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The present study was conducted to characterize a collection of plant growth-promoting (PGP) Rhizopseudomonas isolated from date palm roots for their biosurfactant production ability, heavy metal tolerance, and antimicrobial susceptibility. A collection of 36 bacterial strains was evaluated for several plant growth-promoting abilities including indole acetic acid (IAA) production, mineral phosphate solubilization, siderophores and ammonia release, and protease and cellulase activity. Biosurfactant produc- tion was screened throughout hemolytic activity, bleu agar test, and drop collapse method. Strains exhibiting tolerance to heavy metals at high concentration were subjected to PCR for the detection of heavy metal gene tolerance. Moreover, antimicrobial susceptibility patterns were determined using the disk-diffusion method. High rates of plant growth promotion activities were registered among the isolated rhizopseudomonads, mainly, the indole acetic acid production (88.8%), phosphate solubilization (63.8%), siderophore release (83.3%), and ammonia synthesis (52.7%). Furthermore, biosurfactant production was recorded, using three distinct methods, on 77.7% of the tested strains. Particularly, two strains affiliated to Pseudomonas vancouverensis and Pseudomonas brassicacearum harboring copA-arsR and arsB genes, respectively, and producing biosurfactants, were selected. The evaluation of antibiotic resistance dissemination risk analysis in the environment revealed a low rate of resistance among the analyzed strains and the absence of known antibiotic resistance genes. This investigation could provide the basis for the development of microbial inoculums showing multifarious properties for biotechnological application while caring human health. . . . . Keywords Pseudomonas sp. PGP Biosurfactant Heavy metal Antimicrobial resistance Introduction inhabiting this interface are well-known for enhancing plant growth and were referred as plant growth-promoting (PGP) Plant rhizosphere supports wide range of active microbial rhizobacteria (Kloepper et al. 1980). population influenced by root exudate release. Rhizobacteria PGP rhizobacteria have the ability, as an alternative to chemical fertilizers, to promote growth and yield of crop plants via direct and indirect mechanisms. They directly en- hance crop fitness by plant growth regulator production such * Hadda-Imene Ouzari as phytohormones, the improvement of plant nutrient uptake firstname.lastname@example.org through nitrogen fixation, phosphate solubilization, Laboratoire de Microorganismes et Biomolécules Actives siderophore release, and 1-aminocyclopropane-1-carboxylate (LR03ES03), Faculté des Sciences de Tunis, Université Tunis El (ACC) deaminase synthesis, which can lower plant ethylene Manar, Campus Universitaire, 2092 Tunis, Tunisie levels. Indirect impacts are related to production of metabo- Department of Biochemistry and Molecular Biology, University of lites (e.g., antibiotics, siderophores, cellulases, HCN) and La Rioja, Madre de Dios 51, 26006 Logroño, Spain competition for nutrients that prevent the growth of phyto- Present address: Institute of Food, Nutrition and Health (IFNH), pathogens and other deleterious microorganisms (McCully ETH, Zurich, Zurich, Switzerland 2005;Saharan andNehra 2011). Laboratory of Biotechnology and Bio-Geo Resources Valorization, The study of beneficial association between microorgan- Higher Institute for Biotechnology, University of Manouba, Sidi isms and plants has been extended to environmental Thabet Biotechpole, 2020 Sidi Thabet, Ariana, Tunisia 52 Ann Microbiol (2019) 69:51–59 bioremediation. Indeed, the increase of industrial activity mobilizes them by pseudosolubilization and emulsification. causes environmental contamination by a wide variety of haz- On the other hand, during the application of biosurfactants ardous chemicals, which present a serious threat to human on heavy metals, they create complexes with metals at the soil health (Soccol et al. 2003). One of the emerging strategies to interface, followed by desorption of the metal and removal cope with chemically polluted soil is plant-assisted bioreme- from the soil surface; such process is facilitated by the chem- diation or phytoremediation, an eco-friendly and cost- ical interactions between the amphiphiles and the metal ions effective technology, but the derived processes are rather slow (Banat 1995;Okoro andAkpabio 2015). with lower efficiency compared to other