinez et al.2012). The appearance of vancomycin-resistant strains of this pathogen, which have already been reported in Mexico, as well as resistance to other drugs, highlights the need for new, safe and effective first-line treatments against this pathogen (de Leon-Rosales, Hernandez and Vidal 2015). Vibrio parahaemolyticus causes human gastroenteritis worldwide, with rare cases of lethal septicemia associated with seafood consumption. It was also found to be responsible for 20–30% of food-poisoning cases in Japan and for seafood-borne diseases in many Asian countries. Pandemic V. parahaemolyticus strains have also been isolated in the USA, South America, Africa and Europe, with 45 000 cases annually (Nair et al.2007; Xu et al.2017). These bacteria have been reported scarcely in epidemiological outbreaks in Mexico. A small number of tdh+ or tdh (thermostable direct hemolysin or thermostable related hemolysin, respectively, which are considered major virulence factors, and which are highly cytotoxic to human gastrointestinal cells) environmental strains have been isolated from water and fish (Cabrera-Garcia, Vazquez-Salinas and Quiñones-Ramirez 2004). During September and October 2004, more than 1230 cases of gastroenteritis were reported due to strains of V. parahaemolyticus O3:K6 in inhabitants of southern Sinaloa: all of the cases were attributed to the consumption of raw or undercooked shrimp collected from the Huizache-Caimanero lagunary system (Cabanillas-Beltran et al.2006). Antimicrobial susceptibility tests showed that most strains (93.8%) were resistant to ampicillin, but sensitive to chloramphenicol (98.8%). Multiple drug resistance has increased significantly from 8.6% (2004–10) to 22.93% (2011–13) (Hernandez-Diaz et al.2015). Zavala-Norzagaray et al. (2015) evaluated the presence of Vibrio species and their resistance to antibiotics in 64 marine turtles from the states of Baja California Sur and Sinaloa. From the isolated strains, V. parahaemolyticus (94.1%) was resistant to at least one commonly prescribed antibiotic (mostly ampicillin). Marine bacteria, isolated from several sea ecosystems, are an inexhaustible natural resource for novel antibacterial compounds to control human pathogen growth (Jayanth, Jeyasekaran and Shakila 2002). Although products with antimicrobial, antiviral, immunosuppressive, antitumor and anticoagulant activities have been known for a long time (Rosenfeld and Zobell 1947), the number is increasing rapidly, exceeding 18 000, with hundreds of new compounds being discovered annually from them (Pabba, Samatha and Prasad 2011). In Mexico, this subject has been scarcely addressed, although some bioactive marine strains isolated from different substrates have been reported: (i) a Pseudoalteromonas strain from the Campeche coasts on the Gulf of Mexico (Cetina et al.2010); (ii) extracts produced by Firmicutes and Actinobacteria from seaweed from Baja California showed antitumor and antimicrobial activity against S. aureus, Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa (Villarreal-Gómez et al.2010); and (iii) methyl acetate extracts from Streptomyces, Micromonospora and Salinospora strains collected from the Gulf of California displayed antitumor properties against cervical and breast cancer cell lines (Torres-Beltrán et al.2012). Among the Firmicutes, many members of the genus Bacillus have been described, such as B. subtilis and B. pumilus, who produce diverse biomolecules with effective antimicrobial activity (Stein 2005; Collins et al.2016; Ravindran et al.2016; Zidour et al.2017). Thus, the present study's main goals were to isolate Bacillus species from seaweed and invertebrates from Mexican coasts and identify their in vitro antagonistic properties against S. aureus and V. parahaemolyticus, including their biofilm formation capacity; because of the interrelationships among bacterial strains that are good biofilm producers, they are also good producers of antibacterial compounds in cells attached to surfaces (like agar), and, as occurs in nature, this ability enhances the exopolymer synthesis (Yan, Boyd and Burgess 2002; Yan et al.2003). MATERIALS AND METHODS Isolation of the marine strains Samples were collected from three Mexican coasts to search for antagonistic bacteria in algae, saline sediment, crustaceans and mollusks (Table 1): Bahía de Lobos (27°15΄42.0 N, 110°25΄34.6 W) and Bahía de Guasimas (27°15΄42.0 N, 110°25΄34.6 W) in Sonora and Playa del Carmen (20°40΄05.3 N, 87°01΄40.7 W) in Quintana Roo (Fig. 1). Samples were processed as follows: Figure 1. View largeDownload slide Study area: locations sampled from (A) Bahía de Lobos, (B) Bahía de Guasimas, Sonora and (C) Playa del Carmen, Quintana Roo. Figure 1. View largeDownload slide Study area: locations sampled from (A) Bahía de Lobos, (B) Bahía de Guasimas, Sonora and (C) Playa del Carmen, Quintana Roo. Table 1. Collected samples and the number of bacterial strains isolated from different substrates. Locality Sample Scientific name Strain codea Number of isolates Bahía de Lobos Green seaweed Enteromorpha sp. BLGS 1 Brown seaweed Gracilariopsis sp. BLBS 4 Brown sediment − BLBSe 4 Dark sediment − BLDSe 4 Wild crab Callinectes arcuatus BLCI 1 Wild crab Callinectes arcuatus BLCG 3 Wild crab Callinectes arcuatus BLCHP 2 Marine snail Natica chemnitzii BLMS 1 Guasimas Chinese clam Veneridae iliochione GUCHC 3 White clam Veneridae dosinia GUWC 1 Chinese pink snail Phyllonotus erythrostoma GUCHS 1 Oyster Crassostrea sp. GUO 2 Hermit crab Clibanarius panamensis GUHC 4 Playa del Carmen