4.7. DISEÑO DE LOS RESORTES DEL SISTEMA
5.1.7 ARMADO
culture type specific growth rate (Aihr ’ ) division time (dthr ’ ) length lag phase (hr) span of exponentia 1 phase (hr)
cell mass % cell
death within first hour of exposure to Ag wet weight (mg/ml) dry weight (mg/ml) (1) Ag-r cell alone 0.92db0.013 0.7510.12 110 2010.47 3.310.047 2.810.047 010 (2) Ag-r cells i/p lOpg/ml Ag 0.83±0.008 0.581 0.0047 110 2010.47 2.110.047 1.610.02 1010.94 Zn:+ + (1) 0.01 ± 0.0047 6.231 0.0047 1010.24 1410.24 0.081 0.0047 0.0610.002 5.5510.04 Zn + (2) 0.013± 0.0002 8.671 0.0082 710 810.235 0.0751 0.002 0.051 0.00047 8.310.047 Cu"" + (1) 0.3±0.008 0.2110.008 510.24 1510.24 0.0821 0.0009 0.0631 0.00047 010 Cu + (2) 0.0181 0.0002 38.11 0.0047 910.24 1010.235 0.0611 0.0014 0.0481 0.00047 10.610.9 Fe"" + (1) 0.0810.002 8.661 0.0047 310.24 1510.235 2.810.047 2.6510 010 Fe + (2) 0.5161 0.0026 1.3410.008 510 2010.235 2.410.047 2.1410.25 010
Table 4.3. Effect of Zn Cu and Fe on the growth curves of
Ag-r cells
From Table 4.3 it can be seen that on addition of 0.05mM ZnSO^ to the
Ag-r cells alone there is an initial cell death of 5.55%, the lag length is 10 hours with a low specific growth rate and a fairly high division time. When lOpg/ml silver is added to these cells, the initial cell death is slightly higher, at 8.3% and this culture also experiences the same growth pattern as the culture with Ag-r cells alone. The overall numbers o f viable cells is lower than the control
Chapter 4
cultures o f Ag-r cells grown in presence and absence o f silver. In Ag-r cells
incubated alone the cells tend to be unaffected by Cu^+ ions, although the
specific growth rate is low compared to the culture and the resulting
overall number o f viable cells is low. However, in presence o f silver, the
combined effects o f silver and copper tend to cause an initial cell death o f
10.6% within the first hour. Iron ions did not have an inhibitory effect on the
Ag-r cells alone, however the overall number o f viable cells in this culture were
reduced when compared with the control cells (Figure 4.30).
6 0 g < 20 - ■ -o- 4 0 lin ic (hours)
(Agi in Ag-r cells grown in 0 2ug'mi Ag [Ag] in Ag-r cells grown in 1 25ug ml Ag [Agj in Ag-r cells grown in 2 Sug ml Ag [ Ag] in Ag-r cells grown in 5ug,ml Ag [Ag] in .Ag-r cells grown in I Oug/ml Ag [Ag] in Ag-r cells grown in 20ug. ml .Ag [ Ag] in Ag-r cells grown in 50ug, ml Ag [Ag] in Ag-r cells grown in lOOug'ml Ag [Ag] in Ag-r cells grown in 500ug'ml .Ag [Ag] in Ag-r cells grown in I OOOug/'ml Ag I Ag] in Ps231 cells exposed to 0 2ug ml Ag
Figure 4.23 Ps231 cells subcultured in 2.5pg/m l Ag in nutrient broth and plated out on SAM with 2.5pg/m l Ag with zone sizes to silver metal discs o f 30, 32 and 34mm . x 1.3
Figure 4.24 Ag-r cells subcultured in lOpg/ml Ag in nutrient broth and plated out in SAM
Chapter 4
f c
Figure 4.25 Ag-r cells o f Ps.aeruginosa subcultured in 50pg/m l Ag in nutrient broth and plated out on SAM with 50pg/m l Ag with zones sizes to silver metal discs o f 13, 26 and 22mm . X / %
Figure 4.26 Ag-r cells subcultured in lOOpg/ml Ag in nutrient broth and plated out on SAM with lOOpg/ml Ag with zone sizes to silver metal discs of 18, 19 and 20mm M ^ - X 1 3
Figure 4.27 Ag-r cells subcultured in 500pg/ml Ag in nutrient broth and plated out on
Chapter 4
s
Figure 4.28 Ag-r cells subcultured in lOOOpg/ml Ag in nutrient broth and plated out on SAM with lOOOpg/ml Ag with zone sizes to silver metal discs o f 6 , 6 and 7mm Mag . x FI
%
Figure 4.29 Ag-r cells subcultured in lOOOpg/ml Ag in nutrient broth and plated out on
a 2H £ y I o c <cr - - - -K--- ^ 40 - X - Tim e (hours)
Effect o f Zn ions on Ag-r cells (N o Ag)
Effect o f Zn ions on Ag-r cells with lOpg/ml Ag Effect o f Cu ions on Ag-r cells (N o Ag)
Effect o f Cu ions on Ag-r cells with 1 Opg/ml Ag Ag-r cells with lOpg/ml Ag (Control))
Ag-r cells (no Ag in medium Control culture) Effect o f Fe ions on Ag-r cells (N o Ag) Effect o f Fe ions on Ag-r cells in 1 C^g/ml Ag
Figure 4.30 Effect of Zinc. Copper and Iron ions on the Growth Cycle of Ag-r cells of Ps. aeruginosa.