conventional tech- Among Gram-negatives, soil bacteria Pseudomonas is con- niques such as soil excavation and disposal to landfill sidered as the most abundant genus in the rhizosphere; they (Harbottle et al. 2007; Xuliang et al. 2007). are able to colonize a wide variety of ecological environments. Synergistic use of plants and microbes has been reported to Their successful application as crop inoculants for prompting play a significant role in accelerating phytoremediation plant growth and phytoremediation is well documented, pre- (Kuiper et al. 2001, 2004; Al-Awadhi et al. 2009). Root sys- senting them as a suitable alternative for chemical applications tem forms a dynamic root-soil interface influenced by root (Patten and Glick 2002; Jangu and Sindhu 2011; Praveen et al. exudates, as mentioned previously, allowing associated micro- 2012; Kaur and Sharma 2013). organisms to increase the availability of contaminant com- Nevertheless, the problem of antibiotic gene resistance dis- pounds; in turn, plants help adsorbing and removing the semination among environment has been omitted. We know resulting pollutants (Compant et al. 2010). for a fact that bacterial resistance to antibiotics is with increas- Among these pollutants, heavy metals (e.g., Mn, Cu, Cr, ing concern, the environment offers a reservoir of antibiotic Hg, Zn, Fe, Cd, Pb, As, etc.) are probably the most toxic to gene resistance which might be transferred to other bacterial living organisms. In fact, they are easily assimilable, and at strains and confer novel resistance mechanisms (Meireles high concentration, they are likely to interfere with key meta- et al. 2013). Pseudomonas species are naturally resistant to a bolic pathways (Narayanan and Devarajan 2012). Owing to wide range of antibiotics as ampicillin and derivatives, ceph- the wide range of metabolites released into the rhizosphere alosporins (many of them with some exceptions as ceftazi- (e.g., siderophores, biosurfactant, organic acids, plant growth dime or cefepime), chloramphenicol and trimethoprim, regulators, etc.), microorganisms can alter the bioavailability among others. Nevertheless, Pseudomonas can also acquire of heavy metals, indirectly, through their effects on plant mechanisms of resistance to other antibiotics, and resistance growth dynamics, and directly, through precipitation, acidifi- genes can be included in integrons and mobilized by different cation, chelation, complexation, and enzymatic detoxification genetic elements (Viedma et al. 2014). So, keeping this in and reduction (Silver 1996; Ben Tekaya et al. 2014). view, the application of Pseudomonas inoculums into the en- In addition to heavy metals, rhizospheric microorganisms vironment may be a hidden hazard of antibiotic gene resis- can act directly on organic pollutants such as petroleum hy- tance emergence that has to be evaluated. drocarbons, chlorinated solvents, polycyclic aromatic hydro- Against these backdrops, this investigation aimed to select carbons, and pesticides via their own degradative abilities un- efficient Pseudomonas inoculums for plant growth promotion der the right selection of plant/bacteria partnerships (Chin and and phytoremediation experiments caring out of antibiotic re- Dekkers 2000). sistance evaluation. The application of bacterial biosurfactants in the field of environmental bioremediation has gained importance due to their high biodegradability, low toxicity, and ecological ac- ceptability. Biosurfactants are microbial surface active agents Materials and methods containing hydrophobic (non-polar) and hydrophilic (polar) parts that reduce surface tension and interfacial tensions be- Origin of Pseudomonas strains tween individual molecules at the surface and interface, re- spectively. They are excellent emulsifiers, foaming and dis- A collection of 36 Pseudomonas strains affiliated to 15 differ- persing agents regarding their surface activity (Banat et al. ent species were isolated from rhizospheric soil of date palm 2010; Pacwa et al. 2011). (Phoenix dactylifera L.) from 7 different oases in the southern Biosurfactants produced by microbes such as part of Tunisia. Bacterial strains were retrieved from three rhamnolipids, sophorolipid, and surfactin have the ability to culture media: TSA (tryptic soy agar), YEM (yeast extract effectively solubilize, emulsificate, and mobilize both heavy mannitol), and KB (King’B agar), incubated during 48 h at metals and organic compounds. 