CHAPTER 5
PHYSICOCHEMICAL FACTORS AFFECTING THE GROWTH OF ENTEROCOCCI
5.1. INTRODUCTION
Enterococcus species are wide-spread Gram-positive bacteria and are
natural inhabitant of the gastrointestinal tract (GIT) of humans and animals.
They are also commonly found in soil, sewage, water and food. The ability
to survive in unfavorable conditions is responsible for their ubiquitous nature
and persistence in the environment. In order to survive in the human GIT,
bacteria must overcome several adverse environmental stresses such as
changes in the pH, temperature, nutrient availability, elevated osmolarity and
the deleterious actions of bile.
Another important parameter which can affect growth and survival is
disinfectant. Though disinfection procedures help to reduce the infection risk
by lowering the microbial load, many of the bacteria develop resistance
against the commercially available disinfectants. Biocide resistance will
result in inadequate decontamination of medical devices. Contaminated
environmental surfaces and medical devices can serve as a vehicle of
infectious agents (Manangan et al., 2001) and is associated with risk of
hospital-acquired infection (Boyce, 2007).
Although Enterococcus species have been reported as nosocomial
pathogens their profile of resistance to biocides has been hardly studied.
Similarly heavy metal ions of mercury, silver, copper, lead, zinc and other
metals show antimicrobial effect even in relatively low concentrations.
Studies have shown that heavy metals are inhibiting the adhesion of bacteria
98 Chapter 5
on biofilm (Kielemoes and Verstraete, 2001). In order to survive in the wild
and contaminated environment, bacteria develop different mechanisms to
confer resistance to these heavy metals (Karamanisal et al., 2008).
Since the Enterococcus is recognized as serious nosocomial
pathogens, it is of great significance to understand such responses in
enterococci which enable them to survive in a wide range of environments.
The study was done to investigate the effects of different factors like
temperature, pH, different salt concentrations and presence of bile salts on
the growth of enterococcal strains. Another aim of the work was to test the
efficacy of several commonly used disinfectants on the isolates of
Enterococcus and also to characterize the variations in the disinfectant
susceptibilities of biofilms and planktonic cells. Yet again this study was
focused to determine the effectiveness of heavy metal on Enterococcus
isolates and to find the minimum bactericidal concentration of different
heavy metals.
5.2. MATERIALS AND METHODS
Isolates from four different sources comprising water, clinical
samples, healthy human faeces and chicken faeces were subjected to study
the influence of different physicochemical parameters on the growth. Twenty
E. faecium, twenty E.faecalis and twenty other miscellaneous enterococci
including E.gallinarum, E.avium, E.raffinosus and E.durans were selected to
study the effect of physical factors on the growth.
5.2.1. Effect of incubation temperature on growth
Isolated colonies of Enterococcus strains were inoculated into brain
heart infusion broth and incubated at 37o C for 24 hours and turbidity was
adjusted to a final concentration of approximately 10 8 cells(0.5 McFarland
Physicochemical Factors Affecting the Growth of Enterococci 99
standard). 0.01 mL of the broth was inoculated into BHIB and allowed to
grow at temperatures ranging from 10o C to 40o C for 2 days and at 4o C for
one week, and optical density at a wavelength of 600 nm was checked by
using spectrophtometer (Hitachi F-2700) and was subcultured on BEA and
examined for enterococcus like colonies.
5.2.2. Effect of pH on growth
0.01 mL of Enterococcus culture in brain heart infusion broth culture
of about 108 CFU/mL was transferred into BHIB adjusted to different pH
levels. The optical density of the broth at 600 nm was determined after 48
hours and was subcultured as described above.
5.2.3. Influence of various concentrations of sodium chloride on the growth of Enterococcus
0.01 mL of Enterococcus culture in BHIB with a turbidity adjusted
to 108 CFU/mL was inoculated into BHIB containing different
concentrations of NaCl (Merck). After 48 hours, growth was monitored by
determining optical density at 600 nm and was subcultured as described
above.
5.2.4. Heat resistance of Enterococcus
Test tubes containing sterile BHIB were inoculated with the test
strain followed by incubation at 37o C for 24 hours and turbidity was
adjusted to a concentration of approximately 108 cells/ mL (0.5 McFarland
standards). One set of tubes were kept in a water bath adjusted to 63oC for
30 minutes and others at 72oC for 20 seconds. After this the presence of
viable cells was tested by subculturing, 0.1 mL sample on to BHIA plates.