________________________________________________________ Chapter 4 4.4 Discussion
The lag phase is a period during which cells enlarge in size without cell division. A lag phase is ‘true’ only when it is experienced by the whole population. That the lag length was reduced during repetitive subculturing at one concentration o f silver was indicative of cells adapting to silver.
From results obtained in the growth curves in this study, it can be seen from the length o f the long lag phase that only a small population o f cells appear to be capable o f cell division on exposure to silver. This small population may possibly represent the genetic variants being selected out. Copper resistance in
Escherichia coli determined by plasmid pRJ1004 was found to be inducible by pregrowth at 0.4mM copper sulphate with the level o f growth proportional to the
inducing dose of copper (Rouch et al. 1985).
The lag phase o f silver adapted Pseudomonas aeruginosa cells in my study for
each successive culture is true as all cells survive after the initial exposure to silver. The long lag phase exhibited during the growth cycle may represent several factors responsible for the cells to survive on increasing concentrations of silver. These factors could range from chelation o f silver ions in order to inactivate or reduce its concentration from the growth medium to the exclusion of silver ions from the cell itself.
All cultures showed slower growth rates initially in presence of Ag^ which increased on repeated subculturing at the same concentration o f silver indicative of an inducible resistance. Long lag phases are indicative o f presence of
(Mitra and Bernstein 1984) growing in presence of cadmium and in marine
Pseudomonads (Kdi]m\ et al. 1992).
Studies on silver resistant Enterobacter cloacea (Annear et al. 1976) revealed
that silver resistance was expressed after acclimatization o f the cells and only when silver ions were present in the medium. Similar observations were made in
my study with the Ag-r Pseudomonas aeruginosa cultures. The cultures also
showed slower growth rates in the presence o f silver and the number o f viable cells decreased after a long lag phase. It has been proposed by Deshpande and
Chopade (1994) that specific surface receptors on Acinetobacter baumannii
BL8 8 may be binding silver ions and that the resultant lag phase may be
essential to dilute out the sensitive surface receptors to be replaced by those inhibiting the entry of Ag^ by chelating them. The occurrence o f extended lag phases was observed in cultures o f Ag-r cells at the lower concentrations of silver of 0.2 to 5pg/ml silver. This may possibly be due to saturation of surface receptors by silver ions which then may explain the slow growth rate and the
inducibility o f Pseudomonas aeruginosa in the presence o f silver in my study. A
similar phenomenon has been explained by Smith (1967) for resistance in bacteria to mercury, nickel and cobalt due to alteration in membrane
permeability. Thiobacillus species are known to accumulate large quantities of
silver by mere surface deposition (Pooley 1982).
In my study the accumulation o f silver measured as ng/mg dry weight o f cells increased sharply with an increase in number of viable cells. The cultures grown in lower concentrations o f silver between 0.2 to lOOpg/ml silver all accumulated
________________________________________________________ Chapter 4 silver at a rapid rate during the growth cycle. At concentrations o f 500 and
lOOOpg/ml silver, the amount of silver accumulated decreased even though the number o f viable cells increased during the growth cycle indicating that silver ions are prevented from entering the cells. Immediate accumulation of silver after addition of silver nitrate to the growth medium has also been observed in fungi (Pumpel and Skinner 1986).
