Spectral , Thermal and Antibacterial Studies for Bivalent Metal Complexes of Oxalyl , Malonyl and Succinyl-bis-4-phenylthiosemicarbazide Ligands

The thermogravimetry (TG) and derivative thermogravimetry (DTG) have been used to study the thermal decomposition of some oxalyl (H4OxTSC), malonyl (H4MaTSC) and succinyl-bis-4-phenylthiosemicarbazide (H4SuTSC) ligands and their metal complexes using Horowitz-Metzger (HM) and Coats-Redfern methods. The kinetic thermodynamic parameters such as: E*, ΔH*, ΔS*and ΔG* are calculated from the DTG curves. The isolated complexes have the general composition [M2(L) (H2O)6], where M=Cu(II), Zn(II), L=MaTSC and M=Co(II), Cu(II) or Sn(II) and L=Su TSC and [M2(L) (H2O)n]·nH2O where M=Cu(II), Co(II) or Sn(II), L=OxTS or Ma TSC. The tested compounds show a good activity against four strains of bacteria Gram negative Escherichia coli, Pseudomonas aeruginosa species and gram-positive Bacillus cereus and Staphylococcus aureus.


Introduction
Thiosemicarbazide and its derivatives have received considerable attention because of their pharamacological properties [1].Thiosemicarbazide complexes show a broad spectrum of anticancer activity [2] [3].Also, thiosemicarbazide derivatives are of current interest with respect to their uses as analytical reagents for separations of metal(II) ions [4]- [7], analytical determination of metal ions [8] [9], and clinical analysis [10].Most of these compounds have antifungal [11]- [12], antimicrobial [13] and antitumor activity [14]- [16], as well as radiopharmaceuticals applications [17].Continuing our studies for the chemical and electrochemical synthesis of new metal complexes of ligands containing N, S and O atoms through the reaction of metal ions scarified from the anodic dissolution of metals [18] [19].Our aim work in this paper to report novel complexes prepared from the reaction between bisthiosemicarbazi decompounds which have a good ability to form chelate complexes with transition metal [18]- [20].We report here the thermal, spectral and biological evaluations of Co(II), Cu(II), Zn(II) and Sn(II) complexes for 1,1-oxalyl, malonyl and succinyl-bis-4-phenylthiosemicarbazide ligands.The modern spectroscopic investigations are used to elucidate the structure of the prepared materials.The thermal decomposition is also used to infer the structure of the metal complexes and to calculate the different thermodynamic activation parameters.

The Organic Compounds
1) Preparation of 1,1-Oxalylhydrazide: 1,1-oxalyldihydrazine was prepared by adding oxalyl chloride (7 gm, 0.05 mol) to alcoholic solution of hydrazine hydrate (5 gm, 0.1 mole).The reaction mixture was exothermic and left to cool with stirring.A white crystal precipitate was formed and washed with ethanol diethyl ether and left to dry.
2) Preparation of 1,1-Oxalylbis (4-phenylthiosemicarbazide): It was prepared by adding phenylisothiocynate (2.8 gm, 0.02 mol) to an alcoholic solution of oxalic acid dihydrazide (1.18 gm, 0.01 mole).The reaction mixture was refluxed for 1 hour and left to cool with stirring.The resulting white crystals were collected and washed with ethanol and diethyl ether, respectively.The resulting solids were filtered hot, washed with hot dist.water, EtOH and dried by Et 2 O and finally dried in vacuum over silica gel (Figure 1).
3) Preparation of 1,1-Malonylbis-phenylthiosemicarbazide: 1,1-Malonyl bis-4-phenylthiosemicarbazide) was prepared by adding phenylisothiocynate (1.8 gm, 0.02 mol) to an alcoholic solution of malonic acid dihydrazide (1.32 gm ≈ 0.01 mole).The reaction mixture was refluxed for 1 hour and left to cool with stirring.The resulting white crystals were collected and washed with ethanol and diethyl ether, respectively.The resulting solids were filtered hot, washed with hot dist.water, EtOH and dried by Et 2 O and finally dried in vacuo over silica gel.