The plates were incubated at 37°C for 24h and the colonies were counted by
using colony counter (Joshibha) .Untreated tubes served as control and were
100 Chapter 5
also subcultured and counted. The log10 of number of viable count was
calculated.
5.2.5. Bile tolerance
The Isolated enterococcal colonies were inoculated into BHIA
medium containing 40% of bile salt (HiMedia) and incubated at 37°C. After
24h the plates were examined for bacterial growth.
5.2.6. Survival on dry cotton swabs
Isolates were grown overnight at 37°C in brain heart infusion broth.
After growth, 1 mL from each culture was added to a sterile centrifuge tube
and was centrifuged (10000g 5’ at 5°C). Cell pellets were resuspended in
PBS and cell density was adjusted to 108 CFU/mL by using Mc Farland
turbidity standard. Using a micropipette, cotton swabs were inoculated with
10µl of suspension. Inoculated swabs were inserted into different tubes, and
incubated at room temperature. One swab each of a strain was inoculated on
to Brain heart broth every day and after 48 hours; one loopful was
subcultured on to BEA plates and incubated at 37°C for 48 hours. This was
repeated until cultures no longer showed growth.
5.2.7. Bactericidal effect of disinfectant on planktonic cells:
The bactericidal effect of disinfectant on the isolates was measured
by suspension test (Cremieux et al., 1991). The solutions of disinfectants
(Appendix-2) at different concentrations in 1000 mL volumes were made in
distilled water. Overnight grown TSB culture of the bacteria (1mL) was
centrifuged (10000g 5’ at 5°C) and cell pellets were suspended in saline. 1
mL of the cell suspension containing approximately108 CFU/mL was added
into the 9 mL disinfectant solution. After a contact time of 1, 5 & 10
minutes, serial 1:10 dilutions were performed in neutralizing medium
Physicochemical Factors Affecting the Growth of Enterococci 101
(Appendix-2). 0.1 mL samples were then inoculated on TSA and the
bacterial growth was examined. A bacterial suspension treated with PBS
instead of a disinfectant was used as the control.
5.2.8 Effect of disinfectants on biofilm
Bacterial suspensions standardized to 108 CFU/mL in TSB were
prepared and 200 µl of these suspensions were taken in the sterile wells of
polystyrene micro titre plates and incubated at 37oC for 72 hours so that
biofilm was formed inside the wells. Wells were washed with PBS so as to
remove the non adherent bacteria. Uninoculated well containing trypticase
soy broth was used as control. The disinfectants at different concentrations
were added to the wells. After 1, 5 & 10 minutes, the contents were
discarded. The wells after disinfectant treatment were washed with PBS for 3
times and 200 µl of the fresh TSB was added into each well and biofilm was
detached by scraping the wells. After mixing the contents10 µl of the
suspension was then transferred to TSA and incubated at 37oC for 18 hours
and examined for bacterial growth. The microtitre plates were further
incubated overnight and subcultured to assess the viability of bacteria
remaining if any.
5.2.9. Determination of bactericidal concentration of heavy metal on Enterococcus in planktonic form
The effect of heavy metal ions on enterococci isolates was
determined by the plate-dilution method as described by Malik and Jaiswal,
(2000). Heavy metal salts like Pb (NO3)2, CoCl2, CdCl2, and ZnCl2, HgCl2
and AgNO3 (Merck) were used. Concentrations in the range of 0.1 to 20
mg/mL were used. Metal salts were dissolved in distilled water, filter
sterilized and added to MHA medium in the required concentrations. The
bacterial isolates were grown and 0.01 mL of the inoculum containing
102 Chapter 5
approximately 108enterococci cells were spot inoculated on MHA containing
graded concentrations of heavy metals. MHA medium without metal served
as control. The inoculated plates are incubated at 37oC for approximately 48
hours before reading the results. Minimum bactericidal concentration was
noted when the isolates failed to grow on plates.
