For a better understanding of a disinfectant's effectiveness and standardization of use in purification system programs, the standard bacterial strains analyzed were established as test microbial suspension. The use of the test microbial suspension is to monitor the disinfection procedure and its performance is dependent on both the initial test microbial suspension population (N0
) and the D-value [9
]. The overkill approach to exposure by a disinfectant agent is based on the premise that the extent of treatment will inactivate the initial bioburden (≥ 106
CFU/mL) and provide an additional safety factor [9
]. Decimal reduction times (D- values), the number of decimal logarithm reduction (n) for the period of application of every disinfecting solution and bacteria tested. The exposure time for n = 6 reduction for every chemical agent and bacteria are shown in Table .
Table 3 Decimal reduction times (D- values), and level of confidence (n = number of decimal logarithm reduction) for the period of application of every disinfecting solution and bacteria tested. The exposure time for n = 6 reduction for every chemical agent and (more ...)
In table the decimal reduction times (D-values, min) are presented for the in house and standard strains, respectively, in contact to the chemical agent. The total contact time (t, min) is a multiple of the D-value (min), considering the following relation t = n*D, where n is the number of decimal logarithmic reduction in the initial population (log N0) of the microorganism, after contact with the chemical agent.
Citric acid (0.5%) when applied to in house strains during 30 min/25°C was able to theoretically (predicted based on the average D-value) reduce 15 log10
cycles of A. lowffi
(D = 1.77 min), the most sensitive bacteria, and reduce theoretically 10 log10
cycles of P. alcaligenes
(D = 2.99 min), they were not supposed to survive the sanitation procedure of the system. This contact time was enough to reduce (5–8 log10
cycles) the following microorganism populations: P. aeruginosa
, P. picketti
, P. alcaligenes
INCQS, P. fluorescens
ATCC 3178, P. picketti
ATCC 5031. However, F. aureum
, P. diminuta
and P. fluorescencens
showed resistance to the contact with citric acid similar to B. subtillis
ATCC 9372 (reduction of 2–3 log10
cycles). Although citric acid is effective against some of the tested strains (gram-negatives), after 30 minutes of contact it is still possible for these microorganisms to survive in the system. To be effective as a sanitizer the suggested contact time would be 3h30 min to achieve n = 6 log10
. Citric acid is also used with heated water (100 – 105°C) for 20 hours in dialyser reprocessing, in these conditions all infective agents including spores are destroyed and depyrogenation may occur, however these temperatures may result in structural damage, limiting the use [12
]. Citric acid is used for cleaning and adjustment of reverse osmosis pH membrane.
Hydrochloric acid is used for cleaning and adjustment of pH on continuous de-ionization of the unit. When in contact to hydrochloric acid (0.5%) the more resistant strains were P. picketti
and B. subtilis
ATCC 9372, both showing reductions lower than n = 3 log10
cycles, this is a result that should be highlighted, considering that a wild strain is as resistant as spores of B. subtilis
ATCC 9372, considered standard strain in high level disinfection procedures [9
]. The most sensitive strains were E. coli
ATCC 25922 and P. diminuta
ATCC 11568 (n≈3).
Alcohol is used to clean the outer surface of sampling points. Alcohols exhibit rapid broad-spectrum antimicrobial activity against vegetative bacteria (including mycobacteria), viruses, and fungi but are not sporicidal. They are, however, known to inhibit sporulation and spore germination [13
], but this effect is reversible [14
]. Because of the lack of sporicidal activity, alcohols are not recommended for sterilization but are widely used for both hard-surface disinfection and skin antisepsis [16
Considering the ethanol contact time of 1 minute, the reduction achieved for the tested strains were not enough to reduce the initial population. D-values, were all higher than 1 minute, the lowest being 2.74 min (P. fluorescens ATCC3178) therefore the contact time should be at least 16.44 min to avoid sampling cross contamination.