30 °C. A total of 36 strains were characterized by 16S rRNA For organic compounds such as hydrocarbons, the applica- gene sequencing. The 16S rRNA gene sequences were sub- tion of biosurfactant-producing microbes increases the bio- mitted to the NCBI nucleotide database (accession number availability of organic compounds such as hydrocarbons and summarized in Table 1)(Ferjanietal. 2015). Ann Microbiol (2019) 69:51–59 53 Plant growth-promoting activities metals (arsenic (Na2AsO4H·7H2O), copper (CuSO4), cadmi- um (CdCl2), zinc (ZnCl2), and cobalt (CoCl2)), plates being The 36 bacterial strains were assessed for their PGP abilities incubated at 30 °C for 48 h according to Narasimhulu et al. through the screening of indole acetic acid (IAA) production, (2010). Pseudomonas strains that were able to grow in pres- mineral phosphate solubilization, siderophores and ammonia re- ence of 100 μg/ml of a heavy metal were further grown at lease, and protease and cellulase activity. Quantitative production higher metal level by gradually increasing the concentration of IAA was determined in the original liquid medium of tested of heavy metal to 200 μg each time. Strains exhibiting toler- strains supplemented with L-tryptophan (100 mg/l) as described ance to heavy metals at high concentration were subjected to by Bric et al. (1991). The ability to solubilize inorganic phos- PCR for the detection of heavy metal gene resistance and phate was evaluated on Pikovskaya’s agar medium amended sequencing of positive amplicons for confirmation. The con- with tricalcium phosphate according to Mehta and Nautiyal ditions for PCR amplification were as follows: an initial de- (2001). To detect the production of siderophores, bacterial strains naturation step of 95 °C for 3 min, followed by 30 cycles of were spotted on nutrient agar plates. After incubation at 30 °C for 95 °C for 1 min; 55 °C for 1 min; 72 °C for 1 min plus a final 48–72 h, the grown strains were overlaid with CAS medium extension of 10 min at 72 °C for ars operon (arsC, arsB, arsC, supplemented with agarose (0.9% w/v). Positive strains produc- arsD, arsR) and an initial denaturation step of 94 °C for 3 min, ing siderophores were noted when a color modification around followed by 30 cycles of 94 °C for 1 min; 58 °C for 1 min; colonies from blue to orange was observed (Miranda et al. 2007). 72 °C for 1 min plus a final extension of 5 min at 72 °C for Ammonia synthesis was determined by inoculation of bacterial copper (copAgene). strains in 10 ml of peptone water and using Nessler’sreagent (0.5 ml). Ammonia-producing strains were identified when Antimicrobial susceptibility testing brown to yellow color was developed as reported by Ahmad et al. (2008). Protease and cellulase activities were evaluated by Susceptibility to 11 antimicrobial agents was performed using spot inoculation of tested strains on skimmed milk and CMC the disk-diffusion method in accordance with the Clinical and agar media, respectively. A clear halo around the colonies indi- Laboratory Standards Institute recommendations using the cates the ability of the strains to release the degrading enzymes breakpoints of P. aeruginosa (CLSI, 2013). Antimicrobial (Cattelan et al. 1999). agents tested were (charge in μg/disk) ticarciline (75), pipera- cillin (100), piperacillin-tazobactam (100/10), ceftazidime Screening of biosurfactant production (30), imipenem (10), meropenem (10), gentamicin (10), tobramycin (10), amikacin (30), ciprofloxacin (5), and Biosurfactant production was screened using three distinct colistine (10). methods. Hemolytic activity was determined by streaking the strains on blood agar plates containing 5% (v/v) blood and incu- Detection of antimicrobial resistance genes bated for 48 h at 37 °C; clearing zone around the colonies is indicative of the presence of hemolytic activity (Youssef et al. Beta-lactamase genes of the TEM, SHV, and CTX-M types were 2004). Blue agar test involves methylene blue complexation by tested by PCR in isolates showing resistance to beta-lactams. The adding cetyltrimethylammonium bromide (CTAB) (0.02%) and annealing temperature was 60 °C for TEM and 52 °C for both methylene blue (0.0005%) in tryptic soy agar medium. Bacteria SHVand CTX-M genes (Vinue et al. 2008). Moreover, the pres- from overnight TSB cultures were spot inoculated onto the above ence int1, int2, and int3 genes encoding integrase of class 1, 2, mentioned medium. Plates were incubated at 37 °C for 72 h. and 3 integrons were tested by PCR using an annealing temper- Biosurfactant production is confirmed by the formation