5.2.10 Determination of bactericidal concentration of heavy metal on Enterococcus in biofilm
Enterococcus was grown overnight in TSB at 37°C. 200µl of the
culture (108 CFU/mL) was used to inoculate sterile 96 well-polystyrene
microtitre plates. After incubating for 24 hours at 37°C, the wells were gently
washed 3 times with 200 µl phosphate buffered saline (PBS), dried in an
inverted position. The heavy metals at different concentrations (Stocks of the
metal salts prepared in distilled water) were added to the wells. After 30
minutes, the contents were discarded. The wells after heavy metal treatment
were washed with PBS for 3 times and 100 µl of the fresh PBS was added
into each well and biofilm was detached by scraping the wells.10 µl of the
suspension was then transferred on to TSA and incubated at 37oC for 18
hours and examined for bacterial growth. The microtitre plates were further
incubated overnight and subcultured to assess the viability of bacteria
remaining if any.
5.3. STATISTICAL ANALYSIS
Influence of different factors on growth of the Enterococcus species
was analyzed by Chi square test and by ANOVA by using SigmaStat
software (Sigma-Aldrich, St. Louis USA). The level of significance was set
up at P <0.05.
Physicochemical Factors Affecting
5.4. RESULTS
The growth of different species of
optical density ± SD at 4°C
Figure.5.1. Growth of
The effect of different temperatures on
studied and growth was noticed in
these temperatures affected
same manner with heavy growth
greater OD values than all
environmental sources. Error bars indicates the Standard deviation.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
E.faecium E.faecalis
Me
an
OD
60
0
Physicochemical Factors Affecting the Growth of Enterococci
of different species of Enterococcus measured as mean
4°C is given in Figure 5.1.
rowth of enterococci at low temperature
effect of different temperatures on growth of enterococci was
was noticed in all tested strains (data not shown). E
affected the growth of different enterococci species
with heavy growth except at 4°C. At 4o C, E.faecium
all other enterococci species from both human
Error bars indicates the Standard deviation.
E.faecalis E.gallinarum E.avium E.raffinosus E.durans
Human Environmental
103
measured as mean
growth of enterococci was
Each of
different enterococci species in the
yielded
human and
E.durans
104
Effect of pH 3 on the growth of enterococci is presented in figure 5.2
Figure
Enterococcal isolates from all sources
low pH conditions.
enterococcus species was
shown) except at pH 3
than all other enterococci
difference in the mean
(P>0.05).
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
E.faecium
Me
an
OD
60
0
pH 3 on the growth of enterococci is presented in figure 5.2
Figure 5. 2. Growth of enterococci at pH 3
Enterococcal isolates from all sources were evaluated for
conditions. At each of this pH the growth shown by different
enterococcus species was in the same way with high OD values
except at pH 3. At pH 3 E.faecium produced a higher growth rate
enterococci species. There was no statistical
mean OD values between environmental and human strains
E.faecium E.faecalis E.gallinarum E.avium E.raffinosus
Human Environmental
Chapter 5
pH 3 on the growth of enterococci is presented in figure 5.2.
were evaluated for growth at
pH the growth shown by different
with high OD values (data not
produced a higher growth rate
no statistically significant
between environmental and human strains
E.raffinosus E.durans
Physicochemical Factors Affecting
Effect of pH 10 on the growth of enterococci is presented in
Figure 5.
Enterococcal isolates from all sources were evaluated for growth at
alkaline pH conditions. Growth was obtained up to
not shown by the isolates
shown to have a better growth in alkaline conditions than others.
slight increase in growth was observed in
E.faecium, E.faecalis and
found (P>0.05). Human isolates
more growth than environmental isolates
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
E.faecium E.faecalis
Me
an
OD
60
0
Physicochemical Factors Affecting the Growth of Enterococci
pH 10 on the growth of enterococci is presented in figure 5.3
3. Growth of enterococci at pH 10
Enterococcal isolates from all sources were evaluated for growth at
Growth was obtained up to pH 10 and growth was
shown by the isolates at pH 13 (data not shown). E.faecium isolates were
shown to have a better growth in alkaline conditions than others. Though a
rease in growth was observed in environmental isolates
and E.durans, a significant difference could not be
Human isolates of E.avium and E.raffinosus showed slightly
than environmental isolates.
E.faecalis E.gallinarum E.avium E.raffinosus E.durans
Human Environmental
105
figure 5.3.
Enterococcal isolates from all sources were evaluated for growth at
10 and growth was
isolates were
Though a
environmental isolates of
a significant difference could not be
showed slightly
E.durans
106
The effect of
figure 5.4.
