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
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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
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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
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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