Introduction

 

The genus Pantoea is ubiquitous, it is well known for more than ten years as the genus belonged to Enterobacteriaceae and it resembles species of the genera Erwinia and Enterobacter (Akshita et al., 2020; Mirza et al., 2020; Suhartati et  al., 2021). Pantoea is facultative anaerobic Gram-negative bacilli with peritrichous flagella (AbdAlhussen & Darweesh, 2016). The cultural and biochemical characters and phylogenetic approach are still mainly used to identify various Pantoea spp. Some species of the genus Pantoea have beneficial effects as a producer of antibiotic like pantocin A and B while others infect some plants, but the diversity of Pantoea strains and their possible association with hosts and disease are still to be demonstrated (Cruz et al., 2007; Walterson & Stavrinides, 2015). Its role in phosphorus solubilization which is an important macronutrient, needed for all plant growth is well known. Thus, the solubilization of organic and inorganic insoluble soil phosphate by the eco-friendly bacterium is necessary for agricultural uses (Hegyi et al., 2021; Madrid-Delgado et al., 2021; Zhang et al., 2022).

Pantoea species have been isolated from soil, water, eucalyptus leaves (as epiphytes or endophytes bacteria) and plant seeds and fruits, and, four Pantoea species were isolated and showing symptoms of blight and die-back on the plant shoot while Pantoea allii was recovered from onion leaf and seed (De Maayer et al., 2014; Sheibani-Tezerji et al., 2015; Achbani et al., 2016; Ma et al., 2016; Palmer et al., 2016). Other members of the genus Pantoea have been isolated from animals and human wounds and may be opportunistic pathogens. Some species were isolated from human and animal gastrointestinal tracts, feces, dairy products, human blood, and urine. Also, three new Pantoea species were isolated from infant formula. They cause some human infections like septic arthritis and osteomyelitis. They can live as opportunistic pathogens in the immunocompromised human, causing a wound, blood, and urinary tract infections (Brady et al., 2010). The species of the genus Pantoea included P. agglomerans, P. ananatis, P. citrea, P. dispersa, P. punctata, P. stewartii, and P. terrea were isolated and identified (Schmid et al., 2003). In recent years, a variety of human diseases due to Pantoea species are increased and nosocomial infections by this organism were reported in different hospital intensive care units, burn, hemodialysis, and oncology unit. Pantoea spp. was isolated during an outbreak in a Malaysian neonatal intensive care unit in 2006 (Van Rostenberghe et al., 2006; Liberto et al., 2009). In India, Tiwari and Beriha (2015) isolated P. agglomerans from neonatal sepsis and it is difficult to treat this bacterium. In Iraq, different antibiotics resistance Pantoea spp were isolated from different clinical and environmental sources (AbdAlhussen & Darweesh, 2016).

During the past decade, increasing antibiotic resistance to currently used drugs cause high morbidity and mortality throughout the world, and novel alternative antibiotics with the heterocyclic structure are needed. Actinomycetes, the filamentous Gram-positive, aerobic spore-forming bacteria had DNA rich in GC content (60-80 %) and is the most attractive bacteria with abilities to produce unique or novel bioactive compounds, beneficial to humans against some pathogens like multidrug-resistant bacteria, Serratia marcescens, Staph aureus and MRSA (Khamna et al., 2010; Genilloud, 2017; Kemung et al., 2018; Takahashi & Nakashima, 2018). The soil had microbial diversity of Actinomycetes which form extensive branching substrate and aerial mycelia and play significant geochemical roles in soil. They are highly valued for their capacity to form biological secondary metabolites with antimicrobial activities and today, most of the used antibiotics are Actinomycetes natural secondary products (Barton, 2006). On another hand, unexplored soil areas have a significant potential for exploring new bacterial isolates that present under increased competition in the environment like limited nutrient content and producing antimicrobial substances against each other. Some Streptomyces isolates produce many secondary metabolites with excellent inhibitory activities against different human microbial pathogens (Sadoway et al., 2013; Al-Ansari et al., 2019). Production of bioactive agents or antibiotics by actinobacteria with antibacterial and/or antitumor by members of the genus Streptomyces, Amycolatopsis, Micromonospora, and Nocardia were documented (Nakaew et al., 2009; Arifuzzaman et al., 2010; Rangseekaew & Pathomaree, 2019). Out of 22,000 bioactive secondary metabolites, 16,000 compounds were selected as bioactive antibiotic activities and about 45% of these compounds were from Actinomycetes, and more than 8000 useful bioactive compounds were from the genus Streptomycetes (Berdy, 2005; Khamna et al., 2010). From soil, S. coelicolor and S. violaceoruber produce an important antibiotic actinorhodin with a wide range of applications in the medical sector (Palanichamy et al., 2001). Streptomyces sp. SM01 was isolated from Indian soil and it produced a novel antibiotic picolinamycin which was effective against multi-drug resistant bacteria (Maiti et al., 2020). The present study was focused on the isolation and identification of Pantoea species from soil and its biocontrol by soil actinomycetes.