Figure 5. 4
The growth of enterococci at different salt concentrations was
studied. Growth was obtained at a s
not shown). No growth was shown by the isolates at a salt concentration of
15%. At each of these
enterococci species was
E.faecium produced
isolates of all the isolates demonstrat
isolates (P>0.05)
0
0.05
0.1
0.15
0.2
0.25
0.3
E.faecium
Me
an
OD
60
0
The effect of 11% of NaCl on the growth of enterococci is shown in
4. Growth of enterococci at 11% salt concentration
growth of enterococci at different salt concentrations was
Growth was obtained at a salt concentration of 0.5 to 11% (data
No growth was shown by the isolates at a salt concentration of
At each of these concentrations the growth shown by different
enterococci species was comparable except for salt at 11%.
produced a better growth than other species.
isolates of all the isolates demonstrated slightly more growth
(P>0.05).
E.faecium E.faecalis E.gallinarum E.avium E.raffinosus
Human Environmental
Chapter 5
terococci is shown in
salt concentration
growth of enterococci at different salt concentrations was
alt concentration of 0.5 to 11% (data
No growth was shown by the isolates at a salt concentration of
the growth shown by different
11%. At 11% NaCl,
Environmental
more growth than human
E.raffinosus E.durans
Physicochemical Factors Affecting the Growth of Enterococci 107
The bacteria before and after heat treatment were counted and log
CFU/mL± SD formed by them are presented in table 5.1.
Table.5.1. Heat resistance of enterococcal isolates
log CFU/mL±SD
Enterococcus tested Before heating After heating
63 for 30minutes 72 for 20seconds
E.faecium 5. 6628±3.33 2.8633±1.59 1.8451±0.69
E.faecalis 5.0792±3.95 2.8062±1.64 1.6902±0.84
E,durans 5.1761±3.85 2.7993±1.36 1.5911±0.90
E.gallinarum 5.3222±3.84 2.6021±1.64 1.3010±0.34
E.avium 5.1461±3.22 2.7781±1.22 1.6021±0.48
E.raffinosus 5.0414±3.13 2.6990±1.25 1.4150±0.60
The cultures survived heat treatment at 63°C even though there was a
reduction in the viable count (P<0.05). Exposure to heat at 72 °C resulted in
a notable reduction in bacterial count than at 63°C for all isolates. E.faecium
culture produced more viable cells after heat treatment. E.gallinarum
produced least number of viable cells after heat treatment.
108 Chapter 5
The survival of enterococci strains at room temperature is presented
in table 5.2.
Table 5.2. Survival of various Enterococcus species in dry cotton swabs incubated at room temperature
Enterococcus species
No.of isolates tested
Average period (in days) of survival of the isolates from various sources
Water Chicken Human Clinical
E.faecium 8 12 10 9 13
E.faecalis 10 12 11 9 12
E.gallinarum 3 10 11 9 12
E.avium 3 11 9 9 12
E.raffinosus 3 10 11 9 12
E.durans 2 11 11 9 11
All enterococcal strains were shown to survive in dry cotton swab.
Isolates from clinical sources showed a significantly higher resistance than
the isolates from other sources (P<0.05) .The isolates from human faecal
samples showed the least days of survival.
MBCs of disinfectants against planktonic and biofilms of
E.faecium(n=51)and E.faecalis(n=31) were observed in a range and are
summarized using the box-plot method (Figures 5.5 and 5.6).
Physicochemical Factors Affecting the Growth of Enterococci 109
Figure 5.5. MBC of different disinfectants on biofilm and planktonic forms of E.faecium
On planktonic forms of different E.faecium isolates cresol showed
MBC in the range of 1.25- 10mg/mL (median MBC of 5mg/mL). But on
biofilms 20-30mg/mL was the MBC required (median MBC30mg/mL). The
MBC of BK was in the range of 1.25-2.5mg/mL (median MBC of
2.5mg/mL) on planktonic forms, whereas on biofilm, it was 10-50mg/mL
(median 20 mg/mL).
MBC of glutaraldehyde on planktonic forms was ranging from 1.25-
5mg/ml (median 2.5mg/mL). Nevertheless 20-30 mg/mL (median of
20mg/mL) was needed to achieve bactericicdal activity on biofilm. MBC of
NaOCl and iodine ranged from 0.3125-1.25mg/mL (median 0.3125) and
0.625-2.5(median 0.625) respectively. And on biofilms the MBC of NaOCl
were10-20mg/mL and MBC of iodine was only10mg/mL (median
10mg/mL).
