Production and Extraction of Siderophores-Catecholate- from -MDR-Acinetobacter baumannii

Siderophores are low molecular weight organic compounds produced by microorganisms growing under low iron concentration.In this study we describe the detection, production and extraction of siderophores secreted by Acinetobacter baumannii (Multiple-drug resistant ) pathogens. One hundered twenty Gram –negative non lactose fermenter bacilli isolates have been collected from three hospitals at Baghdad city over three months. Primary identification of these isolates is performed by standard diagnostic methods (biochemical tests and API 20 NE); 19 clinical isolates of A. baumannii are cultured on CHROMagar (highly selective medium for detection of MDR Acinetobacter) as well as diagnoses is documented by using Vitek 2 system. Isolates are examined towards 11 different antibiotics. High resistance is recognized for most isolates. Detection of siderophore has been done by examining the isolates on M9 minimum medium; 5 isolates (26%) are producers for siderophore, the highest producing one is isolated from sputum and chosen to extract siderophore catecholate . (Ab5S) isolate is examined on specific synthetic medium for production then siderophore molecules are extracted by ethyl acetate .Weight of dried extract is determined (115 mg/ml) and siderophore chemical nature has been assessed which appeared as catecholate.


Introduction:
Siderophores are low molecular weight (<14 kDa) iron chelating compounds synthesized in large quantity under iron limitation conditions. There are three major types of siderophores; hydroxamate, catecholate and carboxylate [1]. Iron is a necessary element for the growth of bacterial cells because it acts as a catalyst in enzymatic processes, electron transfer, DNA, RNA syntheses and oxygen metabolism [2]. Iron is also essential for biofilm production because it stabilizes the polysaccharide layer and arranges surface motility [3]. One of the most important steps in initiating an infection is the availability of iron [4]. There are different methods to acquire iron by microorganisms; production of siderophores represents the first and more important method, iron adheres to the bacterial cell by specific receptors and moves inside by common transport techniques [5]. Genes of siderophore biosynthesis are responsible for bacterial infection in Open Access mouse, they activate exotoxins formation, affects cell movement and biofilms maturity [6]. Few studies show the ability of clinical isolates of A.baumannii to grow and produce siderophore compounds under iron-deficient condition [7].
A novel siderophore, called acinetobactin, with both catecholate and hydroxamate functional groups are isolated from low-iron cultures of A. baumannii ATCC 19606 [8]. Aim of the study: Siderophores have many medical applications , the most important one when act as avechile for transport antibiotics inside bacterial cell so extraction of these molecules should be investigated.

Materials and Methods:
1-Bacterial Isolates : One hundered twenty specimens belonging to non fermenter Gram-negative bacilli have been collected from three hospitals at Baghdad city over three months, 19 clinical isolates have been identified as A. baumannii, the diagnosis is confirmed by using highly selective medium CHROMagar Acinetobacter and Vitek 2 system .
The medium is then supplemented with 0.1g thiamine. C-The components are well mixed and poured in disposable sterile plates,. D-After being solidified, the plates are inoculated with tested isolates (touch by sterile woody stick) and incubated at 37°C for 24 hrs. E-If the isolate is siderophore producing, the growth will appear as small,single and seperated colonies on M9 medium [8]. 4-Siderophore production: A synthetic medium with the following components per liter is used: pH was adjusted to 7 and autoclaved [11].
In order to avoid iron contamination ,inoculation of the of producer isolate is performed by sterile woody stick and incubating the culture for 20hrs. at 35°C. Note: All the flasks and glassware materials are soaked with acid, rinsed several times with water before using to minimize iron concentration (8).

5-Extraction of siderophore:
According to the method of Jadhav and Desai (1992); bacterial suspension is centrifuged at 8000 rpm/20 min. The supernatant is acidified to pH=2 ,and immediately siderophore is extracted by adding equal volume of ethyl acetate ,shaked in 50°C water bath to evaporate ethyl acetate layer, then the extract is placed in an oven at 50°C in open petri dishes to obtain dried extract. 6-Estimation of the dry weight of crude extract. 7-Determining the chemical nature of siderophore molecules; bacterial supernatant is used for assay by adding 1 ml of 2% of aqueous FeCl 3 to 1ml of sample. The result is positive by appearance of wine color absorbed at 490 nm in UV spectrophotometer [12].

