Isolation of a broad spectrum antibiotic producer bacterium, Pseudoalteromonas piscicida PG-02, from the Persian Gulf

Materials and Methods

Sample collection

Different samples including coastal water, surface water, deep water, coastal sediment, bed sediment and mangrove forest sediment were collected from 21 study sites in some northern area of Persian Gulf, during February 2008 to November 2009 (Figure 1 and Table I). Water samples were harvested by strelized-niskin bottle (using 70% ethanol prior to sampling) and subsequently samples were collected in sterilized glass bottles. Sediments were collected by sterilized van veen grab into sterilized plastic bags. All of the samples were kept at 4°C till to delivering to the laboratory.

Figure 1: Map of sampling sites (1: Bahrakan port, 2: Mahshahr port, 3: Bushehr port, 4: Qeshm island) in the Persian Gulf

Table I: The geographical characteristics and physicochemical properties of the major location of sampling sites in the Persian Gulf

Isolation of bacteria from marine samples

For isolation of marine bacteria from water samples, 8 uL from each sample was inoculated into Marine Agar 2216 (MA, Highmedia). Sediment samples were serially diluted using sterilized sea water (1:10 v/v ratio) and spread on the entire surface of marine agar 2216. Also, isolation of marine bacteria from both water and sediment samples was performed using pour plate technique. After incubation at 30°C for 3 days, developed colonies were purified by repeatedly subculturing on marine agar and finally, bacterial colonies with distinct characteristics such as pigmentation, size, opacity, elevation, margin and surface appearance (yeon et al., 2005) were chosen for further process.

Production medium and preparation of extract

For screening of the antibiotic producing bacteria, pure colonies were transferred to Erlenmeyer flasks containing Marine Broth (MB, Laboratorios CONDA) medium and were incubated at 30°C on a rotatory shaker (140 rpm) to produce antibiotic compounds. After 2-5 days, the broth culture was centrifuged at 10,000 rpm for 15 min at 4°C, and then supernatant was extracted using same volume of ethyl acetate. Solvent was removed at 37°C.

Selected human pathogens

A broad spectrum of human pathogenic bacteria including Standard (reference) and clinical isolates was selected for this study. Gram-positive bacterial species were Staphylococcus aureus (ATCC 6538), Staphylococcus epidermidis (ATCC 12228), Bacillus subtilis (ATCC 12711), Listeria monocytogenes (ATCC 19112), MRSA (Methicillin Resistant Staphylococcus aureus) (clinical), Staphylococcus epidermidis (clinical), Bacillus anthracis (clinical), Bacillus cereus (clinical), Bacillus pumilus (clinical), L. Monocytogenes (clinical), and Streptococcus pyogenes (clinical). Gram negative bacterial species were Escherichia coli (ATCC 11303), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (ATCC 19430), P. aeruginosa (clinical), E. coli (clinical), Proteus mirabilis (clinical), Brucella melitensis (clinical), S. typhi (clinical), Bordetella bronchiseptica (clinical) and Klebsiella pneumoniae (clinical). Standard strains were prepared from Iranian Research Organization for Science and Technology (IROST); and clinical species were originally isolated from clinical specimens and identified by standard biochemical reactions.

Screening for antibacterial activity

Freshly grown colonies of tested bacterial pathogens were used to inoculate Muller Hinton Broth (MHB, Merck) medium at 37°C for 5-8 hours until turbidity of the suspension was adjusted to the Mc Farland 0.5 turbidity standard (10⁸ cfu/mL) (Mulks et al., 1990; Asthana et al., 2009). Antibacterial activity was assessed two times by disc diffusion method. A total of 0.1 mL of bacterial suspension was poured on each plate containing Muller Hinton Agar (MHA, Merck). The lawn culture was prepared by sterile cotton swab and allowed to remain in contact for 1 min. The dried crude extract was dissolved in ethyl acetate to a concentration of 50 mg/mL. The sterile filter paper discs (6 mm diameter) (Kanoh et al., 2008) were saturated by 35 uL of this concentration of each extract and then were placed on lawn cultures. The Petri dishes were subsequently incubated at 37°C for 24 hours and the inhibition zone around each disc was measured in mm. Nine different antibiotics including vancomycin 30 ug, erythromycin 15 μg, methicillin 5 ug, carbenicillin 100 μg, nitrofurantoin 300 ug, penicillin 10 ug, oxacillin 1 ug, nafcillin  1 ug and streptomycin 10 ug were used as control. All these synthetic antibiotic discs were produced by Difco.

MIC and MBC determination

The MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) of ethyl acetate extract from the most effective bacterial strain were determined against two somewhat important and more sensitive bacteria. MIC was determined by the macro broth dilution assay method (Motamedi et al., 2010). In the tube dilution assay, standard bacterial suspension and different concentrations of extract (5, 10, 20, 40, 80, 160, and 200 mg/mL) were added to tubes containing 1 mL MHB medium. These tubes were incubated at 37°C for 24 hours. The first tube in the above series without sign of visible growth was considered as the MIC. MBC was determined by culturing one standard loop from tubes with no apparent growth on MHA and subsequent incubation at 37°C for 24 hours. The least concentration that inhibited colony formation on agar was considered as MBC for the extract.