0
5
10
15
20
25
30
35
40
45
50
55
60
MB
C (
mg
/mL)
P-Plaktonic, B-biofilm
Q1 Min Median Max Q3
110 Chapter 5
Figure 5.6. MBC of different disinfectants on biofilm and planktonic forms of E.faecalis
On planktonic forms of E.faecalis, cresol showed MBC in the range
of 1.25-10 mg/mL (median 5mg/mL). And on biofilm bacteria 30mg/ml was
required to produce a 100% kill. MBC shown by BK was in the range of
1.25-2.5 mg/mL (median 1.25mg/mL). MBC demonstrated by NaOCl ranged
from 0.3125-0.625 mg/mL (median 0.625mg/mL). Iodine showed a very
high bactericidal activity even at a low MBC ranging from .3125-2.5 mg/mL
(median 0.3125mg/mL). 10 mg/mL concentration of Iodine, NaOCl and BK
killed biofilm of E.faecalis. For planktonic forms glutaraldehyde MBC was
in the range of 1.25-5 mg/mL (2.5 median). Glutaraldehyde of 20mg/mL was
required to produce a 100% kill of biofilm bacteria.
Comparison of action of disinfectants on planktonic and biofilm
forms of other Enterococcus species is shown in Table 5.3.
0
5
10
15
20
25
30
35
MB
C (
mg
/mL)
P-planktonic, B-biofilm
Q1 Min Median Max Q3
Physicochemical Factors Affecting the Growth of Enterococci 111
Table 5.3. MBC of various disinfectants in respect of planktonic and biofilm forms of miscellaneous Enterococcus
Range of MBC (mg/mL) of disinfectants
Species tested* Cresol BK NaOCl Iodine Glutaraldehyde
E.gallinarum (4) planktonic 1.25 1.25 0.3125 0.3125 1.25
Biofilm 20 10 10 10 20
E.durans (4) planktonic 1.25 1.25 0.3125 0.3125 1.25
Biofilm 20 10 10 10 20
E.avium (5) planktonic 2.5 1.25 0.3125 0.3125 1.25
Biofilm 20 10 10 10 20
E.hirae (1) planktonic 1.25 1.25 0.3125 0.3125 1.25
Biofilm 20 10 10 10 20
E.raffinosus (5) planktonic 1.25 1.25 0.3125 0.3125 1.25
Biofilm 20 10 10 10 20
E.mundtii (1) planktonic 1.25 1.25 0.3125 0.3125 1.25
Biofilm 20 10 10 10 20
*No. of strains tested is given in brackets
Among the miscellaneous bacteria E.avium planktonic form was
found to be more resistant when treated with cresol. Biofilm bacteria were
killed by 20 mg/mL of cresol and glutaraldehyde respectively. All the
miscellaneous bacteria exhibited similar pattern of resistance. When BK,
iodine and NaOCl were used, biofilm forms of all these bacteria were
removed by 10 mg/mL of concentration.
The resistance of enterococcal strains to six different heavy metals
such as cobalt, lead, zinc, cadmium, silver and mercury were determined.
112 Chapter 5
Comparative analysis of MBCs on planktonic and biofilm E. faecium is
shown in tables 5.4.
Table 5.4: Minimum bactericidal concentrations of heavy metals required to kill 100% of the tested isolates of E.faecium (n=5)
Metal
Concentration (mg/mL) of heavy metal required to kill 100% of the tested isolates
Planktonic Biofilm
Mercury 0.1-0.25 0.25
Silver 0.1-0.25 0.25
Zinc 0.5 5-20
Cadmium 0.5 2.5-10
Lead 0.5 5-20
Cobalt 0.5 5-20
Silver and mercury were highly effective against E.faecium and 80%
of them were removed at 0.1mg/mL (data not shown). Though 80% of
bacteria in biofilm were killed by 5% of zinc, lead and cobalt, resistant strain
was eliminated only after treating with 20mg/mL of these metals. Ag and Hg
were found to be more effective on planktonic and biofilm than all metals
tested (P<0.05).
Comparative analysis of MBCs on planktonic and biofilm of E.
faecalis are shown in tables 5.5.