The In-Organic Compounds
The preparative results show that the direct electrochemical oxidation of the metals in the presence of a ligand solution is a one-step process and represents a convenient and simple route to a variety of transition metal complexes.The apparatus used in the electrochemical reaction consists of a tall-form 100 mL Pyrex beaker containing 50 mL of the appropriate amount of the organic ligand dissolved in acetone solution.The cathode is a platinum wire of approximately 1 mm diameter.In most cases, the metal (2 -5 g) was suspended and supported on a platinum wire.Measurements of the electrochemical efficiency, Ef, defined as moles of metal dissolved per Faraday of electricity, for the M/L system (where L = ligand used) gave E f = 0.5 ± 0.05 mol•F −1 .

Synthesis of Metals Complexes
Electrolysis of cobalt metal into 60 ml of anhydrous acetone solution of 1,1-oxalaylbis (4-phenylthiosemi-carbazide)ligand as an example, (1.2 gm, 5 mmol), 0.5 mg Et 4 NClO 4 dissolved in two drops of water and 20 V current led to dissolution of 116 mg of Co during 120 min.(E f = 0.5 mol•F -1 ).Since, most of the products are insoluble in the reaction mixture, the collection procedure involved filtration, after which the solid was washed with diethyl ether.The resulting green powder was collected.By the same way Cu, Zn, and Sn complexes were isolated and all the data for carbon, hydrogen and nitrogen were gathered in Table 1.

IR, Raman and 1H-NMR Spectra
Infrared spectra for the three ligands and their metal complexes were recorded by Perkin Elmer FTIR 1605 using KBr pellets (Figures S1-S3).Also, Raman spectra for the ligands, Zinc(II) and Sn(II) metal complexes were recorded in the solid state on Thero Nicolet FT-Raman (USA) with a wavelength 1064 nm power according sample resolution was 8 cm −1 at National Research Center, Cairo, Egypt (Figures S4-S6).The 1 H NMR spectra were recorded on an Varian Mercury VX-300 NMR spectrometer. 1 H-NMR spectra were run at 300 MHz and 13 C-NMRspectra were run at 75.46 MHz in deuterated dimethylsulphoxide (DMSO-d 6 ).

Electronic and Mass Spectra
The electronic spectra for all the ligands and the metal complexes solutions were measured in UV/Vis range (190 -1100) nm using Helios UV Spectrometer at Center Photo energy, Ain-Shams University.Mass spectra were recorded at SHIMADZU GC MS-QP 1000 EX Micro analytical Center, Cairo Universal, Giza and Al-Azher University, Egypt (Figures S7-S9).

Magnetic Molar Conductance Measurements
Magnetic measurements were carried out on a Sherwood scientific magnetic balance using Gouy method.Molar conductivities of freshly prepared 1.0 × 10 −3 mol•L −1 DMSO solutions were measured using Jenway 4010 conductivity meter.

Microanalytical and Magnetic Measurements
Carbon and hydrogen contents were determined using a Perkin-Elmer CHN 2400 analyser.Magnetic measurements were carried out on a Sherwood scientific magnetic balance using Gouy method.

Thermal Investigation
Thermogravimetric analysis (TGA and DTG) were carried out in dynamic nitrogen atmosphere (30 ml/min) with a heating rate of 10˚C/min using a SchimadzuTGA-50H thermal analyzer (Figures S10-S12).