Red seaweed Rhodophyta PCRS1 7 Red seaweed Rhodophyta PCRS2 3 Locality Sample Scientific name Strain codea Number of isolates Bahía de Lobos Green seaweed Enteromorpha sp. BLGS 1 Brown seaweed Gracilariopsis sp. BLBS 4 Brown sediment − BLBSe 4 Dark sediment − BLDSe 4 Wild crab Callinectes arcuatus BLCI 1 Wild crab Callinectes arcuatus BLCG 3 Wild crab Callinectes arcuatus BLCHP 2 Marine snail Natica chemnitzii BLMS 1 Guasimas Chinese clam Veneridae iliochione GUCHC 3 White clam Veneridae dosinia GUWC 1 Chinese pink snail Phyllonotus erythrostoma GUCHS 1 Oyster Crassostrea sp. GUO 2 Hermit crab Clibanarius panamensis GUHC 4 Playa del Carmen Red seaweed Rhodophyta PCRS1 7 Red seaweed Rhodophyta PCRS2 3 aThe first two digits indicate the locality: BL, Bahía de Lobos; GU, Guasimas; PC, Playa del Carmen. The next part of the code corresponds to the sample: GS, green seaweed; BS, brown seaweed; BSe, brown sediment; DSe, dark sediment; CI, crab intestine; CG, crab gills; CHP, crab hepatopancreas; MS, marine snail; CHC, Chinese clam; WC, white clam; CHS, Chinese pink snail; O, oyster; HC, hermit crab; RS1, red seaweed sample 1; RS2, red seaweed sample 2. View Large Table 1. Collected samples and the number of bacterial strains isolated from different substrates. Locality Sample Scientific name Strain codea Number of isolates Bahía de Lobos Green seaweed Enteromorpha sp. BLGS 1 Brown seaweed Gracilariopsis sp. BLBS 4 Brown sediment − BLBSe 4 Dark sediment − BLDSe 4 Wild crab Callinectes arcuatus BLCI 1 Wild crab Callinectes arcuatus BLCG 3 Wild crab Callinectes arcuatus BLCHP 2 Marine snail Natica chemnitzii BLMS 1 Guasimas Chinese clam Veneridae iliochione GUCHC 3 White clam Veneridae dosinia GUWC 1 Chinese pink snail Phyllonotus erythrostoma GUCHS 1 Oyster Crassostrea sp. GUO 2 Hermit crab Clibanarius panamensis GUHC 4 Playa del Carmen Red seaweed Rhodophyta PCRS1 7 Red seaweed Rhodophyta PCRS2 3 Locality Sample Scientific name Strain codea Number of isolates Bahía de Lobos Green seaweed Enteromorpha sp. BLGS 1 Brown seaweed Gracilariopsis sp. BLBS 4 Brown sediment − BLBSe 4 Dark sediment − BLDSe 4 Wild crab Callinectes arcuatus BLCI 1 Wild crab Callinectes arcuatus BLCG 3 Wild crab Callinectes arcuatus BLCHP 2 Marine snail Natica chemnitzii BLMS 1 Guasimas Chinese clam Veneridae iliochione GUCHC 3 White clam Veneridae dosinia GUWC 1 Chinese pink snail Phyllonotus erythrostoma GUCHS 1 Oyster Crassostrea sp. GUO 2 Hermit crab Clibanarius panamensis GUHC 4 Playa del Carmen Red seaweed Rhodophyta PCRS1 7 Red seaweed Rhodophyta PCRS2 3 aThe first two digits indicate the locality: BL, Bahía de Lobos; GU, Guasimas; PC, Playa del Carmen. The next part of the code corresponds to the sample: GS, green seaweed; BS, brown seaweed; BSe, brown sediment; DSe, dark sediment; CI, crab intestine; CG, crab gills; CHP, crab hepatopancreas; MS, marine snail; CHC, Chinese clam; WC, white clam; CHS, Chinese pink snail; O, oyster; HC, hermit crab; RS1, red seaweed sample 1; RS2, red seaweed sample 2. View Large Seaweed and sediments: three samples of seaweed (Enteromorpha sp. [Chlorophyta] and Gracilariopsis sp. [Rhodophyta] from Bahía de Lobos and Rhodophyta from Playa del Carmen) were collected in sterilized plastic bags and maintained at 4°C. The tissues were washed several times in sterilized seawater and ground under aseptic conditions. Serial dilutions were made and accomplished according to the method of Singh et al. (2011, 2015). Ten samples of saline sediment (50 g) were collected aseptically with a spatula from salt mines at low tidal periods in sterile bottles and transported immediately at 4°C to the laboratory. Each sample was mixed with sterile seawater (1:1) and disintegrated with mortar and pestle to homogenize them. The bacteria were released from these samples by filtration, and differential centrifugation was used to separate the microorganisms and the remaining sediment as described (Anas et al.2016). Finally, the samples were serially diluted in sterile saline solution (10−1–10−6) and 100 μL was spread over the surface of culture media with some modifications according to previous work (Anas et al.2016); Crabs (Callinectes arcuatus): ten randomly sampled animals were collected and transported in sterile hermetically sealed plastic containers. These crabs were found only in Bahía de Lobos. Once at the laboratory, the crabs were submerged in 10% formalin for 30 s, bathed with fresh water and washed in sterile demineralized water to eliminate the surface microflora (Talpur et al.2011). Intestines, gills and hepatopancreas were removed with sterile dissecting materials and mixed with sterile saline solution; each organ was pulverized separately under aseptic conditions with mortar and pestle to prepare inocula by following procedures described by Talpur et al. (2011) and Sivasubramanian, Ravichandran and Rajan (2017); Fifteen marine snails (Natica chemnitzii), Chinese pink snails (Phyllonotus erythrostoma) and hermit crabs (Clibanarius panamensis) were collected and transported using the same process described above. Specimens were immobilized at 5°C for 30 min, washed in tap water and then washed in sterile water. Afterwards, the surface was sterilized with 70% ethanol (v/v), and their shells were carefully crushed to expose the soft body; the whole gastrointestinal tracts were dissected with sterilized scissors and tweezers in aseptic conditions (Pawar et al.2012; Hu et al.2018) and ground in sterile saline solution. Serial dilutions and inocula were prepared as described above; and