Physicochemical Factors Affecting the Growth of Enterococci 113
Table 5.5. Minimum bactericidal concentrations of heavy metals required to kill 100% of the tested isolates of E.faecalis (n=5)
Metal
Concentration (mg/mL) of heavy metal required to kill 100% of the tested isolates
Planktonic Biofilm
Mercury 0.1 0.25
Silver 0.1 0.25
Zinc 0.5 5
Cadmium 0.25 5
Lead 0.5 5
Cobalt 0.1—0.25 5
Silver and mercury were very effective on E.faecalis. Mercury and
silver removed all planktonic bacteria at 0.1mg/mL concentration. Zinc and
lead were less effective and all cells were removed only at 0.5mg/mL
concentration. Resistance of biofilm was more compared to planktonic forms
of bacteria. A concentration of 5mg/mL of zinc, lead, cadmium and cobalt
was required to kill E.faecalis biofilm.
5.5. DISCUSSION
While investigating the growth and survival strategies of enterococci,
the growth of Enterococcus was obtained in all the temperatures tested.
When growth was analyzed at 4o C in this study all isolates were found to be
growing. At this low temperature the total count was less in all the isolates
except in E. faecium. E. faecium was shown to have more growth rate than
other enterococci. Minimum growth temperature for E. faecium was reported
to be 0.1o C (Zanoni, 1993). Earlier studies support this by stating E faecium
114 Chapter 5
as a psychrotolerant species (Biomine news). It can be assumed that from
food microbiology point of view, Enterococcus species are important
because of its growth potential at refrigeration temperature.
The findings of this study show the ability of enterococci to grow in
different pH conditions. Growth of enterococci in a wide range of pH was
observed. At pH 3 all species survived and yielded growth and at pH 2, none
of the isolates survived except one strain of E.faecium [data not shown in the
table]. Growth of enterococci at pH 3 was observed in an earlier study (Galaz
et al., 2004). Acid resistance in enterococci helps to establish Enterococcus
of animal origin in the human gut. Enterococcal growth was also obtained at
pH 10 in this study. This is in conformity with a previous study by Flahaut et
al., (1996). Many factors contribute to the tolerance of alkaline pH found in
enterococci, including activation of specific proton pumps and specific
enzyme or buffer devices that helps to keep internal pH constant (Nagao et
al., 1990). This help in the survival of enterococci in alkaline environments
resulting from pollution of waters by industrial effluents or agricultural
wastes and in some foods (Rowbury et al., 1996).
Up to a concentration of 11% of NaCl, all enterococci produced
growth. And E.faecium was the more salt tolerant species. Though there is a
lack of earlier reports in this regard, Enterococcus is well known for its
extraordinary ability to survive in salt water. Taking this into consideration,
EPA recommends enterococci as the best indicator of health risk in salt water
used for recreation. NaCl has a role in the induction of general stress protein
in Enterococcus ( Rincé et al.,2002). Enterococcs has a cation homeostasis
which is thought to be contributing to its resistance to pH, salt, metals and
desiccation.
Physicochemical Factors Affecting the Growth of Enterococci 115
Enterococci, showed the ability to resist pasteurization temperatures
in this study. Consistent results have been reported in previous studies
(Ahmad et al., 2002). E.faecium was more heat resistant than other
enterococci. Similar results were found in many other studies (Kearns et al.,
1995; Bradley and Fraise, 1996). These results confirm that enterococci are
highly thermo resistant bacteria as mentioned in a previous study by Perez et
al., (1982), Heat resistance property is attributed to be responsible for
contamination and spoilage of meat products (Gordon &Ahmed, 1991).
There are several explanations regarding the temperature resistance.
Temperature resistance depends on the membrane structure and has been
related to lipid and fatty acid content (Ivanov, et al., 1999). Enterococci have
been found to respond to sudden increases in temperature by synthesizing the
heat shock proteins (Parsell & Lindquist, 1993).
In this study all enterococci survived and growth occurred in the
presence of 40% bile salts (not shown in table). This capability of
enterococci shows that this pathogen is adapted to grow in the
gastrointestinal tract.
The length of survival of these organisms in a non nutrient medium
may have significant infection control implications. Up to 13 days,
Enterococcus survived on cotton swabs. This is supported by the results
obtained by Wendt et al., (1998) and Neeley and Maley, (2000) who found
similar survival on various dry surfaces (Wendt et al., 1998; Neeley and
Maley, 2000). All enterococcal species had more or less similar survival
period. Compared to other sources, isolates from clinical sources had more
ability to survive in non nutrient dry surface. This obviously contributes to
the enterococcal prevalence and their dissemination in hospital environments
to cause nosocomial infection.