Antibacterial Investigation
Bacterial cultures and growth conditions: Gram negative Escherichia coli, Pseudomonas aeruginosa species and gram-positive Bacillus cereus, Staphylococcus aureus species and fungal Aspergillus fumingatus, Candidaalbicans were used as test microorganisms.The surface of the medium was inoculated and covered with the tested organisms.The agar surface was allowed to dry from 3 to 5 minutes before applying disks.The disks were dipped into a beaker of the chemicals using sterile forceps and placed them in the previous medium.Cultures plates of bacteria were incubated for grown at 37˚C for 48 hours.Chloramphenicol was used as a standard antibacterial agent and Terbinafin was used as a standard antifungal agent.
The bands due to ν(C=S) and ν(C=N) groups appeared at 802 and 1533 cm −1 .On complexation, the bands of the thiosemicarbazide moiety respect to ν(C=S) and ν(C=N) are shifted towards higher wave numbers and notice that the very strong peak of ν(C=S) may be disappeared or decreasing in its intensity.The bands due to ν(C=S), ν(N-N) and ν(C=N) groups appeared at 835, 1101 and 1602 cm −1 (Figure 1).The IR spectra of Copper complex Ia compared with ligand H 4 OxTS, indicates that bands due to ν(NH), ν(C=O) and ν(C=S) are absent, but new bands appear at ca. 1651 and 831 cm −1 due to ν(N=C) and ν(C-S), respectively, suggesting removal of both the hydrazinic protons via enolisation and thioenolisation and bonding of the resulting enolic oxygen and thiolato sulfur takes place with Co(II), Cu(II), Zn(II) and Sn(II).Furthermore, the ligand bands due to thioamide I, thioamide II and ν(N-N) undergo a positive shift of in the range (20 -41 cm −1 ), (20 -56 cm −1 ) and (22 -39 cm −1 ) respectively.Some new appear bands in the range (755 -777cm −1 ) assigned to groups (C-S) vibrations.This is also confirmed by the appearance of bands in the range of 395 -417 cm −1 , this has been assigned to the ν(M-N) [28], and the appearance of bands in the range of 490 -505 cm −1 , this has been assigned to the ν(M-O).A strong band found at 902 cm −1 is due to the ν(N-N) group of the 1,1oxalylbis(4-phenyl-thiosemicarbazide.Thus the ligand behaves as tridentate chelating agent coordinating through azomethine nitrogen, thiolate sulphur andenolic oxygen (Figure 2, Figure 3).

Raman Spectra
The Raman spectrum shows bands at 3201 cm −1 for the NH groups present in H 4 MaTS ligand.The bands occurring at 1635, 1405, 1355, 1088 and 824 cm −1 are assigned to ν [32] (Figure 4).An exhaustive comparison of the Raman spectra of the ligand and complexes gave information about the mode of bonding of the ligand in metal  oenolisation and bonding of the resulting enolic oxygen and thiolato sulfur takes place with Zn(II).Furthermore, the ligand bands due to thioamide I, thioamide II and ν(N-N) undergo a positive shift of (39 cm −1 ), (40 cm −1 ) and (2 cm −1 ) respectively.Ramanbands of complexes are appear of bands at (779 cm −1 ) assigned to groups (C-S) vibrations.It indicates that thione sulphur and also the enolic oxygen coordinates to the metal ion [33]- [35].Thus, it may be concluded that the ligand behaves as hexadentate chelating agent coordinating through azomethine nitrogen and thiolate sulphur.The Raman spectrum of [H 4 SuTS] shows bands at 3201, 3095 and 3063 cm −1 for the two-NH groups present in the ligand.The bands occurring at 1650, 1405, 1355, 900 and 824 cm −1 are assigned to ν(C=O), thioamide I [β(NH) + ν(CN)], thioamide II [ν(CN) + β(NH)], ν(N-N) and ν(C=S), respectively [33]- [35].Raman spectral data of all the ligands and the metal complexes are summarized in Table 2.

Electronic Spectra
The electronic spectrum of , Ia, has bands characteristic for an octahedral geometry [35].The spectrum shows (Table 3) two bands at 20,600 and 31,950 cm −1 assigned to the 4 T 1 g → 4 A 2 g (ν 2 ) and 4 T 1 g → 4 T 1 g (P) (ν 3 ) transitions, respectively, in an octahedral structure.These bands were used to calculate the third spin-allowed band, 4 T 1 g → 4 T 1 g [20].The other ligand field parameters, B, β and the ν 2 /ν 1 values were calculated to be 1060 cm

Magnetic Susceptibility
The observed values of magnetic moment for complexes are generally diagnostic of the coordination geometry about the metal ion.Co(II) has the electronic configuration 3d * and should exhibit a magnetic moment higher than that expected for two unpaired electrons in octahedral (1.5 -3.3 BM).The magnetic moment observed for the Co(II) complexes lies in the value of 3.2 BM which is consistent with the octahedral stereochemistry of the complexes.Room-temperature magnetic moment of the Cu(II) complexes lies in the range of 1.5 BM, corresponding to one unpaired electron.

1 H-NMR Spectra
The 1 H-NMR spectra of compounds Ic and IIc on comparing with that of the ligands indicates that the ligands acts as a hex dentate through the nitrogen atom of C=N oxygen atom of C=O and sulfur atom of C=S. 1 H-NMR spectrum of zinc (II) complex is in agreement with the suggested coordination through the C=N and C=S groups by the presence of the signals of (two from 2NH amine groups and two protons from 2NH amide groups).