Clam (Veneridae iliochione, Veneridae dosinia) and oyster (Crassostrea sp.): samples were collected randomly, and their surfaces were washed in tap water and sterile water; the surfaces were then sterilized with 70% ethanol. Their shells were opened with sterilized knives, and the gills, digestive glands (stomach, gut and digestive diverticula) and residual tissues (mantle and adductor muscle) were dissected under sterile conditions. Each tissue (5 g) was mixed with 20 mL (1:4) sterile saline and homogenized in sterile grinders. Serial dilutions of the homogenate were spread onto culture media (Castro et al.2002; Wang et al.2014). In some cases, serial dilutions were optimized until the appropriate dilution was reached, and samples were subcultured several times by repeated streaking until pure colonies were obtained. The dilutions were cultured in tryptic soy broth (TSB, Dibico, Mexico) with 2% NaCl and marine broth 2216 (MB, Becton, Dickinson and Company, Sparks, MD, USA) and incubated at 33°C for 48 h. Isolates were subcultured and incubated for 24 h on tryptic soy agar (TSA, Dibico) with 2% NaCl and marine agar (MA, Becton, Dickinson and Company). The resulting bacterial colonies were selected for identification based on each strain's colonial morphology to obtain axenic cultures. Gram staining and microscopic observation were performed to differentiate the microorganisms’ morphology, and oxidase and catalase tests were performed (Dunlap et al.2016). Codes for each strain were determined by the sampling area: Bahía de Lobos (BL), Bahía de Guasimas (GU) and Playa del Carmen (PC). The other part of each code corresponded to the individual sample, as shown in the strain code column in Table 1. Assessment of antimicrobial activity based on the cross streak method (CSM) The antagonistic response against human pathogenic bacteria was evaluated using the CSM (Haber and Ilan 2014; Saha and Santra 2014). S. aureus and V. parahaemolyticus (both isolated from human patients in the Laboratory of Medical Microbiology, Universidad Autónoma de Nuevo León) were used for this experiment. Bacterial isolates from marine coasts were cultured in TSA with 2% NaCl for 18–24 h, then a sample was taken with a sterile swab, streaked longitudinally in a single middle line, dividing the plate into two halves on a fresh plate with TSA-2% NaCl and incubated for 48 h at 28–32°C, until a well-grown bacteria middle line was observed. Next, bacterial suspensions of the pathogens were adjusted to 1 on the McFarland scale (measured at 600 nm, with ∼3 × 108 CFU mL−1) for challenge tests (Fooladi et al.2014), and the pathogens were streaked at a 90° angle, crossing the well-grown bacterial isolate as described (Haber and Ilan 2014) and incubated for 24 h at 28–32°C (Fig. 2). After incubation, a vernier scale was used to measure the bacterial isolate inhibition length to determine the antagonism (Saha and Santra 2014). Three replicates were performed for each experimental set. The results were compared using one-factor ANOVA to assess differences between isolates, followed by a Tukey's HDS test using IBM SPSS v21.0 statistical software. Figure 2. View largeDownload slide Antagonism test by the cross streak method. Bacterial isolates from marine coasts (i.e. PCRS1-07) were streaked longitudinally in a single middle line. After 48 h the pathogens were streaked at a 90° angle, crossing the well-grown bacterial isolate. Figure 2. View largeDownload slide Antagonism test by the cross streak method. Bacterial isolates from marine coasts (i.e. PCRS1-07) were streaked longitudinally in a single middle line. After 48 h the pathogens were streaked at a 90° angle, crossing the well-grown bacterial isolate. Amplification and sequencing of bacterial 16S ribosomal DNA (16S rDNA) of bioactive strains Isolates positive for antagonistic activity were grown overnight in TSA with 2% NaCl at 32°C. One bacterial colony was removed using a sterile loop and re-suspended in phosphate-buffered saline (PBS). Genomic DNA was isolated using the PBS 1X + Tween 20 0.05% extraction protocol (López and Mejía 2012). DNA quality was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). DNA purity was calculated at an absorbance of 260–280 nm, where an optical density (OD) 260/280 ratio of 1.8–2.0 indicated high purity. The DNA extracts were PCR amplification templates targeting a region in the 16S rRNA gene of the antagonist strains using the universal primers, 27F (5΄-AGA GTT TGA TCM TGG CTC AG-3) and 1492R (5΄-TAC GGY TAC CTT GTT ACG ACT T-3) with MyTaq DNA polymerase kit (Bioline, MA, USA), in a MultiGene thermal cycler (Labnet International Inc., Edison, NJ, USA). PCR reactions were conducted under the following conditions (Balqadi, Salama and Satheesh 2017): initial denaturation at 94°C for 2 min followed by 35 amplification cycles at 94°C for 35 s, 55°C for 1 min and 72°C for 1 min. A final extension was performed at 72°C for 5 min. DNA extracts were also amplified and sequenced by Macrogen (Seoul, South Korea; http://foreign.macrogen.co.kr/eng/business/seq_16s_rRNA_sequencing.html) using their protocol. The Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) was used. PCR products were sequenced in the 3730xl automated DNA sequencing system (Applied Biosystems) using the universal primers (Lane 1991; Kim Balaraju and Jeon 2017). The nucleotide sequences of the 16S rRNA gene fragments (∼600 bp) were analyzed using BLAST on the NCBI server (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A homology of > 99% identity was the criterion