In all the cultures the effect o f treating the cells with the uncoupling agent, FCCP resulted in a high increase in the accumulation of silver by the Ag-r cells. This was also observed for the Ps231 cells although the cells died due to
poisoning effects o f silver and FCCP. The de-energisation o f the pH gradient
across the cytoplasmic membrane which allows the to exchange for cations,
on treatment with FCCP increased the levels of silver in both Ag-r and the parent strain Ps231 indicative o f an efflux activity in these cells. That the Ag-r cells accumulated low levels o f silver up to 7 hours o f growth which then increased drastically on addition o f FCCP to these cultures, was also indicative
of an efflux pump operating in these cells. Li et a i (1997) found that in their
cultures of Ag-r Escherichia coli the de-energisation o f cells by CCCP resulted
in an increase in the accumulation o f silver in these cells. These researchers also added glucose to their ‘starved’ cultures which activated the efflux process in these cells. They also observed that the differences in accumulation levels in susceptible and resistant strains became smaller as the external concentration of silver was increased. This was also observed in my study in which the
that o f cells cultured in low concentrations o f silver. This can possibly be
explained by the proposal made by Li et a i (1997) that the influx o f silver may
overwhelm the efflux o f silver with the silver efflux system becoming saturated at high external concentration of silver. The isolation of a silver resistant 54kb
plasmid (pUPI199) from an environmental isolate o f Acinetobacter baumannii
BL8 8 transformed to cultures of Escherichia coli DHa showed an immediate
accumulation of silver after addition of silver nitrate in the transformant
Escherichia coli DHa The growth rate o f the transformant Escherichia coli
D//ûf(pUPI199), however, was slower when compared with Escherichia coli
DHa. (Deshpande et a i 1994). These workers also reported that while
Acinetobacter baumannii BL8 8 accumulated and retained the silver, the
Escherichia coli DHa effluxed 63% of the accumulated silver. This action was attributed to the possible presence o f plasmid coded specific surface receptors in
these cells (Deshpande et a i 1994).
Pseudomonas aeruginosa FAO4068 has also been shown to accumulate four to fivefold more silver on pretreatment with lOOpM CCCP (Li and Williams 1997,
unpublished data; personal communication). Li et a i (1997) have suggested an
active efflux system o f Ag^in wild type strains of Pseudomonas aeruginosa
which is independent of the known multidrug resistance (MDR) efflux pumps o f
Pseudomonas aeruginosa. In my study, the data indicated that an efflux process
for the Ag-r cells o f Pseudomonas aeruginosa may exist and that the plasmid
identified in Chapter 3 could be responsible for this process.
________________________________________________________ Chapter 4 part achieved at very high external concentrations o f silver by active efflux of
the silver ions. Such resistance has also been reported to occur in Pseudomonas
stutzeri (Slawson et al 1994). Multidrug efflux resistance pumps (MDR) involving effluxing o f different substrates may be operational in the bacteria in my study.
On the effect of other heavy metal ions on the growth o f Ag-r Pseudomonas
aeruginosa, there was immediate cell death on exposure o f Ag-r Pseudomonas aeruginosa cells to zinc alone (Figure 4.30) although the synergistic effect o f zinc and silver was found to be more toxic to these cells than with either o f the
metal alone. Inhibition o f the respiratory chain of Escherichia coli has been
reported by Kasahara and Anraku (1972) who showed that ImM zinc ions inhibited succinate dehydrogenase activity by more than 70%. The presence of silver ions may prevent zinc ions from totally inhibiting the enzyme activity but may also slow down the metabolic activity o f the cells. Zinc ions have been known to inhibit uptake and oxidation o f citrate, glucose and alanine in intact
cells o f Pseudomonas aeruginosa (Eagon and Abseil 1969). Silver is known to
have a less toxic effect than zinc and in this study may offer ‘protection’ to Ag-r cells from zinc ions by binding these sites competitively.
On the effect o f copper ions on the Ag-r cells, the synergistic effect o f silver and copper ions caused an initial cell death o f 10.6 3% in the first hour. The cells that do survive on exposure to copper ions may be genetic variants o f increased silver resistance and may possibly be afforded ‘protection’ by the silver ions. Another explanation is that silver which has the smaller ionic radii o f
0 .126nm may competitively bind the cell surface receptors in favour o f copper ions which have a larger ionic radii of 0 .196nm. Silver may also compete with copper ions for binding sites on Omp C which is a copper binding and utilising
protein o f the outer membrane in Pseudomonas aeruginosa (Yoneyama and
Nakae 1996).