Results and Discussion: Identification and Antimicrobial Susceptibility
Nineteen isolates of A. baumannii from several clinical sources; (5 from sputum; 5 from wound swab; 4 from blood; 3 from urine and two isolates from tracheal secretion) are identified by growing of red colonies on CHRO Magar and depending on the identification results of Vitek 2 system (7). The results of the antimicrobial susceptibility are shown in Table(1).  The highlest producing isolate is Ab5S from sputum. Clear correlation is noticed between antimicrobial resistance and siderophore formation. Table (1) shows that the producer isolates are; Ab5S, Ab6W, Ab11B, Ab14B, Ab18TS with high resistance to antimicrobials. On M9 medium as mentiond earlier, the producer isolates colonies appear circular wrinkled and dried (Fig.1):

Colonies of Siderophore Producing Isolates on M9 Minimum Medium
Each colony in the Figure above represents the growth resulted from the stick touch, little and weak growth may be attributed to the poor and limited nutrients and depleted iron in M9 minimum medium. Not all the isolates are siderophore producers and this agreed with the results of Yamamoto et al., (1994) who report that 4 of 12 clinical A.baumannii strains examined are siderophore producers, indicative of strain -to-strain variation in the ability of acinetobactin production. [13] Sokol et al.,(1992) describe a novel siderophore from Pseudomonas cepacia (recently Burkholderia cepacia) cultures named azurechelin, and indicat that 88% of pathogenic isolates produced it. This compound correlates to bacterial virulence and may increase morbidity and mortality in patients of cystic fibrosis. [14] Bnyan et al., (2010) showing that siderophore production is in 76.6% of uropathogenic Escherichia coli (UPEC) compared to 5% in E.coli fecal isolates thus siderophore production has been shown to be more frequent in E. coli from patients with UTI than in fecal isolates and is suggests that siderophore production positive isolates can be considered as UPEC. Other isolates have shown no growth on M9 minimum medium that may suggest variation in efficiency of siderophore production or they may form different type of siderophore other than catecholate according to Yamamoto et al.,(1994) who detect and extract the acinetobactin (catecholate and hydroxamate functional groups) from A.baumannii . The isolate Ab5S is inoculated in a specific minimum liquid medium and care is taken to use metal-free glass ware. Flasks and other glassware are kept in acid to remove all traces of metals from medium, inoculation have been done by sterile woody stick. The optimum conditions for maximum production occurs after 20hrs.at 30°C, pH 7, where no iron contamination was found. Previous studies indicate that the presence of iron can inhibit siderophore production as well as results indicating that iron-binding proteins, which may play a role in chelating the siderophorebound iron, are produced under ironstarved conditions [16,17] Iron-binding proteins are present in membranes of cultures grown under iron limitation. Siderophores chelate iron and supply to bacterial cell by outer membrane receptors, iron is an important nutrient element for growth and maintenance, hence the siderophore molecules after 20 hrs. Become outside the bacterial cells -in the medium. The concentration of siderophores in the culture supernatant is maximal after 20 hrs. of growth which means that siderophore production occurrs in parallel with growth. Therefore extraction of these molecules should be done on bacterial filterate after precipitating of bacterial cells. Because ethyl acetate layer is evaporated to dryness by shaking water -bath at 50° C, care should be taken from high temperatures which may denature the amino acids conjugated with the phenolates in catecholate siderophores. Weight of crude extract is estimated which is equal to 115mg/l, it is considered low when compared to the result got by Hussien et al.,(2013) where the weight of pyoverdin extract from P.aeruginosa is equal to 235 mg/l that may be attributed to the difference in the extraction medium and other experiment parameters as well as the producing microorganism. In another study [18], the weight is 200 mg/l also they extracted siderophores from P.aeruginosa .Chemical nature of the extracted molecules indicates catecholate (phenolates) structure because of wine colour of extract after adding 2% aq.FeCl 3 indicator absorbed at 490 nm in UV spectrophotometer. Actis et al., (1993) show in their work that different A. baumannii isolates are able to grow under iron-depleted conditions. The bacterial growth is accompanied by the formation of ironregulated catechol siderophores, independently of the bacterial plasmid content. Goel et al.,(1998) [20]. [21] Pal and Gokarn (2010) have concluded that there is no significant difference occuring in the production of siderophore in commensal and clinical bacterial isolates. They suggested that siderophore production may be a necessary factor of virulence but not a determinant of virulence. [22] Al-Muhanna et al., (2014) have discussed the correlation between siderophore and aerobactin gene. Isolates of K. pneumoniae that produce aerobactin are more virulent, but non siderophore producing isolates are less virulent. Also, they find that K. pneumoniae isolates totally produce siderophores are expressed aerobactin genes.
[23] Naik and Dubey (2011) document that low lead nitrates concentrations up to 0.5mM may enhance siderophore production in P. aeruginosa. We conclude from our study that MDR-A.
baumannii could produce siderophores but in variable amounts among isolates. It is apparent that highly antimicrobial resistant isolates are siderophore producers.The extracted siderophore compound is from catecholate type.