116 Chapter 5
This study evaluated the efficacy of conventional antimicrobial
products like povidone iodine, sodium hypochlorite, cresol, glutaraldehyde
and benzalkonium choride on enterococci from different sources in
planktonic and biofilm forms. MBCs of disinfectants did not vary with
respect to source. There are also some reports with results demonstrating
similar observation (Anderson, 1975).
Chlorine is used extensively in the disinfection of drinking waters to
reduce the problem of bacterial growth in distribution systems and in health
care environments. In this study low concentrations of NaOCl and iodine
effectively killed enterococci. Effectiveness of povidone iodine was
previously proved against vancomycin resistant and sensitive enterococci
(Block et al., 2000). Similarly the efficacy of sodium hypochlorite in
eradicating enterococci has already been reported by Abdulla et al., (2005).
In contrast, there is a report on Enterococcus exhibiting resistance to sodium
hypochlorite (Kearns et al., 1995).
Benzalkonium chloride and glutaraldehyde are common hospital
disinfectants. In this study benzalkonium chloride at a low concentration
(1.25 mg/mL) eradicated most strains of enterococci. The resistant strains
were inhibited, at 2.5mg/mL concentration. It is appealing to note that there
are also reports on benzalkonium chloride resistant genes in enterococci
(Braga et al., 2010). It has been suggested that the widespread use of
quaternary ammonium compounds [QAC] may impose a selective pressure
and contribute to the emergence of QAC resistance. In this study
glutaraldehyde at a low concentration (1.25 mg/mL) eradicated most of the
strains of E.faecalis and E.faecium and all other enterococci. Those resistant
strains of E .faecalis and E.faecium were killed, at a higher concentration.
The effectiveness of glutaraldehyde was reported in another study by
Physicochemical Factors Affecting the Growth of Enterococci 117
Bovallius and Anas, (1977). There is even report on the intrinsic resistance to
glutaraldehyde in enterococci (Fraise, 2002). All enterococcal strains showed
identical elevated pattern of resistance to Cresol. Phenolics were found to be
resistant in many of the studies (Bean and Walters, 1961).
As explained by other authors, this study also supports the
observation that the bacteria sequestrated in biofilms are shielded and are
often harder to be killed than their free floating counterparts (Gilbert et al.,
2002). It was observed in this study that sodium hypochlorite and iodine
were found to be effective in eradicating the bacteria in the biofilm. Paulson,
(2005), was also of opinion that enterococcal biofilm could be removed by
the action of povidone iodine and alcohol. And the usefulness of sodium
hypochlorite against enterococcal biofilm was reported by Dunavant et al.,
(2006). In the present study on planktonic and biofilm, povidone-iodine
treatment even at low concentrations resulted in the elimination of a massive
number of bacteria. Thus, the use of povidone-iodine solution for local
treatment of biofilm may be effective.
Benzalkonium chloride and glutaradehyde are often used in hospitals
for disinfection purpose. They were effective against E.faecium biofilm, at
higher concentrations than the recommended in use concentrations in this
study.
The mechanism of biofilm resistance may be due to the delayed
penetration of disinfectants through the biofilm matrix or the reaction
between the two. Other factors that may be responsible for the increased
resistance of the biofilm cells are the presence of extracellular polymeric
substances, alteration of the target in the bacterial cell, enzymatic
modification or active efflux mechanisms. In order to increase the efficacy
118 Chapter 5
of antiseptic and disinfectants on bacterial biofilms, Takeo et al., (1994)
recommended an increase in the concentration and contact time. But in this
study contact time did not have much influence in the MBC of disinfectant.
Only a slight decrease in the count could be noticed after 1 minute contact
time (data not shown).
In the present study E.faecalis and E.faecium strains showed high
resistance to the disinfectants, while other species were annihilated at lower
concentrations of the disinfectants. Since there is species wise difference in
susceptibility to disinfectants, it is possible that the mechanism of resistance
can also be different in different species. It can be inferred from the
observations in this study that sodium hypochlorite and iodine are the most
effective disinfectants on all enterococcal strains in comparison with other
disinfectants tested. Laboratory experiments have demonstrated that
biocides, present at low concentrations in the environment due to discharge
after use, may lead to an increased selective pressure towards disinfectant
and antibiotic resistance. These observations raise concerns over the
indiscriminate and often inappropriate use of biocides, whereby contributing
to the development of microbial resistance. There are reports suggesting a
possible link between the disinfectant resistance and antibiotic resistance at
the genetic level Codling et al., (2004).
