MICROBIOLOGICAL EVALUATION OF DIFFERENT ANTISEPTIC POVIDONE-IODINE AND CHLORHEXIDINE FORMULATIONS AFTER INTENTIONAL CONTAMINATION OF CONTAINERS

This study aimed to evaluate the survival rate of microorganisms within different antiseptic formulations – povidone-iodine (PVP-I) and chlorhexidine (CHX) – after intentional contamination, and to establish the minimum care necessary to ensure sterilization of non-disposable antiseptic solution containers. A laboratory study was performed with 180 antiseptic containers, which were contaminated with Serratia marcescens [1 x 105 UFC/ mL]. The containers were closed and stored, at room temperature, during seven days and shaken daily. The antiseptic cultures were evaluated to be 100% negative to Serratia marcescens in all of the non-disposable containers. These results suggested that antiseptic solutions inactivate microorganisms [1 x 105 UFC/mL]. Since cleaned antiseptic containers have around 102 UFC coming from tap water, it can be inferred that cleansing is a safe minimum procedure to ensure reuse of containers for distribution of CHX and PVP-I solutions in aqueous, detergent and alcoholic formulations.


INTRODUCTION
Antiseptics are anti-microbial substances used on skin and mucosa to reduce bacterial counts (1) .
Attempts to use these agents to prevent infections and reduce their complications originated in Hippocrates' time.Currently, the main use of antiseptics in health institutions is: to cleanse hands, to prepare patients' skin before surgery, to degerm hands and forearms of the surgical team, and in some invasive procedures like central and arterial venous puncture, vascular and vesical catheterism, among others, where the individual's normal barriers of defense are broken (1)(2) .
Reports found in the literature regarding antiseptic products infected by microbiological contamination are a cause of concern for professionals in the area of infection control related to health care, because these contaminations have been the cause of outbreaks in hospitals (1)(2) .
Despite uncertainties regarding microbiological resistance to biocides, concentrations currently used in health services are considerably higher that the Minimum Inhibitory Concentration (MIC) of bacteria able to grow in antiseptic solutions (10)   .
The contamination of these solutions is mainly associated to exogenous causes.Inadequate storage and failure in keeping the stability of the product formulation can partially contribute to the apparent contamination found in practice (11)   .
Contamination related to the processing of containers, whose practice is largely discussed in health services with no scientific basis, was not found among publications on antiseptic contamination.
Therefore, this study aimed to evaluate the survival rate of microorganisms within different formulations of antiseptics -CHX and PVPI-I -after intentional contamination of containers with Serratia marcescens and, based on laboratory results, to establish minimum care to be delivered to non-disposable containers used to store antiseptics.

MATERIAL AND METHOD
Experimental After one month, the study continued with group II, using the same work method, in which the PVP-I solutions were tested in the three different formulations (alcoholic, detergent and aqueous).
The media were prepared by adding a specific neutralizer for chlorhexidine (tween 80 0.5%) and for the PVP-I (sodium sulphite 0.1%).
Positive control: growth of test microorganism in 10 mL of TSB to control the strain viability and equivalence to the prepared media (two tubes).
Negative control: two limpid TSB (two tubes) incubated at 22ºC for 72 hours indicated absence of microorganisms.