Assignments
The compounds

Mass Spectrum
The electronic impact mass spectrum of the ligand I shows a molecular ion (M+) peak at m/z = 243 amu corresponding to species C 9 H 7 N 3 OS, which confirms the proposed formula.It also shows series of peaks at 70, 88, 111, 127 and 170 amu corresponding to various fragments.The intensities of these peaks give the idea of the stabilities of the fragments.The electronic impact mass spectrum of the Ia complex 1,1-oxalayl-bis (phenylthiosemicarbazide) cobalt monoacetone dehydrate shows a molecular ion (M+) peak at m/z = 758 amu corresponding to species [C 22 H 30 Co 2 N 6 O 8 S 2 ], which confirms the proposed formula.It also shows series of peaks at 39, 75, 90, 111, 127, 138, 169, 184, 201, 226, 243, 271 and 336 amu corresponding to various fragments.
The complex Ia was thermally decomposed in five successive decomposition steps within the temperature range 25˚C -1000˚C.The first step (obs.= 6%, calc.= 6.6%) at 25˚C -175˚C, may be attributed to the liberation of the 3 water molecules.The second step at 175˚C -390˚C (obs.= 29.2%,calc.= 28.6%), is accounted for the removal of 2 acetone, 4 water and N 3 H 3 fragment.The decomposition third step at 390˚C -707˚C (obs.= 18.3%, calc = 18.9%) is accounted for the removal of (C 4 N 3 S 2 ) fragment.The fourth step at 707˚C -990˚C (obs.= 22.8%, calc = 22.8%) is accounted for the removal of (C 9 H 7 ) fragment.The rest of the ligand molecule was removed and fifth the decomposition of the Co(II)/L complex molecule ended with a final 2CoO and residual carbon 3/2C 2 fragment (obs.= 23.7%,calc = 22.9%).The TG curve of Ib complex indicates that the mass change begins at 25˚C and continuous up to 1000˚C.The first and second mass loss corresponds to the liberation of the 12 water molecules and two (HCN) fragment (obs.= 34.4%,calc = 33.9%)at 25˚C -342˚C.The third step occurs in the range 342˚C -475˚C and corresponds to the loss of (CN 4 S) (obs.= 12.8%, calc = 12.6%).The fourth and fifth decomposition step are final decomposition organic ligand to the C 13 H 8 , 1/2S 2 , O 2 fragments and Cu 2 metal residual atoms (obs.= 52.8%,calc = 53.4%).
Ic complex was thermally decomposed in mainly five decomposition steps within the temperature range 25˚C -700˚C.The first decomposition step (obs.= 20.64%,calc = 20.64%) at 25˚C -245˚C, may be attributed to the liberation of two water and two acetone molecules.The second step at 245˚C -386˚C (obs.= 23.4%,calc = 23.6%) is accounted for the removal of the 2(HCN), 2N 2 and S 2 fragments.The third step found within the temperature 386˚C -700˚C (obs.= 19.7%,calc = 19.96%).The rest of the ligand molecule was removed and fourth the decomposition of theligand molecule ended with a final residue of (C 8 H 4 ), (ZnO) and zinc metal (obs.= 36.3%,calc = 35.7%).
Ligand II was thermally decomposed in mainly decomposition steps within the temperature range successive   The complex IIa was thermally decomposed in five steps within the temperature range 25˚C -1000˚C.The first step (obs.= 5.2%, calc.= 4.94%) at 25˚C -188˚C, may be attributed to the liberation of the two H 2 O molecules.The second step at 188˚C -448˚C (obs.= 33.1%,calc.= 33.5%), is accounted for the removal of 6H 2 O, 2N 2 , 2(HCN), and C 2 H 2 fragments.The decomposition third step at 448˚C -760˚C (obs.= 23.4%,calc = 22.9%) is accounted for the removal of S 2 and O 2 molecules.The fourth step found at 760˚C -885˚C (obs.= 21.8%,calc = 22.5%) is accounted for the removal of CH 4 and C 12 H 4 fragments.
The TG curve of IIb complex indicates that the mass change begins at 25˚C and continuous up to 1000˚C.The first and second mass loss corresponds to the liberation of the 6 H 2 O molecules (obs.= 16.4%, calc = 15.4%) at 25˚C -245˚C.The third step occurs in the range 245˚C -475˚C and corresponds to the loss of N 2 , 2(HCN), N 2 H 2 , and O 2 (obs.= 20.6%,calc = 20.5%).The fourth step at 475˚C -765˚C (obs.= 42.4%,calc = 42.5%) is accounted for the removal of (C 13 H 8 , S 2 ) fragments.The fifth steps are final decomposition organic ligand to the C 2 and Cu 2 residual (obs.= 20.6%,calc = 21.5%).
The complex IIc was thermally decomposed in mainly four steps within the temperature range 25˚C -700˚C.The first decomposition step (obs.= 15.5%, calc = 15.3%) at 25˚C -224˚C, may be attributed to the liberation of 6 H 2 O.The second step at 224˚C -338˚C (obs.= 26.5%,calc = 26.9%) is accounted for the removal of 2N 2 , S 2 , 1/2O 2 and 2(HCN) fragment.The decomposition third step found within the temperature 338˚C -643˚C (obs.= 18.6%, calc = 18.2%) is accounted for the removal of 1/2O 2 , CH 4 and C 2 H 2 fragments.The rest of the ligand molecule was removed and fourth the decomposition of the ligand molecule ended with a final residue metal of Zn 2 and C 12 H 4 fragment (obs.= 39.4%, calc = 39.5%).
Ligand III was thermally decomposed in mainly decomposition steps within the temperature range successive decomposition steps within the temperature range 25˚C -700˚C (Figure 5).The first decomposition step (obs.= 36%, calc.= 35.8%)within the temperature range 25˚C -234˚C, may be attributed to the liberation of the 2(HCN), 2N 2 and S 2 fragments.The second decomposition steps found within the temperature range 234˚C -334˚C (obs.= 32.1%,calc.= 32.3%),which is reasonably accounted by the removal of O 2 and C 4 H 6 .The decomposition of the ligand molecule ended with a final C 12 H 10 residue (obs.= 31.86%,calc = 31.7%).
The complex IIIa was thermally decomposed in four successive decomposition steps within the temperature range 25˚C -1000˚C.The first decomposition step (obs.= 31.9%,calc.= 32%) within the temperature range 25˚C -332˚C, may be attributed to the liberation of the 6water molecules, 2(HCN) and N 2 H 2 fragments.The second decomposition steps found within the temperature range 332˚C -550˚C (obs.= 16.9%,calc.= 16.8%), which is reasonably accounted by the removal S 2 , O 2 and C 2 H 2 fragments.The rest of the ligand molecule was removed and fourth the decomposition of the Co(II)/L complex molecule ended with a final 3/2C 2 and Co 2 metal is cobalt residue (obs.= 26.6%,calc = 26.8%).
The TG curve of complex IIIb indicates that the mass change begins at 25˚C and continuous up to 1000˚C.The first mass loss corresponds to the liberation of the 12 water molecules (obs.= 17.8%, calc = 17.8%) within the temperature range 25˚C -465˚C, (Figure 6).The second decomposition steps found within the temperature range 465˚C -700˚C (obs.= 15%, calc.= 14.8%), which is reasonably accounted by the removal of 2(HCN), 2N 2 and 2(CH 2 ) fragments.The decomposition fourth and fifth decomposition step are final decomposition organic ligand to the found within the temperature 910˚C-more than 1000˚C (obs.= 33.3%,calc = 32.6%)which is reasonably accounted for by the removal of carbon and 4(CuO), all the thermal diagrams in Figure S12.