used to identify the isolates to the species level. Biofilm production test The biofilm production assay was performed on microorganisms with the best antagonistic activity from the cultures incubated overnight at 30–33°C in TSB with 2% NaCl and diluted 1:100 in TSB with 2% NaCl. Diluted samples (120 μL) were transferred to a 96-well U-bottom plate, with 10 replicates per strain. The plate was incubated aerobically at 30–33°C for 24 h. The cultures were removed, and the wells washed twice with 200 μL of PBS (pH 7.4) to eliminate non-adherent cells. The plates were dried in an inverted position. The adherent bacteria were fixed with 200 μL of ethanol (90%) for 2 min, decanted, dried at room temperature and stained with 100 μL of crystal violet. After 10 min the stain was removed and washed three times with sterile distilled water (200 μL). Finally, the water was removed, and the samples were air dried at room temperature. Absorbance was read at 595 nm (Knobloch et al.2001) in a spectrophotometer (Multiscan, MS; Thermo Labsystem, MA, USA). Adhesion ability was classified as strong (OD ≥ 1), moderate (0.1 ≤ OD < 1) or weak (OD < 0.1) (Lopez-Leon et al.2016). Bioactivity of spent media from the strains cultured as biofilm The antimicrobial activities of spent media were carried out as described (Yan et al.2003) with some modifications. Bacteria with the best inhibitory activity were grown in marine broth at 30°C for 4 days and then they were cultivated on the surfaces of sterile semipermeable membranes of nylon discs (diameter 35 mm; Whatman, Brentford, Middlesex, UK). Each membrane was placed over the edges of petri dishes (diameter, 35 mm; height, 13 mm; Corning Glass Works, Corning, NY, USA) prefilled with TSB with 2% NaCl, allowing the membranes to be in contact with the air on the upper side and in contact with the culture media on the lower side. Next, each petri dish with the inoculated membrane was placed into a bigger petri dish (diameter, 60 mm; height 18 mm) and incubated at 30°C. After incubation for 5 days the membranes were removed and the spent media was centrifugated (6000 g, 10 min, 4°C) and the supernatant filtered twice (pore size 0.2 μm, Whatman). Antimicrobial activity was performed in petri dishes with TSA inoculated with pathogenic S. aureus or V. parahaemolyticus. Wells of a diameter of 8 mm were made on these cultures and filled with 100 μL of the supernatant (Aoudia et al.2016). Petri dishes were incubated (30°C for 24 h) and inhibition zones were measured. Also, cell-free supernatant of antagonist planktonic bacteria were cultivated in TSB with 2% NaCl (30°C for 24 h) to test their antimicrobial activity. Spent media of the cell-free supernatants were also centrifugated and filtrated as described. Fresh TSB was included as a negative control. RESULTS Isolation of the marine strains Forty-two strains were isolated from a variety of marine samples (Table 1). These isolates were composed of three Gram-positive coccobacilli (7.14%), four Gram-positive cocci (9.5%) and 35 rods (11 Gram-negative [26.2%] and 24 Gram-positive [57.14%]). The colonial morphology was also recorded, as shown in Table 2. Table 2. Physical and morphological characteristics of microorganisms isolated from Mexican coasts. Identities of strains with antagonist activity (genus and species) were determined by bacterial 16S ribosomal DNA analyses. Straina Gram Morphology Cat/Oxi+ Colonial morphology Identity of the strains with antagonistic activity BLGS-03 − Rod +/− Round, big, opaque NAA BLBS-01 + Rod +/− Flat, crater-like appearance, opaque white Bacillus safensis BLBS-07 + Rod +/− Round, diffuse, crater-like appearance, cream Virgibacillus senegalensis BLBS-08 + Coccobacilli +/− Creamy, round, opaque white NAA BLBS-09 + Coccobacilli +/− Creamy, round, opaque white NAA BLBSe-01 + Rod +/− Small, round, opaque NAA BLBSe-03 + Rod +/− Small, round, opaque Bacillus pumilus BLBSe-05 + Rod +/− Small, round, opaque, lobulated Bacillus oryzicola BLBSe-09 + Rod +/− Light orange, opaque, flat growth NAA BLDSe-01 + Cocci +/− Round, opaque white NAA BLDSe-02 + Cocci +/− Round, small, opaque yellow NAA BLDSe-03 + Rod +/− Flat, transparent, round Bacillus boroniphilus BLDSe-04 + Rod +/− Round, opaque cream Bacillus pumilus BLCI-01 + Cocci +/− Small, round, opaque white NAA BLCG-01 + Cocci +/− Small, round, opaque white NAA BLCG-02 − Rod +/− Creamy, indefinite growth, opaque white NAA BLCG-05 + Rod +/− Flat, crater-like appearance, opaque white Bacillus pumilus BLCHP-01 + Rod +/− Light orange, opaque, indefinite growth NAA BLCHP-02 + Rod +/− Small, round, opaque NAA BLMS-06 + Rod +/− Small, round, opaque Bacillus paralicheniformis GUJS-02 + Rod −/− Light orange, opaque, flat growth NAA GUCHC-01 + Rod +/− Light orange, opaque, flat growth NAA GUCHC-02 − Rod −/+ Round, big, opaque NAA GUCHC-03 + Rod −/− Flat, transparent, round NAA GUWC-01 + Rod +/− Crater-like, striated appearance, opaque cream NAA GUCHS-01 + Rod +/− Round, diffuse, crater-like appearance, cream NAA GUO-01 + Rod +/− Round, opaque cream Bacillus pumilus GUO-02 + Rod +/− Small, round, opaque. Bacillus altitudinis GUHC-03 + Rod +/− Round, opaque cream Bacillus altitudinis GUHC-04 + Rod +/− Cream, irregular, small Bacillus pumilus GUHC-06 − Rod +/− Light red, irregular, small NAA GUHC-07 + Rod +/− Flat, crater-like appearance, opaque white Bacillus altitudinis PCRS1-01 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS1-02 − Rod +/− Round, diffuse, opaque orange NAA PCRS1-03 − Rod +/+ Small, round, opaque NAA PCRS1-04 + Coccobacilli +/+ Creamy, indefinite growth, opaque white NAA PCRS1-05 − Rod −/− Round, diffuse, crater-like appearance, cream NAA PCRS1-06 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus pumilus PCRS1-07 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus aerius PCRS2-02 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-03 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-05 − Rod +/− Round, diffuse, crater-like appearance, cream NAA Straina Gram Morphology Cat/Oxi+ Colonial morphology Identity of the strains with antagonistic activity BLGS-03 − Rod +/− Round, big, opaque NAA BLBS-01 + Rod +/− Flat, crater-like appearance, opaque white Bacillus safensis BLBS-07 + Rod +/− Round, diffuse, crater-like appearance, cream Virgibacillus senegalensis BLBS-08 + Coccobacilli +/− Creamy, round, opaque white NAA BLBS-09 + Coccobacilli +/− Creamy, round, opaque white NAA BLBSe-01 + Rod +/− Small, round, opaque NAA BLBSe-03 + Rod +/− Small, round, opaque Bacillus pumilus BLBSe-05 + Rod +/− Small, round, opaque, lobulated Bacillus oryzicola BLBSe-09 + Rod +/− Light orange, opaque, flat growth NAA BLDSe-01 + Cocci +/− Round, opaque white NAA BLDSe-02 + Cocci +/− Round, small, opaque yellow NAA BLDSe-03 + Rod +/− Flat, transparent, round Bacillus boroniphilus BLDSe-04 + Rod +/− Round, opaque cream Bacillus pumilus BLCI-01 + Cocci +/− Small, round, opaque white NAA BLCG-01 + Cocci +/− Small, round, opaque white NAA BLCG-02 − Rod +/− Creamy, indefinite growth, opaque white NAA BLCG-05 + Rod +/− Flat, crater-like appearance, opaque white Bacillus pumilus BLCHP-01 + Rod +/− Light orange, opaque, indefinite growth NAA BLCHP-02 + Rod +/− Small, round, opaque NAA BLMS-06 + Rod +/− Small, round, opaque Bacillus paralicheniformis GUJS-02 + Rod −/− Light orange, opaque, flat growth NAA GUCHC-01 + Rod +/− Light orange, opaque, flat growth NAA GUCHC-02 − Rod −/+ Round, big, opaque NAA GUCHC-03 + Rod −/− Flat, transparent, round NAA GUWC-01 + Rod +/− Crater-like, striated appearance, opaque cream NAA GUCHS-01 + Rod +/− Round, diffuse, crater-like appearance, cream NAA GUO-01 + Rod +/− Round, opaque cream Bacillus pumilus GUO-02 + Rod +/− Small, round, opaque. Bacillus altitudinis GUHC-03 + Rod +/− Round, opaque cream Bacillus altitudinis GUHC-04 + Rod +/− Cream, irregular, small Bacillus pumilus GUHC-06 − Rod +/− Light red, irregular, small NAA GUHC-07 + Rod +/− Flat, crater-like appearance, opaque white Bacillus altitudinis PCRS1-01 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS1-02 − Rod +/− Round, diffuse, opaque orange NAA PCRS1-03 − Rod +/+ Small, round, opaque NAA PCRS1-04 + Coccobacilli +/+ Creamy, indefinite growth, opaque white NAA PCRS1-05 − Rod −/− Round, diffuse, crater-like appearance, cream NAA PCRS1-06 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus pumilus PCRS1-07 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus aerius PCRS2-02 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-03 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-05 − Rod +/− Round, diffuse, crater-like appearance, cream NAA +Cat/Oxi, catalase/oxidase; NAA, no antagonistic activity. aFor abbreviations, see Table 1. View Large Table 2. Physical and morphological characteristics of microorganisms isolated from Mexican coasts. Identities of strains with antagonist activity (genus and species) were determined by bacterial 16S ribosomal DNA analyses. Straina Gram Morphology Cat/Oxi+ Colonial morphology Identity of the strains with antagonistic activity BLGS-03 − Rod +/− Round, big, opaque NAA BLBS-01 + Rod +/− Flat, crater-like appearance, opaque white Bacillus safensis BLBS-07 + Rod +/− Round, diffuse, crater-like appearance, cream Virgibacillus senegalensis BLBS-08 + Coccobacilli +/− Creamy, round, opaque white NAA BLBS-09 + Coccobacilli +/− Creamy, round, opaque white NAA BLBSe-01 + Rod +/− Small, round, opaque NAA BLBSe-03 + Rod +/− Small, round, opaque Bacillus pumilus BLBSe-05 + Rod +/− Small, round, opaque, lobulated Bacillus oryzicola BLBSe-09 + Rod +/− Light orange, opaque, flat growth NAA BLDSe-01 + Cocci +/− Round, opaque white NAA BLDSe-02 + Cocci +/− Round, small, opaque yellow NAA BLDSe-03 + Rod +/− Flat, transparent, round Bacillus boroniphilus BLDSe-04 + Rod +/− Round, opaque cream Bacillus pumilus BLCI-01 + Cocci +/− Small, round, opaque white NAA BLCG-01 + Cocci +/− Small, round, opaque white NAA BLCG-02 − Rod +/− Creamy, indefinite growth, opaque white NAA BLCG-05 + Rod +/− Flat, crater-like appearance, opaque white Bacillus pumilus BLCHP-01 + Rod +/− Light orange, opaque, indefinite growth NAA BLCHP-02 + Rod +/− Small, round, opaque NAA BLMS-06 + Rod +/− Small, round, opaque Bacillus paralicheniformis GUJS-02 + Rod −/− Light orange, opaque, flat growth NAA GUCHC-01 + Rod +/− Light orange, opaque, flat growth NAA GUCHC-02 − Rod −/+ Round, big, opaque NAA GUCHC-03 + Rod −/− Flat, transparent, round NAA GUWC-01 + Rod +/− Crater-like, striated appearance, opaque cream NAA GUCHS-01 + Rod +/− Round, diffuse, crater-like appearance, cream NAA GUO-01 + Rod +/− Round, opaque cream Bacillus pumilus GUO-02 + Rod +/− Small, round, opaque. Bacillus altitudinis GUHC-03 + Rod +/− Round, opaque cream Bacillus altitudinis GUHC-04 + Rod +/− Cream, irregular, small Bacillus pumilus GUHC-06 − Rod +/− Light red, irregular, small NAA GUHC-07 + Rod +/− Flat, crater-like appearance, opaque white Bacillus altitudinis PCRS1-01 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS1-02 − Rod +/− Round, diffuse, opaque orange NAA PCRS1-03 − Rod +/+ Small, round, opaque NAA PCRS1-04 + Coccobacilli +/+ Creamy, indefinite growth, opaque white NAA PCRS1-05 − Rod −/− Round, diffuse, crater-like appearance, cream NAA PCRS1-06 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus pumilus PCRS1-07 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus aerius PCRS2-02 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-03 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-05 − Rod +/− Round, diffuse, crater-like appearance, cream NAA Straina Gram Morphology Cat/Oxi+ Colonial morphology Identity of the strains with antagonistic activity BLGS-03 − Rod +/− Round, big, opaque NAA BLBS-01 + Rod +/− Flat, crater-like appearance, opaque white Bacillus safensis BLBS-07 + Rod +/− Round, diffuse, crater-like appearance, cream Virgibacillus senegalensis BLBS-08 + Coccobacilli +/− Creamy, round, opaque white NAA BLBS-09 + Coccobacilli +/− Creamy, round, opaque white NAA BLBSe-01 + Rod +/− Small, round, opaque NAA BLBSe-03 + Rod +/− Small, round, opaque Bacillus pumilus BLBSe-05 + Rod +/− Small, round, opaque, lobulated Bacillus oryzicola BLBSe-09 + Rod +/− Light orange, opaque, flat growth NAA BLDSe-01 + Cocci +/− Round, opaque white NAA BLDSe-02 + Cocci +/− Round, small, opaque yellow NAA BLDSe-03 + Rod +/− Flat, transparent, round Bacillus boroniphilus BLDSe-04 + Rod +/− Round, opaque cream Bacillus pumilus BLCI-01 + Cocci +/− Small, round, opaque white NAA BLCG-01 + Cocci +/− Small, round, opaque white NAA BLCG-02 − Rod +/− Creamy, indefinite growth, opaque white NAA BLCG-05 + Rod +/− Flat, crater-like appearance, opaque white Bacillus pumilus BLCHP-01 + Rod +/− Light orange, opaque, indefinite growth NAA BLCHP-02 + Rod +/− Small, round, opaque NAA BLMS-06 + Rod +/− Small, round, opaque Bacillus paralicheniformis GUJS-02 + Rod −/− Light orange, opaque, flat growth NAA GUCHC-01 + Rod +/− Light orange, opaque, flat growth NAA GUCHC-02 − Rod −/+ Round, big, opaque NAA GUCHC-03 + Rod −/− Flat, transparent, round NAA GUWC-01 + Rod +/− Crater-like, striated appearance, opaque cream NAA GUCHS-01 + Rod +/− Round, diffuse, crater-like appearance, cream NAA GUO-01 + Rod +/− Round, opaque cream Bacillus pumilus GUO-02 + Rod +/− Small, round, opaque. Bacillus altitudinis GUHC-03 + Rod +/− Round, opaque cream Bacillus altitudinis GUHC-04 + Rod +/− Cream, irregular, small Bacillus pumilus GUHC-06 − Rod +/− Light red, irregular, small NAA GUHC-07 + Rod +/− Flat, crater-like appearance, opaque white Bacillus altitudinis PCRS1-01 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS1-02 − Rod +/− Round, diffuse, opaque orange NAA PCRS1-03 − Rod +/+ Small, round, opaque NAA PCRS1-04 + Coccobacilli +/+ Creamy, indefinite growth, opaque white NAA PCRS1-05 − Rod −/− Round, diffuse, crater-like appearance, cream NAA PCRS1-06 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus pumilus PCRS1-07 + Rod +/− Round, diffuse, crater-like appearance, cream Bacillus aerius PCRS2-02 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-03 − Rod +/+ Round, diffuse, crater-like appearance, cream NAA PCRS2-05 − Rod +/− Round, diffuse, crater-like appearance, cream NAA +Cat/Oxi, catalase/oxidase; NAA, no antagonistic activity. aFor abbreviations, see Table 1. View Large Antagonistic assay using the CSM Fifteen (35.71%) of the 42 strains had antagonistic activity against S. aureus or V. parahaemolyticus (Fig. 2). The strains GUHC-04 (an average of 16 mm of inhibition), BLCG-05 (15.7 mm), GUHC-03 (14.7 mm) and BLBSe-05 (14 mm) showed higher antimicrobial activity against V. parahaemolyticus. Strain PCRS1-07 had the best inhibitory activity against S. aureus, with an average of 20 mm of inhibition, followed by GUO-01 and BLCG-05 (18.3 mm, Fig. 3). One-way ANOVA showed a significant difference in each assay against both pathogens (P < 0.05), and post hoc comparisons using Tukey's HSD test indicated significant differences between the means of each subset (Fig. 3). Figure 3. View largeDownload slide Results of antagonistic assay. Antagonistic activity of the microbial isolates against Staphylococcus aureus and Vibrio parahaemolyticus. Results are expressed in millimetres (mm). Data were analyzed by the Tukey´s HSD test (P < 0.05). Error bars at the top of each column indicate the standard deviation of the mean. For abbreviations, see Table 1. Figure 3. View largeDownload slide Results of antagonistic assay. Antagonistic activity of the microbial isolates against Staphylococcus aureus and Vibrio parahaemolyticus. Results are expressed in millimetres (mm). Data were analyzed by the Tukey´s HSD test (P < 0.05). Error bars at the top of each column indicate the standard deviation of the mean. For abbreviations, see Table 1. Bioactive strain identification by bacterial 16S ribosomal DNA Identifying and comparing the specific ribosomal gene sequences with those deposited in GenBank (homology > 99%) showed that strains belonged to species of the Bacillus and Virgibacillus genus (Table 2). Strain PCRS1-07 was identified as B. aerius (DDBJ/EMBL/GenBank accession number KY973966), and BLCG-05 as B. pumilus (KY970152), isolated from red seaweed and wild crab, respectively. The remaining Bacillus species were B. oryzicola (BLBSe-05, KY970153) isolated from brown sediment, B. safensis (BLBS-01, KY969737) from brown seaweed, B. boroniphilus (BLDSe-03, KY970154) from dark sediment, B. paralicheniformis (BLMS-06, KY974263) from marine snail, B. altitudinis isolated from oyster (GUO-02, KY969737) and hermit crab (GUHC-03, KY970156; GUHC-07, KY970088), five strains of B. pumilus (BLBSe-03, MH54836; BLDSe-04, KY970128; GUO-01, KY969738; GUHC-04, KY969739; PCRS1-06, KY970107) isolated from brown and dark sediment, oyster, hermit crab and red seaweed, respectively, and V. senegalensis (BLBS-07, MH548361) from brown seaweed. Biofilm production test Five strains, as indicated in Fig. 4, showed a moderate capacity to form biofilm, with an OD between 0.23 and 0.57. Strain PCRS1-07 (B. aerius) produced the best adherence, followed by GUHC-07 (Bacillus altitudinis). Figure 4. View largeDownload slide Biofilm production test. Average biofilm production of the five microbial isolates selected by their antagonistic activity. Antagonists’ isolates showed a moderate capacity to form biofilm. Results are expressed in optical density (OD). Error bars at the top of each column indicate the standard deviation of the mean. For abbreviations, see Table 1. Figure 4. View largeDownload slide Biofilm production test. Average biofilm production of the five microbial isolates selected by their antagonistic activity. Antagonists’ isolates showed a moderate capacity to form biofilm. Results are expressed in optical density (OD). Error bars at the top of each column indicate the standard deviation of the mean. For abbreviations, see Table 1. Bioactivity of spent media Inhibition zones from 15 to 30 mm were recorded with the supernatants of spent media from the antagonist strains cultured on the surfaces of sterile semipermeable membranes of nylon (Fig. 5). These results of inhibition were like those gathered with the CSM. In contrast, no inhibition zones were observed with the assays performed with the negative control and supernatants of spent media of antagonist bacteria cultured in liquid media. Figure 5. View largeDownload slide Representative results of bioactivity of spent media. No inhibition zone was shown by negative control (NC) or spent media of cell-free supernatants of antagonistic bacteria cultured in liquid media (CFS). Spent media from the strains cultured as biofilm (for example, GUHC-4 and GUO-01) showed inhibition zones against Staphylococcus aureus and Vibrio parahaemolyticus. Figure 5. View largeDownload slide Representative results of bioactivity of spent media. No inhibition zone was shown by negative control (NC) or spent media of cell-free supernatants of antagonistic bacteria cultured in liquid media (CFS). Spent media from the strains cultured as biofilm (for example, GUHC-4 and GUO-01) showed inhibition zones against Staphylococcus aureus and Vibrio parahaemolyticus. DISCUSSION The present study applied mesophilic and aerobic culture conditions to identify bacterial strains with antimicrobial activity against S. aureus and V. parahaemolyticus, two important agents of food poisoning and antibiotic resistance worldwide (de Leon-Rosales Hernandez and Vidal 2015; Xu et al.2017). Rhodophyta algae were the better source of antagonistic bacteria (B. safensis, B. pumilus, B. aerius and V. senegalensis), thus increasing the list of new antagonists against both pathogens. Although Enteromorpha (Chlorophyta) was previously reported as the best source with the most epiphytic marine bacteria with antimicrobial properties (at 20°C and 7 days of incubation) against pathogenic bacteria of public health and/or veterinary concern (Lemos, Toranzo and Barja 1985), our findings were negative due to the stringency of the temperature and incubation period (33°C for 48 h). Marine sediments demonstrated the same importance as sources of antagonist bacteria with four isolates: two B. pumilus strains, B. boroniphilus and B. oryzicola. Sediment was previously reported to contain antagonistic marine bacterium (Staphylococcus sp. strain MB30) isolated from deep-sea sediment that exhibited broad-spectrum antimicrobial activity against human pathogenic bacteria and in vitro anticancer potential (Lalitha et al.2016). Anas et al. (2016) reported bioactive potential in 131 isolates from superficial sediments of the southeast Arabian sea from the phyla γ-Proteobacteria (63%), Bacillales (34%) and Micrococcaceae (3%), which were bactericidal to the human pathogens, Escherichia coli and Pseudomonas sp., while 20–30% of these were bactericidal to Vibrio sp. and Staphylococcus sp. Eight isolates, belonging to Pseudomonas spp., Bacillus sp. and/or Lysinibacillus sp., displayed strong bactericidal/cytotoxic properties. Marine sediment was followed by hermit crabs with three different isolates of B. altitudinis, and B. pumilus (Table 1), which appeared to be the first report of C. panamensis associated with antagonism against clinically important pathogens. Additionally, only one antagonistic isolate each was obtained from Callinectes sp. and marine snails (B. pumilus and Bacillus paralicheniformis, respectively). The current study is the first report of antagonists associated with these hosts, specifically antagonists of S. aureus and V. parahaemolyticus. The ANOVA and Tukey's HSD test showed highly significant differences between the antagonist and pathogen challenge tests by CSM, which showed reproducibility, independent of the replicate number for both pathogens (P < 0.05). The CSM was chosen as the best technique for screening the microorganism's ability to produce antimicrobial compounds by providing qualitative information on marine microorganismal growth inhibition, to evaluate a large number of different bacterial strains in a short time (Brito de Asis et al.2016). In this survey, Bacillus and Virgibacillus species were the unique isolates. A higher number of species could have been identified by using different culture media, anaerobic