Silver injected into mice has been shown to interfere with copper utilisation in rats for ceruloplasmin synthesis ( Sugawara and Sugawara 1984). Silver may
also inhibit copper activity on Ag-r cells o f Pseudomonas aeruginosa by
possibly interfering with the copper binding proteins o f the electron transfer chain. Studies on azurin, a copper protein involved in bacterial electron
transfer chain in Pseudomonas aeruginosa, showed silver to bind to azurin by
total displacement o f Cu(I) ion from its native binding site (Tordi et a i 1990).
Copper, like silver is known to bind at specific sites on DNA. Antimicrobial activity of copper is related to oxidation o f sulphydryl groups o f enzymes (Hugo 1987) which inhibits enzymatic activity and interferes with cell respiration
(Domek et a i 1984 and 1987). In my study in the Ag-r cells a slight resistance to
copper was observed as the cells were repeatedly subcultured in silver (Chapter 3), indicative o f possible selection o f genetic variants amongst the cells that survived.
Iron does not appear to inhibit the growth o f Ag-r cells but the specific growth rate was lower than that o f the control cells indicating that the rate o f cell division was decreased in the iron exposed cells. The combined action o f iron with silver resulted in a great reduction o f the numbei\viable cells in the culture
________________________________________________________ Chapter 4 exposed to both silver and iron, although the pattern o f bacterial growth was not affected.
Iron may possibly be affecting the zinc enzyme, alkaline phosphatase by displacing it and thus interfering with the normal cell respiration functions. Silver may also indirectly affect iron transport by damaging the absorption site of the iron siderophores. However, further experiments to monitor the levels o f siderophores produced by these cells need to be carried out in cells cultured in high amounts o f silver to verify this suggestion.
4.5 Summary
Silver was found to decrease the number o f viable cells after a long lag phase. The cultures showed slower growth rates in presence o f Ag^ indicative of an inducible property and selection o f genetic variants able to resist toxic levels of
silver ions in the growth medium. Repeated subculturing o f Pseudomonas
aeruginosa cells resulted in a decrease in the length o f the lag phase. The level o f resistance to silver increased on successive subculturing o f silver adapted cells with an increase in concentration o f silver ions in the growth medium. Uptake o f silver ions in cultures o f Ag-r cells is rapid within the first hour. Silver uptake is gradual in Ag-r cells grown in higher concentration o f silver and rapid in lower concentration o f silver in the untreated cells. However as most o f the subcultures experienced a percentage o f cell death during the first hour of exposure, the amount o f silver accumulated decreased with an overall decrease in the number o f viable cell in these cultures. Overall, less silver appears to be accumulated by Ag-r cells. Evidence o f a possible efflux pump operating in the
cells exposed to extremely high concentrations o f silver was observed to be
present in the Ag-r Pseudomonas aeruginosa cells during studies with the
uncoupling agent FCCP. The presence o f silver ions may prevent zinc ions from totally inhibiting the enzyme activity but may also slow down the metabolic activity o f the cells. Silver ions may create iron limiting conditions for cell
growth in Ag-resistant Pseudomonas aeruginosa cells. Silver may also compete
with copper ions for binding sites on Omp C which is a copper binding and
utilising protein of the outer membrane in Pseudomonas aeruginosa.
The morphology o f both the parent Ps231 and the Ag-r cells will be studied in Chapter 5 to determine the action o f silver ions on and within these cells.
Chapter 5
C hapter 5
Morphology of Bacterial Cells 5.1 Introduction
5.2 M aterial and Methods
5.2.1 Transmission Electron Microscopy (TEM) 5.2.1.1 Fixation
5.2.1.2 Dehydration
5.2.2 Scanning Electron Microscopy (SEM) 5.2.3 Negative Staining of the Cells.
5.3 Results
5.3.1 TEM of Bacteria
5.3.1. 1 Control cells Ps231.
5.3.1.2 Control cells Ps231 ‘exposed’ to 0.2pg/ml silver. 5.3.1.3 Ag-r cells C ultured in lOpg/ml silver.
5.3.1.4 Ag-r cells Cultured in lOOpg/ml silver. 5.3.1.5 Ag-r cells Cultured in lOOOpg/ml silver.
5.3.1.6 Statistical Analysis of Cells Associated with Electron Dense particles.
5.3.2 Energy Dispersive X-ray Analysis (EDX) of Electron Dense particles
5.3.3 Negative Staining of Ps231 and Ag-r cells
5.3.4 SEM of Ag-r cells on Ag- coated Titanium Pellets. 5.4 Discussion
5.4.1 TEM of Bacterial Cells
5.4.2 SEM and Negative staining of Ag-r cells 5.5 Sum m ary
Chapter 5
Morphology of Bacterial Cells