For in house (wild) strains, the sodium bisulphite (0.5%) was able to reduce theoretically more than 13 cycles in 90 minutes (recommended contact time), these strains are P. aeruginosa (n = 14); P. diminuta (n = 23); P. fluorescens (n = 13); P. alcaligenes (n = 25); P. picketti (n = 21); F. aureum (n = 24) and A lowffi (n = 18). While on standard strains, P. diminuta ATCC 11568 (n = 13); P. alcaligenes INCQS (n = 16), P.aeruginosa ATCC 15442 (n = 12); P. aeruginosa ATCC 27853 (n = 23); P. fluorescens ATCC 3178 (n = 13); P. picketti ATCC 5031 (n = 21); B. subtilis ATCC 9372 (n = 9); B. subtilis ATCC 6633 (n = 7), and E. coli ATCC 25922 (n = 18). Even though sodium bisulphite is used to preserve and de-chlorine multi-medium filters, softeners and coal filters, it effectively promoted safe level of confidence (n>6) related to the standard and even the wild bacteria isolated from the purified water system, which were not supposed to be found after the disinfection procedure.
Hypochlorites are widely used in healthcare facilities in a variety of settings [17
]. Inorganic chlorine solution is also used for disinfecting of counter tops and floors. Hypochlorites are the most widely used of the chlorine disinfectants and are available in a liquid (e.g., sodium hypochlorite) or solid (e.g., calcium hypochlorite) form. They have a broad spectrum of antimicrobial activity (i.e., bactericidal, virucidal, fungicidal, mycobactericidal, sporicidal), do not leave toxic residues, are unaffected by water hardness, are inexpensive and fast acting, [17
] remove dried or fixed organisms and biofilms from surfaces, [18
] and a low incidence of serious toxicity.
P. aeruginosa (n = 18), P. diminuta (n = 15), P. picketti (n = 13), E. coli ATCC 25922 (n = 11), P. aeruginosa ATCC15442 (n = 13) were more sensitive to the presence of sodium hypochlorite (0.5%) for 60 minutes. Other tested microorganisms decreased between 6–9 log10 cycles, after the contact time. Overall, sodium hypochlorite solution was very effective against the tested stains, keeping a safe level of confidence (n = 6), although it is just used to clean the feeding water tank, the purified water storage tank and distribution loop points.
Sodium hydroxide (0.4%) is used for disinfecting and pH adjustment in reverse osmosis membrane and continuous de-ionization. This solution was able to reduce just 2–3 log10 cycles in 30 minutes of all wild strains and B. subtilis ATCC 9372. However, the initial population of standard strains was reduced more than 5 log10 cycles in 30 minutes.
Minncare™ is used for hygienization of reverse osmosis membranes and continuous de-ionization unit. The association of hydrogen peroxide (2.2%) + peracetic acid (0.45%), Minncare™, was the most effective tested solution against the bacteria strains tested, promoting between 24 and 63 log10 reduction in the initial population of B. subtilis ATCC 9372 (the most resistant strain), and P. picketti ATCC 5031 (the most sensitive strain), respectively.
Hydrogen peroxide (H2
) is a widely used biocide for disinfection, sterilization, and antisepsis. It is a clear, colorless liquid that is commercially available in a variety of concentrations ranging from 3 to 90%. H2
is considered environmentally friendly, because it can rapidly degrade into the innocuous products water and oxygen. Although pure solutions are generally stable, most contain stabilizers to prevent decomposition. H2
demonstrates broad-spectrum efficacy against viruses, bacteria, yeasts, and bacterial spores [19
]. In general, greater activity is seen against gram-positive than gram-negative bacteria; however, the presence of catalase or other peroxidases in these organisms can increase tolerance in the presence of lower concentrations. Higher concentrations of H2
(10 to 30%) and longer contact times are required for sporicidal activity [20
]. Peracetic acid (CH3
COOOH) is considered a more potent biocide than hydrogen peroxide, being sporicidal, bactericidal, virucidal, and fungicidal at low concentrations (0.3%) [19
]. PAA also decomposes to safe by-products (acetic acid and oxygen) but has the added advantages of being free from decomposition by peroxidases, unlike H2
, and remaining active in the presence of organic loads [15
]. Its main application is as a low-temperature liquid sterilant for medical devices, flexible scopes, and hemodialyzers, but it is also used as an environmental surface sterilant. Similar to H2
, PAA probably denatures proteins and enzymes and increases cell wall permeability by disrupting sulfhydryl and sulfur bonds [15