of a dark ature equal to 62 °C (Vinue et al. 2008). halo surlined by white halo surrounding the colony (Siegmund and Wagner 1991). In the drop collapse test, 7 μl of mineral oil was added to each well of a 96-well microtiter plate and covered Results and discussion for 24 h at room temperature. Strains were cultivated on TSB medium supplemented with 2% of glucose. To test the presence Pseudomonads function as predominant constituent among of biosurfactant, 20 μl of the culture supernatant was added on the rhizobacteria that exhibits versatile metabolic capacity; they have oil surface in the microtiter well. SDS with sterile-distilled water gained immense attention in agriculture due to their plant was used as control suspension (Tugrul and Cansunar 2005). growth-promoting and phytopathogen biocontrol activities. Identification of Pseudomonas strains, isolated from rhizospheric Heavy metal tolerance soil of date palm (Phoenix dactylifera L.) from 7 different oases in the southern part of Tunisia, allowed their assignment to 15 Isolate tolerance to heavy metals was evaluated on nutrient different species, dominated by P. brassicacearum (12), agar media supplemented with 100 μg/ml of different heavy P. koreensis (7), and P. putida (3). Other species including 54 Ann Microbiol (2019) 69:51–59 P. segetis (2), P. chlororaphis (2), P. lini (1), P. vancouverensis was the most displayed activity by Pseudomonas species. In fact, (1), P. fluorescens (1), P. argentinensis (1), P. corrugate (1), 88.8% of strains (mainly of P. brassicacearum) were recorded to P. pseudoalcaligens (1), P. xanthomarina (1), P. oryzihabitans produce a phytohormone amount of IAA ranging from 10 to (1), P. stutzeri (1), and Pseudomonas sp. (1) (Table 1). 46 μg/ml. The high frequency of IAA-producing Pseudomonas Pseudomonas strains were subjected to several tests pointing species is in line with several reports (Ouzari et al. 2008;Suresh mechanisms underlying plant growth promotion. Screening re- et al. 2010;Arisetal. 2011;Kaurand Sharma 2013). sults of PGP traits are depicted in Table 1. The majority (83.7%) Phytohormone IAA affects the root system through the contri- of tested strains showed multiple PGP features. IAA production bution in the division and differentiation of tissues enabling the Table 1 Plant growth promotion traits, biosurfactant production, heavy metal genes, and antibiotic resistance phenotypes of Pseudomonas spp. Accession PGP activities Closest described relative for heavy Antibiotic Biosurfactant ** Strains Species affiliation number metal sequences resistance Blue Drop Prot. Sid. NH3 IAA P.sol. PGP a Hem. c phenotypes Ag. C. V1R1A1 P. brassicacearum KJ956613 V16R3A1 P. brassicacearum KJ956621 4 VBR3A3 P. brassicacearum KJ956612 4 VCR3B3 P. brassicacearum KJ956597 5 V16R2Y1 P. brassicacearum KJ956603 4 V9R1B3 P. brassicacearum KJ956605 5 VCR3Y12 P. brassicacearum KJ956602 5 V8R3B4 P. brassicacearum KJ956596 4 arsB : S. epidermis CT V5R3A4 P. brassicacearum KJ956592 5 V5R2Y16 P. brassicacearum KJ956662 V5R2A10 P. brassicacearum KJ956616 VCR1B2 P. brassicacearum KJ956600 5 TLC VCR1A12 P. koreensis KJ956649 5 TLC V9R2B4 P. koreensis KJ956628 4 TLC V9R3B1 P. koreensis KJ956606 4 CT TLC VCR2B4 P. koreensis KJ956598 4 TLC VBR1B1 P. koreensis KJ956609 5 3 CT V1R1B2 P. koreensis KJ956614 TLC VBR2Y2 P. koreensis KJ956647 4 VBR3B2 P. putida KJ956633 4 V8R2B2 P. putida KJ956652 4 VCR3Y1 3 TLC P. putida KJ956668 2CT VCR3A12 P. segetis KJ956601 2CT V5R3B1 P. segetis KJ956639 V8R3Y3 P. chlororaphis KJ956627 V8R3Y2 P. chlororaphis KJ956656 3 3TLC VCR3A8 P. lini KJ956595 3 arsR: S. warneri and copA: S. epidermis V9R1B2 P.vancouverensis KJ956607 3TLC CAZ V9R3Y3 P. fluorescens KJ956658 CT V16R1B7 P. argentinensis KJ956643 1 V16R3B6 P. corrugata KJ956610 5 V5R3A11 P.pseudoalcgenes KJ956617 V5R1Y7 P. xanthomarina KJ956618 1 TLC VBR3Y4 P. oryzihabitans KJ956608 4 V5R1A15 P. stutzeri KJ956671 V5R2Y15 P. sp. KJ956661 Positive activity: * (a): Blue agar test, (b): Hemolytic test, (C): Drop collapse. ** S. : Staphylococcus Ann Microbiol (2019) 69:51–59 55 root surface extension and the increase of nutrient uptake biosurfactant. This assay was rather developed to reveal an- (Phillips et al. 2011; Kochar and Srivastava 2012). ionic surfactants, having the property to form insoluble ion Phosphorus, nitrogen, and iron are the key constituents for pairs with various cationic substances, like CTAB, in aqueous plant nutrition and growth (Schachtman et al. 1998); however, solution (Siegmund and Wagner 1991). The