Kinetic Studies
1,1-Oxalyl, 1,1-malonyl and 1,1-succinyl-bis-4-phenyl-thiosemicarbazide and all the metal Co(II), Cu(II), Zn(II) and Sn(II) complexes thermodynamic activation parameters of decomposition processes of the samples, namely activation energy, E * , enthalpy, ΔH * , entropy, ΔS * , and Gibbs free energy change of the decomposition, ΔG * , were evaluated graphically (Figures S13-S27) by employing the Coats-Redfern and Horowitz-Metzger relations [34]- [36].All the thermodynamic parameters for the rest of materials, malonyl and Succinyl complexes were also calculated,.All the data for Kinetic thermal studies were summarized in Tables 7-9.The high values of the activation energy illustrated to the thermal stability of the complexes.The activation energies of decomposition Cu Cu were in the range 55 -450 kJ•mol -1 .The high values of the activation energy illustrated to the thermal stability of the complexes.ΔG is positive for reaction for which ΔH is positive and ΔS is negative.The reaction for which ΔG is positive and ΔS is negative considered as unfavorable or non spontaneous reactions.Reactions are classified as either exothermic (ΔH < 0) or endothermic (ΔH > 0) on the basis of whether they give off or absorb heat.Reactions can also be classified as exergonic (ΔG < 0) or endergonic (ΔG > 0) on the basis of whether the free energy of the system decreases or increases during the reaction.The thermodynamic data obtained with the two methods are in harmony with each other.The activation energy of all 1,1-oxalyl-bis (4-phenyl) thiosemicarbazide and its Co 2+ , Cu 2+ , Zn 2+ and Sn 2+ complexes is expected to increase in relation with decrease in their radii (Tunali and Ozkar 1993).The smaller size of the ions permits a closer approach of the ligand (H 4 OxTSC).Hence, the E value in the first stage for the Zn 2+ complex is higher than that for the other Sn 2+ , Cu 2+ and Co 2+ complex.The correlation coefficients of the Arrhenius plots of the thermal decomposition steps were found to lie in the range 0.9925 to 0.9995 showing a good fit with linear function.It is clear that the thermal decomposition process of all complexes is non-spontaneous, i.e., the thermal stability of the complexes.The activation energy of Ligand II and its Co 2+ , Cu 2+ , Zn 2+ and Sn 2+ complexes is expected to increase in relation with decrease in their radii.The high values of the activation energy illustrated to the thermal stability of the complexes.The data were calculated and are summarized in Table 8.The smaller size of the ions permits a closer approach of the ligand (H 4 MaTSC).Hence, the E value in the first stage for the Zn 2+ complex is higher than that for the other Sn 2+ , Cu 2+ and Co 2+ complex.The activation energies of III and its metal complexes are summarized in Table 9.The high values of the activation energy illustrated to the thermal stability of the complexes.It is clear that the

