However, soil has scarcely been investigated as a possible source of
As drug-resistant
The
Based on results of a previous investigation (17), we used two soil samples representative of slightly acidic and alkaline soil from Istria, Croatia –
Soil pH value was measured with a WTWSenTix81 electrode (WTW, Weilheim, Germany) after triplicate suspension in distilled water (1:2.5 wt/v). The procedures for chemical (ICP-ES/MS following a lithium borate fusion and dilute nitric digestion) and mineralogical analyses (X-ray powder diffraction of <2 mm and <2 μm fractions) were described in detail in Durn et al. (18).
Natural zeolitised tuff (NZ) was obtained from a Zlatokop sedimentary deposit in Serbia and consisted of 73 % clinoptilolite, 14 % plagioclase, and 13 % quartz (wt). To load NZ with metals, we used the ion-exchange procedure described in our earlier study (14). Briefly, it consisted of the following steps: 1) conversion of NZ into Na-enriched form (NaNZ) by treating NZ with 2 mol/L of NaCl solution at 25 °C for 48 h; 2) treatment of NaNZ with 6 mmol/L of Cu(NO3)2 or AgNO3 solution in a water bath at 25 °C for 24 h; and 3) recovering of the metal-containing products (CuNZ and AgNZ) by filtration.
The leaching of cations from zeolites to soil samples is technically hard to track. Moreover, our previous investigation (13) showed negligible leaching of Cu2+ from CuNZ into the water medium. This is why we investigated only Ag+ leaching in this study. One gram of red palaeosol was supplemented with 1 mg (0.1 wt%) of AgNZ and added to 100 mL of autoclaved commercially available spring water and left in the dark at 25 °C for 1 h. The solid was separated from water by filtration through 0.45 μm syringe filters and the concentration of the leached Ag determined in water solution by atomic absorption spectrophotometer (AAS Varian, Spectra AA 55b, Agilent Technologies, Santa Clara, CA, USA).
The
Both soils were distributed in 100 g quadruplicate samples to laboratory glasses.
At the beginning of the experiment and at specified time points (Figures 1 and 2), the soil samples were mixed well and subsamples taken for bacteriological analysis in triplicate. One gram of wet soil was suspended in sterile physiological solution by vortexing at 45 Hz for 3 min and then passed through sterile membrane filters directly when bacterial concentrations were low or diluted decimally and inoculated on bacteriological media when the concentrations were sufficient for dilution.
Removal of
Removal of
Total native heterotrophic bacteria were counted on nutrient agar (Biolife, Milan, Italy) after incubation at 22 °C for 72 h.
Bacterial colonies were identified on whole cells with matrix-assisted laser desorption/ionisation – time of flight mass spectrometry (MALDI-TOF MS, software version 3.0, Microflex LT, Bruker Daltonics, Billerica, MA, USA). Randomly chosen colonies developed on CHROMagar Acinetobacter were confirmed as
Statistical significance (p<0.05) of the difference between the baseline and end bacterial counts was established with Student’s
The samples of modified zeolites contained similar amounts of metal cations per gram of dry sample: 0.37 mmol Cu2+ (CuNZ) or 0.50 mmol Ag+ (AgNZ).
In
The addition of 1 wt% CuNZ (Figure 1) significantly accelerated the removal of
The addition of 1 wt% of AgNZ (Figure 1) resulted in complete removal within 1 h of contact. The same result was achieved with 0.1 wt% of AgNZ. Although this effect was accompanied by a statistically significant reduction of heterotrophic bacteria, their count was still high (5.2±0.2 log CFU/g) after 24 h of contact.
In red palaeosol supplemented with 1 wt% of NZ (Figure 2)
The difference in
The addition of 1 wt% of CuNZ (Figure 2) completely removed
The addition of 1 or 0.1 wt% of AgNZ (Figure 2) removed the entire
Considering the long survival of
Table 1 shows native heterotrophic bacteria identified in red palaeosol before and after treatment with NZ, CuNZ, or AgNZ. Treatment with NZ and CuNZ left both spore-forming (
Native soil heterotrophic bacterial isolates determined with MALDI-TOF MS in fresh red palaeosol before and at the end of treatment with 1 wt% of NZ or CuNZ or AgNZ
Isolate | Fresh soil | NZ | CuNZ | AgNZ |
---|---|---|---|---|
6 | 1 | 1 | ||
4 | 1 | |||
1 | ||||
1 | ||||
2 | 6 | |||
2 | ||||
1 | ||||
1 | ||||
1 | ||||
1 | ||||
1 | ||||
2 | 2 | |||
1 | ||||
2 | ||||
1 | 1 | |||
1 |
* spore-formers
Ag cations exert bactericidal effects by blocking the enzymatic machinery of bacterial cells (22). There are concerns about
With its almost instant bactericidal effect against
The addition of AgNZ to soil resulted in removal of 6.8 log CFU/g of viable