large distribution many agricultural soils are nutrient-deprived. Consequently, of such biosurfactant in Pseudomonas genus is in correlation farmers relied on chemical sources for plant nutrition and crop with some data reporting the production of glycolipid surfac- yield improvement. tant containing rhamnose and 3-hydroxy fatty acids within Amendment with soil microorganisms to improve mobili- members of this genus (Mata-Sandoval et al. 2002). Equally zation of phosphorus in soils is gaining interest for with hemolysis agar assay, drop collapse test showed 22.2% agrobiology development (Glick 2012). Phosphate solubiliza- of positive strains for biosurfactant production. In hemolysis tion screening is a promising approach for the selection of test, strains causing lysis on blood agar plates are surrounded microorganisms, allowing the increase of phosphorus avail- by a clear zone. In addition to biosurfactant production, this ability to plant roots. In this study, phosphate solubilization halo may be caused by divalent ions and other hemolysins was detected in 63.8% of Pseudomonas species as detailed in produced under the experiment. Indeed, Satpute et al. (2008) Table 1. reported the possibility of biosurfactant production deprived Many Pseudomonas species have been described as efficient of hemolytic activity. phosphate solubilizers and have been used as bio-inoculants re- On the other hand, for the drop collapse test, positive garding their biofertilizing ability to improve soil fertility and to strains collapsed the oil drop and produced a flat drop even enhance nutrient uptake (Rodriguez and Fraga 1999; Gulatietal. for low rhamnolipid or surfactin concentrations in the solu- 2008;Trivedi andSa 2008;Vyaset al. 2009). tions. The drop collapse method is rapid and suitable for Another important PGP trait interfering with plant growth screening low amount of biosurfactant (Youssef et al. 2004; enhancement is ammonia production through nitrogen supply Tugrul and Cansunar 2005;Mahjoubietal. 2013). A total of (Wani et al. 2007). Furthermore, the ammonia released by 14 Pseudomonas species exhibited surfactant activity with an bacterial strains may increase the glutamine synthetase activ- unconformity among the different methods (Table 1), indicat- ity (Chitra et al. 2002). This trait was recorded in 52.7% of ing different structure and concentration of the produced Pseudomonas species associated to date palm root. biosurfactant (Bodour et al. 2003; Mahjoubi et al. 2013). High level of siderophore-producing Pseudomonas (81%) Rhamnolipids produced by P. aeruginosa are the most widely was observed, probably because this PGP trait confers advan- studied surfactants and the most effective for the removal of tages in the colonization ability of bacteria under iron-limiting hydrophobic compounds and pesticides from contaminated soils (Raaijmarkers et al. 1995). Siderophore compounds soils (Mata-Sandoval et al. 2002; Rahman et al. 2002, 2003; exhibiting high binding affinity for iron are known to exert a Das and Mukherjee 2007;Razaetal. 2007). To the best of our biocontrol role in reducing iron-dependent spore germination knowledge, this is the first report describing biosurfactant pro- of phytopathogens (Ribeiro et al. 2012). In addition to duction for several species such as P. brassicacearum, siderophore production, cell wall-degrading enzymes are in- P. segetis, P. lini, P. vancouverensis,and P. corrugata. volved in the suppression of fungal growth and organic matter Biosurfactants produced by PGP bacteria have been also turnover mechanisms (Nagrajkumar et al., 2004). Protease demonstrated to enhance heavy metal mobilization in contam- activity was widely distributed (66.6%) among inated soils (Braud et al. 2006). In fact, various studies have Pseudomonas strains comparing to cellulase production evidenced the feasibility of using biodegradable surfactants to where only one strain displayed this ability. In addition to remove heavy metals from contaminated soils (Mulligan et al. these common PGP traits, biosurfactant production is recently 2001a, b; Dahrazma and Mulligan 2007; Palashpriya et al. receiving attention for their role against fungal phytopatho- 2009; Sankar and Dipak 2012). The combined use of PGPR gens (Mital et al. 2011; Sonica et al. 2013; El-Sheshtawy exhibiting biosurfactant production and tolerance to heavy and Doheim 2014) and for improving soil properties such as metals could improve the phytoremediation efficiency. In this the enhancement of soil water