Aim: To investigate the effect
of neutralization of reactive oxygen species (ROS-neutralized conditions)
on the enumeration of chlorine-injured Escherichia coli and Enterococcus
faecalis using selective and nonselective media.
Methods: Pure cultures of E.
coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute
sodium hypochlorite, at free chlorine levels of 0·6 and 0·9 μg ml 1,
respectively, and then enumerated at 37°C by surface plate counts on nonselective
nutrient (N) agar and on several selective media, either under (i) standard
aerobic conditions; (ii) aerobic conditions using growth medium, supplemented
with 0·05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions
designed to neutralize ROS, using a combination of 0·05%-w/v sodium pyruvate
in the growth medium, together with incubation in an anaerobic jar.
Results: The counts obtained on
the nonselective medium were lowest under aerobic conditions in unsupplemented
medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and
highest for ROS-neutralized conditions. Counts for the selective media
were often lower than those for nonselective N (nutrient) agar, with enhancement
under peroxide-neutralized conditions and a further increase in counts
under ROS-neutralized conditions. Broadly similar observations were made
for three other strains of each organism.
Conclusions: Chlorine-injured
E. coli and Ent. faecalis become sensitive to ROS, giving
higher counts under ROS-neutralized enumeration conditions than under conventional
aerobic conditions.
Significance and Impact of the Study: The
enhancement in counts observed under ROS-neutralized conditions indicate
that the addition of pyruvate to the growth medium may not fully counteract
the effects of sublethal injury under aerobic conditions, which is a novel
observation. Thus, ROS-neutralized conditions may be required for optimal
enumeration of faecal indicator bacteria. Furthermore, the lower counts,
obtained using selective media indicate that the sensitivity of chlorine-injured
bacteria to selective agents is not necessarily reversed under ROS-neutralized
conditions. |