conditions, and different incubation temperatures and times, which would reveal a great diversity of microorganisms as psychrotrophic, psychrophilic or thermophilic bacterial populations (Faghri et al.1984; Talpur et al.2011; Anas et al.2016). Bacillus is frequently reported to have antagonistic activity, as they produce many antimicrobial substances (Sharma, Kapoor and Neopaney 2006), but Vigibacillus sp. has only been reported as an antagonist of S. aureus (Gad 2017). Herein we identified species with antagonistic effects against S. aureus (a Gram-positive) and V. parahaemolyticus (a Gram-negative). Bacillus strains have been screened for their potential antimicrobial activity against pathogenic bacteria and their ability to produce a host of enzymes, antibiotics, antimicrobial peptides, lipopeptides, iturines, polymyxins, fengycins and, mainly, bacteriocins and surfactins; the last two compounds are responsible for reducing Gram-positive and Gram-negative pathogen populations (Cole et al.2006; Line et al.2008; Kim et al.2009; Ravindran et al.2016). Bacillus has further beneficial applications: lipopeptides, lantipeptides and cyclical peptides with antimicrobial activities are reported to be active metabolites of B. pumilus and B. paralicheniformis (Collins et al.2016). Additionally, a strain of B. altitudinis was found to be an excellent antagonist against the oomycete, Phytophthora nicotianae (Jin et al.2011). Bacillus aerius has not been directly reported to have antagonistic activity, but some reports reveal that it produces thermophilic lipase with antitumor properties, making it a possible candidate for therapeutic applications (Saun et al.2014; Saun, Mehta and Gupta 2014). This is the first report from marine ecosystems in Mexico of Bacillus against bacteria of public health concern, where the highest inhibition was demonstrated by the strains PCRS1-07 and BLCG-05 (B. aerius and B. pumilus) against S. aureus and V. parahaemolyticus, respectively. Also, this is the first report of B. aerius, B. altitudinis, B. oryzicola, B. safensis and B. boroniphilus being isolated in Mexico. The presence of microorganisms with antimicrobial properties from Mexican marine ecosystems adds to the scarce information available on this subject by Cetina et al. (2010), Villarreal-Gómez et al. (2010), and Torres-Beltrán et al. (2012). It has been demonstrated that cells attached to agar surfaces enhanced the production of antibacterial compounds that planktonic cells could not produce. Bacterial growth on agar (like in the CSM assay) constitutes a biofilm-like growth. Bioactivity of spent media from the antagonistic strains cultured as biofilm was demonstrated with the inhibition zones observed in the assays, however spent media of bacteria cultured in liquid media did not show any activity (Yan, Boyd and Burgess 2002; Yan et al.2003). Bacterial strains that are good biofilm producers may be used to produce inhibitory substances (Avendaño-Herrera, Lody and Riquelme 2005) in areas such as public health, aquaculture and agriculture, where using substrates from Bacillus strains represents an important approach in these practices that are currently affected by new pathogenic microorganisms (Stein 2005; D’Alvise et al.2014). The biofilm production results in this study showed a potential use for these strains on a larger scale by applying the active metabolites that they produce (Yan, Boyd and Burgess 2002; Yan et al.2003). We reported five B. pumilus strains that had antimicrobial capability, and this is the most important species with previously reported antimicrobial properties against several pathogens (V. parahaemolyticus, V. anguillarum, V. alginolyticus, Listeria innocua, L. monocytogenes and S. aureus) (Leyton et al.2012; Zidour et al.2017). Thus, Bacillus species are excellent candidates with environmental ubiquity, persistence and stability under extreme conditions as their spores can tolerate and proliferate various environmental conditions to inhibit and compete against other potential human pathogens (Prieto, O’Sullivan and Tan 2014). In conclusion, these marine isolates may be a future research option against other clinically relevant bacteria. Using marine microorganisms to fight infectious diseases is a research field that continues to grow. These isolates can inhibit pathogenic bacterial growth, making them candidates for developing new substances that have antimicrobial activity. Their active compounds are currently being studied and tested. Acknowledgements We are very grateful to Dr. Cuauhtémoc Ibarra Gámez for his excellent technical support regarding the samples collected in Sonora, Mexico. We thank Vida Mariel Molina-Garza and Lucio Abraham Galaviz-Molina for their support throughout the proofreading and editing process. FUNDING This work was supported by the PAICyT (Programa de Apoyo a la Investigación Científica y Tecnológica, UANL), grant number SA170-15 (Microbiota from marine ecosystems and their role as antagonist against pathogens) and grant no. 3157 from FOINS-CONACyT (Fondo Institucionala del Consejo Nacional de Ciencia y Tecnología) to LGS. 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TI - Bacillus and Virgibacillus strains isolated from three Mexican coasts antagonize Staphylococcus aureus and Vibrio parahaemolyticus
JF - FEMS Microbiology Letters
DO - 10.1093/femsle/fny202
DA - 2018-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/bacillus-and-virgibacillus-strains-isolated-from-three-mexican-coasts-ax0msk8bKc
VL - 365
IS - 19
DP - DeepDyve
ER -