retention and nutrient-holding regard, Pseudomonas strains were assessed for their ability to ability (Guodong et al. 2013). Surfactant activity of tolerate (100 μg/ml) various heavy metals, i.e., cadmium, rhizospheric Pseudomonas was investigated using three dis- zinc, cobalt, arsenic, and copper. While no strain showed tol- tinct methods. A high proportion (80.5%) of tested strains erance to cadmium, zinc, or cobalt, P. vancouverensis showed biosurfactant production ability by at least one meth- V9R1B2 was tolerant to arsenic and copper and od. Nevertheless, an uneven distribution of biosurfactant pro- P. brassicearum V5R3A4 exhibited only resistance to arsenic. duction among Pseudomonas species by the different method Those two strains were further grown at higher metal level by was recorded. The highest percentage of positive strains gradually increasing the concentration of arsenic and copper (61.1%) was detected by CTAB-methylene blue complexation to 200 μg each time, in order to establish the rate at which the method specific for the detection of rhamnolipid growth was inhibited. Strain V5R3A4 was found to tolerate 56 Ann Microbiol (2019) 69:51–59 2600 μg/ml of arsenic, whereas V9R1B2 supported 1800 and strains involved in heavy metal tolerance showed also multi- 2400 μg/ml of copper and arsenic, respectively. Arsenic- and ple PGP features and present biosurfactant production copper-tolerant strains were subjected to PCR amplification of (Table 1). These strains could be good candidates as bio- ars operon (arsC, arsB, arsR, arsA, and ars D) and copA. inoculants to assist phytoremediation and increase perfor- Strain V5R3A4 was positive for arsB, while strain V9R1B2 mance of plants in contaminated environment. was positive for copA and arsR genes. Rhizosphere habitat could contain antibiotics produced by Sequence comparison of copper PCR fragment showed a associated microbiota or released in the environment from high homology (99%) with copA of Staphylococcus different human practices and might contain toxic com- epidermidis ATCC 12228. Similarly, the comparison of the pounds. The adaptive flexibility of Pseudomonas to colonize sequences of arsenic PCR product indicated also a very high such habitat probably allows them to develop systems to avoid homology with ars genes of Staphylococcus epidermidis the activity of antimicrobials and toxic compounds which strain M13/0453 SCCmec element, complete sequence contributes to the emergence of antibiotic resistance (99%) and arsR of Staphylococcus warneri SG1, complete (Martinez, 2009a). genome (98%) (Table 1). copA and arsB gene sequences were Agricultural soils are increasingly considered as a potential submitted to the NCBI nucleotide database under the acces- source for antibiotic resistance. Moreover, introduction of in- sion numbers MH940213 and MH940214, respectively. oculums or fertilizers which unintentionally contain antibiotic Our findings revealed that P. vancouverensis V9R1B2 carry resistance genes into agriculture could increase the risk of their copA and arsR genes while P. brassicearum V5R3A4, tolerant dissemination. Antibiotic resistance determinants could be only to arsenic, encompasses arsB gene. A research paper on transmitted to human and animal bacteria simply by ingesting comparative genomic analysis of four representative plant them with vegetables or fruits (Qiuzhi et al. 2015). To this growth-promoting rhizobacteria in Pseudomonas reported the regard, the characterization of root-associated bacteria for ag- presence of copA and ars operon in Pseudomonads (Xuemei riculture application also needs an evaluation of antimicrobial et al. 2013). Nevertheless, the detection of only arsR and arsB resistance. In the present study, Pseudomonas species were among operon genes suggests that some other mechanisms are resistant to only 3 antibiotics among the 11 tested (Table 1). involved in arsenic tolerance in the current strains. Resistances were restricted to ticarcillin, ceftazidime, and co- Bacterial tolerance to arsenic and copper reported in our listin (27.7%, 2.7%, and 16.66%, respectively). According to investigation is in agreement with previous surveys dealing our data, Pseudomonas associated to date palm roots with heavy metal tolerance of Pseudomonas genus in the displayed low prevalence of antibiotic resistance and affected environment. Matyar et al. (2010) and Yogendra et al. only to two classes of antimicrobials. The nine ticarcillin- (2013) reported the occurrence of copper-tolerant resistant