MATERIALS AND METHODS

Collection of soil samples

Soil samples were collected in sterilized bags from different depths, 5, 15, and 20 cm, from contaminated soil, collected from Jeddah, Saudi Arabia. Soil samples were kept under 4ºC until used for bacterial isolation on nutrient agar using serial dilutions methods. The obtained bacterial colonies were selected, purified, and preserved on the same medium.

 

Phosphate solubilization and indole production

All the bacterial isolates were screened for phosphate solubilization on phosphate mineral agar containing g/l: 10 glucose, 5 Ca₃(PO₄)₂, 5 MgCl₂.6 H₂O, 0.25, MgSO₄ 7H₂O, 0.2 KCl, 0.1 (NH₄)₂SO₄ (Nautiyal, 1999) and all plates were incubated for 5 days at 30°C. The colonies with clear halo zones were considered to be phosphate-solubilizing bacteria and the mean diameter of the clear zone (mm) around the tested bacterial colony was measured (Janardan & Verma, 2012). The bacterial growth was measured and represented as +++ (high growth) and ++ (moderate growth). The positive bacteria with clear zone were selected for indole acetic acid production (IAA) in a medium supplemented with 2 mg/ml of L-tryptophan and IAA was extracted from the culture filtrate with ethyl acetate and the quantity was detected according to the method described before by measuring the A_{530 nm} and preparing a standard curve of IAA (Bano & Musarrat, 2003; Ahmed et al., 2004). IAA-producing Azotobacter sp. was obtained from the culture collection of the faculty of Science, KAU, Jeddah, Saudi Arabia, and used as the reference strain.

 

The used actinomycete isolate

The isolate Streptomyces coeruleorubidus MM5 was previously isolated from the industrial zone, Jeddah (Aburas et al., 2021) and kindly provided from the culture collection of the faculty of Science, KAU, Jeddah, Saudi Arabia. It was grown on a Starch Nitrate agar medium (Shirling & Gottlieb, 1966), examined under light and scanning microscopes at the KHMR center, KAU, and preserved on the same medium.

 

Preparation of the bacterial suspension

The bacterial suspension was prepared in a sterile Falcon tube containing 3 ml of sterile nutrient broth medium, these tubes were incubated at 30˚C for 24 hrs and growth was measured at OD _{520 nm} using Spectrophotometer and it was adjusted to 0.5.

 

Screening of Actinomycetes for antimicrobial activity

The Actinomycete isolate, Streptomyces coeruleorubidus MM5 was screened for inhibitory substances production against the tested bacterium MO11 using different methods. Primary screening using Cross streak method is applied by inoculating a single streak of the selected isolate on the center of the Mueller Hinton agar plate and incubating for 5 days at 30ºC, then the plates are seeded with the isolate MM11 in a perpendicular arrangement and the plates were re-incubated again for 24 hrs (Oskay, 2009). Also, the Agar plug diffusion method was used to determine the inhibitory activity of the selected actinomycete against the isolate MO11. By sterile cork borer, a disc of 8 mm diameter of bacterial growth was put on the center of the Mueller Hinton agar plate inoculated with the tested pathogen MO11. The antimicrobial activity of the bacterial growth disc was detected by the inhibition zone (mm). Also, the selected actinomycete, S. coeruleorubidus MM5 was grown on Starch Nitrate agar medium at 30˚C for 5 days at 80 rpm and the culture supernatant was filtrated sterilized through bacterial filter 0.22 µm and the filtrate was extracted with ethyl acetate (1:1V/V). The extract was dried and dissolved in DMSO. Then, 100 µl of the extract was used to fill each agar well with dimmer 6 mm on Muller Hinton agar plates or blood agar inculcated by the tested bacterium, and the plates were incubated at 30˚C for 24 hrs. The mean diameter of the produced inhibition zone was determined (Magaldi et al., 2004; Valgas et al., 2007).