RESULTS
In the experiments of groups I and II, 100%

DISCUSSION
The processing of non-disposable containers is a practice not explained in the literature and largely discussed in health services with no scientific basis.
The method used to search for the minimum procedure indicated for the processing of these containers was to verify the antiseptics' capacity to inactivate microorganisms intentionally inoculated in the containers through contamination in the order of 1 x 105 UFC/mL.This inoculum was considered strong due to the potential contamination antiseptics would be exposed to when bottled in containers cleaned only with tap water and detergent.
When the results of this study are extrapolated, cleansing is verified to be the minimum procedure required before safe reuse of containers, because the load of microorganisms present in the cleaned container would certainly be smaller that the inoculum challenge of 1 x 10 5 UFC/mL.Therefore, a new challenge emerges in the processing of these containers: the heterogeneous contamination of the rinse water.Researchers have showed there is low microbiological load in surgical material after cleaning, on the average 10 2 UFC/ material (12-14)   .
The legal parameters for water potability, Rule nº 518 of March 25 th , 2004, issued by the Brazilian Ministry of Health, "establishes the procedures and responsibilities related to the control and surveillance of water quality for human consumption and its potability standards, in addition to other necessary steps" (15) .The expected microbiological standard is found with a tolerance of contamination much lower than the tested inoculum in this study: absence of E.
coli or thermotolerant coliforms in 100mL of water.
This is an original in vitro study, since publications that presented contamination of antiseptics result from outbreaks in care practice.In Brazil, laboratory studies on microbiological action of biocides gained expression as from the 1980s (16) .
The main references consulted on antiseptic contamination appoint issues of handling and inadequate care that lead to product instability and allow microorganisms to grow (3)(4)(5)(6) .
Cases of antiseptic contamination were also reported with findings of organic debris in the solution and biofilm on internal container walls, evidenced by electronic microscopy (7)(8)(9) .Their presence might have provided protection to the contaminant.The formation of biofilm is a process in which microorganisms irreversibly adhere and grow on the surface, producing extracellular polymer that ease adherence and matrix formation.However, physical methods, such as mechanical cleansing or friction, or even ultrasound, are effective in assuring its removal, provided that it is adequately performed (17) .
As previously mentioned, concentrations currently used in health services are substantially higher than the Minimum Inhibitor Concentration of these bacteria (10) .Thus, the contamination of antiseptic solutions is not currently associated to reduced susceptibility of microorganisms.
Deductive reasoning was used in this study to conclude that cleaning is the minimum procedure for processing containers in which antiseptics are bottled.This consideration is strongly based on studies that confirm that the number of microorganisms recovered from only cleaned material is around 10 2 UFC (12)(13)(14) .
, laboratory study, developed in the Laboratory of Microbiology at the Department of medical-surgery nursing at the University of Sao Paulo, School of Nursing.Inoculum A sample of the culture Serratia marcescens ATCC 14756 was kept in BHI (Brain Heart Infusion) culture and undercultivated in Tryptone Soy Broth (TSB).After 10 5 UFC/ml of inoculum size was confirmed (turbidity equivalent in the McFarland scale), the material was used to contaminate the containers.The inoculation of containers was done in 1 mL collected through automated mini-pipette and distributed on the entire internal container wall with rotating movements.Sample size: after the pretest, the experiment sample size was established at 30 samples for each formulation (CHX and PVP-I in aqueous, detergent and alcoholic formulations), totaling 180 containers, with 95% confidence interval (p<0.05).Experimental planning: The investigated antiseptic solutions -CHX and PVP-I -in the aqueous, detergent and alcoholic formulations were divided in two groups and tested at different moments: Group I -chlorhexidine and Group II -PVP-I.First, 90 containers were intentionally contaminated with 1mL of 10 5 UFC/mL of S. marcescens suspension.The three different formulations of chlorhexidine antiseptic (alcoholic, detergent and aqueous) were distributed in 150ml volumes.To make sure the experiments were initiated with no microorganisms, all containers were previously cleaned and sterilized with ethylene oxide (ETO).The containers were lidded and stored at room temperature for seven days and shaken daily, three times a day, at 8AM, 2PM and 8PM, so that the antiseptic solutions would enter in contact with surfaces above the 150mL level.Aliquot parts of 1mL of each of the 90 solutions of CHX in test were pipetted daily and transferred to tubes containing the culture, in order to verify the growth of S. marcescens.Therefore, at the end of seven days, 630 TSB solutions were cultivated (90 per day during seven days) and inoculated with the antiseptic sample contaminated with the test microorganism.The inoculated tubes were incubated at room temperature during 21 days, with daily reading in the first week, and on the 7 th , 14 th and 21 st days.
Considering this deductive reasoning, adequate care with cleaning, rinsing, and drying containers is important for safety control.Residues of detergent can occasionally destabilize chemical formulations, and residues from rinse water can dilute antiseptic concentration.Rev