The presence of bacterial biofilms is one of the main challenges in
terms of antimicrobial resistance and is of public health concern, particularly
when dealing with medical isolates (Donlan and Costerton, 2002) Most
laboratories do not use biofilm disinfection tests to assess the efficacy of
biocides and no standards for the testing of disinfectants against biofilms in
health care applications exist (Cookson, 2005). Resistance or increased
tolerance to disinfectants shown by the biofilms in the present study could
Physicochemical Factors Affecting the Growth of Enterococci 119
explain the ability of Enterococcus to persist in the environment and even in
the body. All these difficulties emphasize the importance of regular and
effective disinfection.
Heavy metal resistance is a widespread attribute among
microorganisms isolated from different environments. Enterococcal growth
was not affected much in the presence of low concentrations of zinc. The
pattern of zinc tolerance in the different enterococcal isolates was identical in
the present study. In agreement with the present finding, a high zinc
tolerance was reported by da Silva et al., (2012) in enterococci.
The tolerance of cadmium by the Enterococcus species was not high.
The observations in this study show that enterococci were inhibited by low
concentration of cadmium. But there are reports indicating the requirement
of higher concentration of cadmium for the bactericidal action against
enterococci (da Silva et al., 2012). Furthermore a high natural resistance to
cadmium exhibited by E. faecalis has been reported by Laplace et al.,
(1996).
Even though several studies revealed the effectiveness of cobalt as a
potent antimicrobial agent (Kimiran-Erdem et al.,2007), this study shows
cobalt resistance in enterococci and is possibly due to variation in isolates
from different geographical areas.
Silver ions are widely used as bactericide in catheters, burn wounds
and dental work (Margaret et al., 2006). Silver salts can still be used as
topical antimicrobial agents. In this study silver killed enterococci at a very
low concentration. The effectiveness of silver on enterococci has been
reported by Day and Russell, (1999) & Jones et al., (2004).
120 Chapter 5
Mercury has no beneficial biological role, and is highly toxic to all
forms of life. The results of the study have shown it was active against all
planktonic and biofilms of enterococci. At an incredibly low concentration, it
inhibited enterococci in a study by da Silva et al., (2012). There are reports
on the plasmid borne resistance to mercury transferred by conjugation or
transduction (Ghosh et al., 2000).
E.faecium demonstrated resistance against most of the heavy metal
tested. All other species were relatively less resistant to the heavy metals.
Biofilms were observed to be more resistant to heavy metals than the
planktonic bacteria. Mercury and silver were found to have more action than
the other heavy metals studied.
Generally, the resistance to heavy metals could be due to plasmid
(Bruins et al., 2000). Several other metal resistance mechanisms have been
identified which include exclusion by permeability barrier, intra and extra
cellular sequestration, active transport, efflux pumps, enzymatic
detoxification, and reduction in the sensitivity of the cellular targets to metal
ions (Poole and Gadd, 1989).
Many investigators have also reported the association between heavy
metal and antibiotic resistance (Bhattacherjee et al., 1988 & Lawrence,
2000). The genes encoding heavy metal resistance are located in plasmids
together with antibiotic resistance genes (Dalsgarrd and Guardbassi, 2002) .
It is therefore, likely that selective pressure by any of these indirectly selects
for the whole set of resistance. Since the resistance to different heavy metals
is brought about by similar mechanism, multiple tolerances are common
phenomena among heavy metal resistant bacteria.
Physicochemical Factors Affecting the Growth of Enterococci 121
This study has demonstrated the amazing ability of enterococci
particularly E.faecium to grow at a wide range of temperatures, pH shifts and
high salt and its survival for extended periods in unfavorable environmental
conditions. These properties in turn may favor its growth and survival. The
biofilm formation capability should be taken into account while carrying out
the disinfection. Disinfectant and heavy metal resistance allows enterococci
to survive in the environments where antimicrobial agents are heavily used
such as a hospital environment. This may result in the selective proliferation
and dissemination of the drug resistant enterococci capable of infections.