 

Characterization of the selected bacterium

The morphological characterization and biochemical tests were carried out for the most active bacterial isolate MO11. The selected isolate was examined on Nutrient, MacConkey, and blood agar plates after incubation at 30˚C under aerobic conditions for 2 days. Cells were examined under a light microscope using an oil immersion lens after Gram staining. Catalase test, oxidase test, starch hydrolysis, coagulate test, blood hemolytic, and antibiotic sensitivity were also recorded. VITEK-2 system at KAUH was used for bacterial identification. Antibiotic susceptibility testing was carried out on Mueller-Hinton agar by "The Kirby-Bauer disc diffusion method. The fresh bacterial suspension was prepared with OD 0.5 and inoculated on Mueller-Hinton plates and antibiotic discs were applied o the agar surface. All the inoculated plates were incubated at 30◦C for 24 hrs, and the susceptibility of the tested isolate to each antimicrobial agent was recorded.

 

Molecular identification of selected isolate

The selected isolate MO11 was grown on Nutrient agar medium for 2 days at 30˚C, then, DNA was extracted, purified, and amplified using twp primers. After PCR, the sequences of the 16S rRNA were determined at Macrogen. The information on the used primer was summarized in Table 1.

 

Table 1. Primer used for molecular identification of the isolate MO11

The information on the used Primers
PCR (primer name and sequence)	Sequence ( primer name and sequence)
27F 5' (AGA GTT TGA TCM TGG CTC AG) 3	785F 5' (GGA TTA GAT ACC CTG GTA) 3'
1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3'	907R 5' (CCG TCA ATT CMT TTR AGT TT) 3'

 

Statistical analysis

Statistical Package for Social Science (SPSS for windows, version 17) was used for comparing the data, a mean value of three reading ± SD was used and a t-test was used to detect any significant difference between the sample and control. Significant results were obtained at P<0.05.

rESULTS AND DISCUSSION

 A bacterium was isolated from soil samples, collected from the contaminated area. Out of 20 bacterial isolates obtained on Nutrient agar, 7 isolates were phosphate-solubilizing bacteria and they had different abilities to produce IAA (Table 2). The isolate MO11 was the most active isolate, thus it was selected for more detailed studies.

Under light microscopic, the selected isolate MO11 appeared as Gram-negative motile bacilli (Figure 1a). It grows well on different agar media, Nutrient agar (Figure 1b), MacConkey agar, and blood agar plates. It appeared as punctuate circle smooth colonies, middle in size, while on blood agar it produce smooth non-hemolytic colonies. It had positive results for catalase, indole, and citrate production and fermentation of Raffinose. It showed negative results to produce H₂S and other biochemical reactions had variable results (Table 3). The isolate MO11 was resistant to Amoxicillin, Amikacin, and Gentamicin while it was sensitive to Imepinem and Meropenem (Figure 1c). The isolate MO11 was identified by an automated VITEK-2 compact system using GN-ID cards as Pantoea spp. A phylogenic analysis was performed to identify the selected isolate MO11using a partial sequence of the 16S rRNA which showed a high level of sequence similarity with member genus Pantoea with a homology level of 97% to P. calida as in the phylogenic analysis

 

Table 3. Biochemical test for identification of Pantoea sp.MO11

Test	Result	Test	Result
Shape	Small rod	Citrate	+
Catalase	+	VP	+
Oxidase	-	Gas	+
Gelatinase	-	H2S	-
Hemolysis	γ	Urease	+
Motility	+	Indole	+
Fermentation of Raffinose	+	MR	-

 

a)

b)

c)

Figure 1. The isolate MO11, a) After Gram stain, b) grown on nutrient agar, c) resistance of the isolate to some antibiotics.

 

 

Figure 2. The phylogenic tree of the isolate MO11 and the most related isolates

 

a)

b)

c)

Figure 3. Streptomyces coeruleorubidus MM5, grow on starch nitrate agar (a) for 7 days, under a scanning electron microscope (b) and after Gram stain (c).