Antimicrobial Activity
Three compounds were tested in vitro for their antibacterial activities against four strains of bacteria Gram nega-tive Escherichia coli, Pseudomonas aeruginosa species and gram-positive Bacillus cereus and Staphylococcus aureus.The bacteria were maintained on nutrient agar media.The minimal inhibitory concentration of some of the tested compounds was measured by a threefold serial dilution method.The screening results indicate that not all the compounds exhibited antibacterial activities.In this study, the tested compounds oxalyl, malonyl, and succinyl bis-4-phenylthiosemicarbazide were active against both Bacillus cereus, Staphylococcus aureus which are Gram-positive bacteria as well as Escherichia coli and Pseudomons aeruginose which are Gram-negative bacteria.However, the antibacterial activity was very pronounced against the Gram-negative bacteria and could be classified in the order of very good activity.

Conclusion
The activation energies of decomposition of 1,1-oxalyl, 1,1-malonyl and 1,1-succinyl-bis-4-phenyl-thiosemicarbazide and all the metal complexes are calculated.The data are summarized in Tables 7-9.The high values of the activation energy are illustrated to the thermal stability of the complexes.It is clear that the thermal decomposition process of all

Table 8 .
1,1-oxalyl-bis-4-phenylthiosemicarbazide (H 4 OxTSC) and its complexes is thermally stable.The activation energy of Ligand II and its Co 2+ , Cu 2+ , Zn2+and Sn 2+ complexes are expected to increase in relation with decrease in their radii.The high values of the activation energy are illustrated to the thermal stability of the complexes.The data are calculated and are summarized in Table7, The smaller size of the ions permits a closer approach of the ligand (H 4 MaTSC).Hence, the E value in the first stage for the Zn 2+ complex is higher than that for the other Sn 2+ , Cu 2+ and Co 2+ complex.The activation energies of III and its metal complexes are summarized in

Table 9 .
The high values of the activation energy are illustrated to the thermal stability of the complexes.It is clear that the thermal decomposition process of compounds I, II, III and Co 2+ , Cu 2+ , Zn 2+ , Sn 2+metal complexes are non-spontaneous, i.e., the materials are thermally stable.The tested compound I, II and III show a good activity against four strains of bacteria Gram negative Escherichia coli, Pseudomonas aeruginosa species and Gram-positive Bacillus cereus and Staphylococcus aureus.