Pseudomonas isolates (one of them also showing ceftazidime resistance) did not carry the beta-lactamase genes Pseudomonas strains in marine environment and plant rhizosphere. As well, Shilev et al. (2006) highlighted the pos- analyzed. This could be due to the presence of novel resistance itive influence of P. fluorescens on sunflower plant growth and determinants or because antibiotic resistance could be intrinsic arsenic accumulation. in some soil bacteria as suggested by Martinez (2009b). Contamination of soil by heavy metals represents a serious Moreover, no integrons were detected in our bacterial environmental problem due to their toxic threats. According to collection. The Agency for Toxic Substances and Disease, arsenic is in the first rank among a list of the 275 hazardous substances that pose a serious risk to human health. Long-term exposure to arsenic can cause lung and skin cancers and nervous and car- Conclusions diovascular problems (Vahter 2007;Glick 2010). Moreover, copper is essential micronutrients, but at higher concentrations In conclusion, the present study is an attempt to evaluate and may cause severe damage to ecosystems and human health characterize different Pseudomonas species associated to date (Lin et al. 2010). palm root for their plant growth promotion abilities and bio- A large number of plant which can accumulate high con- remediation potentialities. Due to multifarious properties re- centration of metals defined as hyperaccumulators were used corded, Pseudomonas strains could be developed as potential to cleanup metalliferous soils. However, the bioavailability of microbial inoculants for agriculture and bioremediation pur- metals to plant roots is a critical requirement for plant metal pose. Moreover, Pseudomonas rhizobacteria expressed low bioaccumulation. Several studies have evidenced a synergistic rate of antibiotic resistance limiting hazardous effects of the use of plants and PGP bacteria to increase phytoremediation dissemination and the evolution of antibiotic resistance in the efficiency by enhancing metal uptake and improving the environment. Further studies are required to evaluate the bio- growth of hyper accumulator plants (Ma et al. 2011). remediation potentiality of Pseudomonas on contaminated en- According to our results, it is interesting to note that the two vironment as well as the involved mechanisms. Ann Microbiol (2019) 69:51–59 57 Acknowledgements The authors thank the Tunisian Ministry of Higher Chitra RS, Sumitra V, Chanda YDS (2002) Effect of different nitrogen Education and Scientific research in the ambit of the laboratory project sources and plant growth regulators on glutamine synthetase and LR03ES03. glutamate synthase activities of radish. Bulg J Plant Physiol 28(3– 4):46–56 CLSI (2013) Clinical and Laboratory Standards Institute. Performance stan- Funding This work was supported by the Tunisian Ministry of Higher dards for antimicrobial susceptibility testing; eighteenth informational Education and Scientific Research (LR03ES03), the project supplement. CLSI document M100-S22. Wayne, Pennsylvania BIODESERT GA-245746 BBiotechnology from desert microbial Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting extremophiles for supporting agriculture research potential in Tunisia bacteia in the rhizo-and endosphere of plants: their role, coloniza- and Southern Europe^ (European Union) and the project SAF2012- tion, mechanisms involved and prospects for utilization. Soil Biol 35474 from the Ministerio de Economía y Competitividad of Spain and Biochem 42:669–678 FEDER. Dahrazma B, Mulligan CN (2007) Investigation of the removal of heavy metals from sediments using rhamnolipid in a continuous flow con- Compliance with ethical standards figuration. Chemosphere 69:705–711 Das K, Mukherjee AK (2007) Crude petroleum-oil biodegradation and Conflicts of interest The authors declare that they have no conflict of efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains interest. isolated from a petroleum-oil contaminated soil from north-East India. Bioresour Technol 98:1339–1345 El-Sheshtawy HS, Doheim MM (2014) Selection of Pseudomonas Ethical approval This article does not contain any studies with human aeruginosa for biosurfactant production and studies of its antimicro- participants performed by any of the authors. bial activity. 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Annals of Microbiology – Springer Journals
Published: Oct 24, 2018
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