Kinetics of growth and antibiotic production

Kinetic of growth and antibiotic production was studied in a batch culture system in a 250 mL Erlenmeyer containing 100 mL marine broth and at 30°C on a rotatory shaker (140 rpm). Kinetics of growth or biomass accumulation measured according to measuring of optical density (OD) at 600 nm. At first, the best antibiotic producing bacterium was grown in marine broth. Serial samples were harvested from production medium at 2 hours intervals from 0 till 6 hours and 4 hours intervals from 6 hours till entering stationary and death phases of growth. Supernatants were harvested  from these samples by centrifugation at 8,000 rpm at 4°C for 10 min and subsequently were sterilized by filtration 0.45 µm and then used for antibacterial assay (Shibl and Sowaygh, 1980). Antibiotic activity of samples was assessed against MRSA using disc diffusion method.

Determination of growth curve interference (GCI)

GCI is a parameter which defined as the lowest concentration of an antibiotic compound that modifies the growth curve of tested pathogen compared with a control without antibiotic (Castillo et al., 2008). Tested pathogen in this study was MRSA. At first, four 250 mL flasks containing 15 mL MHB medium was prepared, and then freshly colonies of MRSA from a 24 hours culture on Nutrient Agar (NA, Merck) was inoculated in medium until turbidity of the suspension adjusted to the Mc Farland 0.5 turbidity standard. Also, 15 mL of crude extract from marine isolate at four concentrations equal to and below the MIC was prepared. A flask containing 30 mL MHB with the Mc Farland 0.5 turbidity of MRSA but without extract was used as control. Finally, each of the prepared extracts were added to one of the flasks and then incubated at 37°C on a rotatory shaker (110 rpm); biomass accumulation measured (every 1 hour) according to measuring optical density (OD) at 600 nm until entering stationary and death phases of growth. Related curves were drawn and GCI was determined.

Morphological and biochemical characterization of antibiotic producer bacterium

The isolated marine bacterial strain with antibacterial activity was identified based on morphological and biochemical characters according to the methods described in Bergey's manual for systematic of bacteriology (Garrity et al., 2006). Definite identification was done based on 16S rRNA gene sequencing.

Identification based on 16 S rRNA gene sequencing

In order to genomic DNA extraction, a single colony of intended bacterium was sub-cultured in MB and incubated at 30°C for 48 hours. The genomic DNA was extracted by a commercial kit (Genfanavaran, Iran), according to the manufacture instruments. Full length sequence of 16S rRNA genes was amplified from the isolated genomic DNA using the following universal bacterial 16S rRNA primers: Forward strand: 5´-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG and reverse strand: CCCGGGATCCAAGCTTA-CGGTTACCTTGTTACGACTT-5 (Papizadeh et al., 2010). Polymerase chain reaction (PCR) was performed in a 50 uL reaction mix (mixture) containing 50 mM MgCl₂, 10 mM each deoxynucleoside triphosphate (dATP, dTTP, dGTP and dCTP) dNTP, 10x PCR buffer, 10 umol/μL of each primer, and 5 U of Taq polymerase. The PCR thermal profile was consisted of denaturation at 94°C for 5 min followed by 30 cycles of amplification, each cycle consisting of a denaturation at 94°C for 1 min, annealing at 62°C for 40 sec, and chain elongation at 72°C for 2.5 min. A final elongation step at 72°C for 20 min was included. The Electrophoresis of PCR product was done on 1% agarose gel for 55 min at 95 v/cm in order to confirmation of the 16S rRNA amplification (Figure 2). PCR product was sequenced by Genfanavaran biotech corp. Alignment of sequences was done by CLC Main Workbench 6.0 software program. The obtained sequences were compared to those available in the Genbank by BLAST searches (http://www.ncbi.gov/blast). Finally, phylogenetic tree was constructed by CLC Main Workbench 6.0 software program using the neighbor-joining algorithm (Saitou and Nei, 1987). Also, the obtained sequence from intended antibiotic producer bacterium was compared to the sequence of its closest phylogenetic neighbor strain by chimera check/pintail program (http://www.bioinformatics-toolkit.org/Web-Pintail/). 

Figure 2: Agarose gel electrophoresis of amplified 16 S rRNA fragment (1500 bp) of PG-02 isolate

Morphological observation of antibiotic producer isolate

Surface morphology study of intended bacterium was done using scanning electron microscopy (SEM). A single colony of antibiotic producer bacterium from 24 hours culture on marine agar was dissolved in 5 mL sterile distilled-water and subsequently 2 uL from this suspension was harvested and dried at room temperature. The prepared sample was coated by silver for 7 min and surface morphology was studied by Leo1455 Vp scanning electron microscopy.